expert opinion on biological therapy

1. Introduction

2. The pros and cons of

TH17-based immunotherapy

3. Can dendritic cells be educated

to drive TH17 responses against

ovarian cancer?

4. Expert opinion

Editorial

Dendritic cell vaccination againstovarian cancer -- tipping theTreg/TH17 balance to therapeuticadvantage?Martin J Cannon†, Hannah Goyne, Pamela J B Stone &Maurizio Chiriva-Internati†University of Arkansas for Medical Sciences, Department of Microbiology and Immunology,

Little Rock, Arkansas, USA

The pathology of ovarian cancer is characterized by profound immuno-

suppression in the tumor microenvironment. Mechanisms that contribute to

the immunosuppressed state include tumor infiltration by regulatory T cells

(Treg), expression of B7-H1 (PDL-1), which can promote T cell anergy and apo-

ptosis through engagement of PD-1 expressed by effector T cells, and expres-

sion of indoleamine 2,3-dioxygenase (IDO), which can also contribute to

effector T cell anergy. Expression of both B7-H1 and IDO has been associated

with differentiation and recruitment of Treg, and clinical studies have shown

that each of these mechanisms correlates independently with increased mor-

bidity and mortality in patients with ovarian cancer. In a remarkable counter-

point to these observations, ovarian tumor infiltration with TH17 cells

correlates with markedly improved clinical outcomes. In this Future Perspec-

tives review, we argue that dendritic cell (DC) vaccination designed to drive

tumor-antigen-specific TH17 T cell responses, combined with adjuvant treat-

ments that abrogate immunosuppressive mechanisms operative in the tumor

microenvironment, offers the potential for clinical benefit in the treatment

of ovarian cancer. We also discuss pharmacological approaches to modulation

of MAP kinase signaling for manipulation of the functional plasticity of DC,

such that they may be directed to promote TH17 responses following

DC vaccination.

Keywords: dendritic cells, ovarian cancer, p38 MAPK, regulatory T cells, TH17 T cells

Expert Opin. Biol. Ther. (2011) 11(4):441-445

1. Introduction

In recent years, it has become increasingly apparent that ovarian tumors avail them-selves of multiple mechanisms of immune evasion, the most prominent of which isrecruitment and infiltration of regulatory T cells that suppress anti-tumor immu-nity. Landmark studies from Curiel and colleagues showed that regulatory T cells(Treg) are recruited to ovarian tumors by the chemokine CCL22 (predominantlyexpressed by ovarian tumors), and that the presence of Treg confers immune priv-ilege and is associated with a poor prognosis and increased mortality [1]. Otherinvestigators have corroborated these observations, showing that high expressionof the forkhead box transcription factor foxp3, which is preferentially expressedby CD4+ Treg, is an independent prognostic factor for reduced overall survival inovarian cancer [2], and that a high CD8+ T cell:Treg ratio is associated with amore favorable prognosis for this disease [3]. These observations support the idea

10.1517/14712598.2011.554812 © 2011 Informa UK, Ltd. ISSN 1471-2598 441All rights reserved: reproduction in whole or in part not permitted

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that depletion of tumor-associated Treg, or inhibition of Tregfunction, may be beneficial, particularly in conjunction withactive tumor-specific immunotherapy.In contrast with the strong evidence that Treg infiltration is

associated with poor outcomes in ovarian cancer (and othermalignancies), the recent observation that TH17 T cell infil-tration in ovarian cancer correlates with markedly more favor-able clinical outcomes provides a striking counterpoint [4].Tumor-infiltrating TH17 cells were positively associatedwith effector cells and negatively associated with Treg infiltra-tion, with the latter relationship arguably being founded onthe known reciprocal regulation of Treg and TH17 differenti-ation [5,6]. Tumor-associated macrophages were shown to beefficient inducers of T cell IL-17 production, through anIL-1b-dependent mechanism [4], an observation that is con-sistent with evidence pointing to a critical role for IL-1b inthe induction of human TH17 responses [7-9]. Furthermore,Kryczek and colleagues found a positive correlation betweenascites IL-17 and the TH1-associated chemokines CXCL9and CXCL10, and provided evidence that TH17 T cellproduction of IL-17 and IFN-g-induced expression ofCXCL10. In turn, the levels of CXCL9 and CXCL10 intumor ascites positively correlated with tumor-infiltratingCD8+ T cells [4].

2. The pros and cons of TH17-basedimmunotherapy

These observations have inevitably led to the question ofwhether TH17 cells could be therapeutically induced orexpanded, either by tumor vaccines or adoptive immunother-apy [10]. Although the current evidence in ovarian cancerappears to present a strong case in favor of TH17-based anti-tumor immunotherapy, this is a controversial issue, sincea number of studies have indicated a role for IL-17 inpromoting tumor growth and invasion [11-16]. On the otherhand, several recent reports have supported the view thatTH17 responses may have therapeutic benefit in promotinganti-tumor immunity and survival. In the B16 mouse modelof melanoma, adoptive T cell therapy with tumor-specificTH17 cells prompted strong activation of tumor-specificCD8+ T cells (which were required for the antitumor effect),thus indicating that TH17-driven inflammation can play apivotal role in antitumor immunity [17]. Induction ofTH17 responses in a mouse model of pancreatic cancer hasalso been shown to delay tumor growth and improve sur-vival [18]. In similar vein, tumor growth and pulmonarymetastasis was enhanced following injection of the MC38colon cancer cell line in IL-17-deficient mice [19], again sug-gesting a protective role for IL-17-expressing T cells. Mostnotably, the pretreatment frequency of CD4+ TH17 cells inprostate cancer patients was found to correlate with the clini-cal response to a whole-cell vaccine [20], suggesting that theassociation of TH17 cells with improved survival may not beunique to ovarian cancer.

Furthermore, and in marked contrast with the prevailingopinion that CD4+ TH1 T cell responses and CD8+ CTLresponses represent an optimal line of attack for antitumorimmunotherapy, recent evidence has suggested that TH17-based cellular immunotherapy may offer the potential forgreater therapeutic efficacy. Groundbreaking studies fromthe National Cancer Institute have clearly shown that adop-tively transferred CD4+ TH17 cells were markedly moreeffective than CD4+ TH1 cells in eradication of advancedB16 melanoma in a mouse model [21]. These investigators fur-ther showed that, compared with TH1 cells, TH17 cells enjoya survival advantage in vivo, suggesting that their improvedpersistence may be a key reason for their greater ability tocontrol disease.

3. Can dendritic cells be educated to driveTH17 responses against ovarian cancer?

This section is based on the premise that active immuno-therapy, and particularly dendritic cell (DC) vaccination,designed to drive a tumor-antigen-specific TH17 T cellresponse holds the potential to be of clinical benefit forpatients with ovarian cancer. Various studies have shownthat TH17 T cell differentiation in vitro can readily bedriven by cytokines, notably IL-1b (see above), suggestingthat tumor-antigen-specific TH17-based adoptive T cellimmunotherapy may be a viable approach for treatmentof ovarian cancer. However, such procedures are cumber-some and complex, and are not readily translated to clinicalpractice. A more practical and efficient alternative may befound with DC vaccination. DC are remarkable for theirplasticity in directing T cell differentiation and effectorfunction, and thus the key to success may reside in ourability to educate DC to drive ovarian tumor-antigen-specific TH17 responses. How could this be achieved?Several recent studies have indicated that regulation of thep38 and extracellular-signal-regulated kinase(ERK)--MAPkinase (MAPK) signal transduction pathways in DC playsa central role in direction of T cell differentiation. Inhibi-tion of MEK 1/2 and ERK MAPK signaling promotesIL-12 production and TH1 T cell responses, whereas inhi-bition of p38 MAPK increases signal transduction throughERK 1/2 and blocks IL-12 production [22]. At face value,these observations suggest that inhibition of p38 MAPKsignaling would be disadvantageous for DC-driven anti-tumor T cell responses, since this would abrogate TH1responses, which are widely held to be important foreffective anti-tumor immunity. However, p38 inhibitionpromotes differentiation and survival of monocyte-derivedDC [23], and p38 inhibition or MEK/ERK MAPK activa-tion restores deficiencies in DC function in myelomapatients [24], suggesting that treatment of DC with pharma-cological inhibitors of p38 signaling may confer some ben-efit. Furthermore, p38 MAPK signaling in DC is associatedwith increased expression of IL-10 and the induction of

Dendritic cell vaccination against ovarian cancer -- tipping the Treg/TH17 balance to therapeutic advantage?

442 Expert Opin. Biol. Ther. (2011) 11(4)

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tolerance in a mouse model of melanoma, thus contributingto the suppression of anti-tumor T cell responses [25].Inhibition of p38 signaling in DC from tumor-bearingmice markedly suppressed expression of IL-10 and restoredthe capacity of DC to stimulate T cells.

It is of particular significance that blockade of thep38 pathway can attenuate Treg induction by DC [26],whereas blockade of the ERK pathway suppresses DC-drivenTH17 responses [27], suggesting that p38 blockade (whichenhances ERK phosphorylation) may favor a switch fromTreg induction to TH17 differentiation and expansion. Theseobservations could have major implications for the rationaldesign of DC vaccines against ovarian cancer.

4. Expert opinion

The proposal that tumor-antigen-specific CD4+ TH17immune responses may benefit cancer patients is a challengingposition to adopt. Based on experimental evidence, there islittle doubt that TH17 responses can drive tumor progression,invasion and angiogenesis. On the other hand, it is equallyevident from experimental models and clinical studies thatTH17 responses can support robust anti-tumor immunityand favor patient survival. How can these apparently oppos-ing observations be reconciled? First, it is probable thatTH17 responses are not homogeneous, and that differingeffector functions under that broad umbrella are likely tohave different outcomes. The ultimate challenge for tumorimmunologists will be to dissect the nuances of TH17 func-tion, and to determine how to drive a response that favorsanti-tumor immunity rather than disease progression [16].

In the case of ovarian cancer, clinical evidence presentsa strong rationale for basing active immunotherapy onstrategies that drive a TH17 response [4]. We propose thatmanipulation of DC function to drive ovarian tumor-anti-gen-specific TH17 responses may afford the best opportunityfor immunological treatment of ovarian cancer through DCvaccination. We have also discussed experimental evidencethat inhibition of the p38 MAPK signaling pathway in DCmay be an appropriate line of investigation to achievethis goal.

Assuming that such a strategy is viable, there remainnumerous barriers to successful DC vaccination for ovariancancer. Immunosuppressive mechanisms operative in the

ovarian tumor microenvironment include infiltrating Treg(discussed above), and expression of B7-H1 (programmeddeath ligand 1 (PDL-1)) by tumor cells and infiltratingmacrophages, resulting in apoptosis and anergy [28,29]. Ofparticular clinical interest, a retrospective analysis of humanovarian cancers revealed that patients with higher B7-H1expression had a significantly poorer prognosis than thosefor whom the tumors had lower B7-H1 expression [30].Expression of indoleamine 2,3-dioxygenase (IDO), whichcan contribute to recruitment of Tregs [31,32], has also beenassociated with poor clinical outcomes in ovarian cancer [33,34].Tumor expression of endothelin-1, which can inhibit effectorT cell migration across vascular endothelium into the tumormicroenvironment, may also reduce the efficacy of immuno-therapy or vaccination [35]. The optimal strategy for DC vac-cination may thus combine adjuvant treatments designed toabrogate immunosuppression in the tumor microenviron-ment. B7-H1 may be blocked with specific antibodies, andIDO function can be blocked with 1-methyl-tryptophan, acompetitive inhibitor of enzyme function that is currentlybeing tested in clinical trials. Small-molecule antagonists ofendothelin receptors are also undergoing clinical tests [36].Last, but not least, various strategies can be applied to abro-gation of tumor-associated Treg activity, notably treatmentwith denileukin diftitox (ONTAK) or low-dose cyclophos-phamide [37]. Paclitaxel, which is commonly used for treat-ment of ovarian cancer, may also have activity againstTreg [38]. Given the current weight of evidence, we wouldadvocate further studies on the potential for treatment ofovarian cancer with DC vaccination formulated to driveTH17 responses, in combination with adjuvant treatmentsdesigned to blockade immunosuppressive mechanisms thatprevail in the ovarian tumor microenvironment.

Declaration of interest

The authors are sponsored by an NIH grantUL1RR029884-01, Arkansas Center for Clinical Transla-tional Research. MJ Cannon is founder of DCV TechnologiesInc, a biotechnology company dedicated to the clinical devel-opement of dendritic cell vaccines for the treatment of cancer.M Chiriva-Internati is founder of Kiromic Inc, a biotech-nology company that seeks to develop therapeutic cancervaccines. The other authors declare no conflict of interest.

Cannon, Goyne, Stone & Chiriva-Internati

Expert Opin. Biol. Ther. (2011) 11(4) 443

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AffiliationMartin J Cannon†1,2 PhD, Hannah Goyne1 MD,

Pamela J B Stone2 MD &

Maurizio Chiriva-Internati3 PhD†Author for correspondence1University of Arkansas for Medical Sciences,

Department of Microbiology and Immunology,

Little Rock, Arkansas, USA

Tel: +1 501 296 1254; Fax: +1 501 686 5359;

E-mail: [email protected] of Arkansas for Medical Sciences,

Division of Gynecologic Oncology,

Department of Obstetrics and Gynecology,

Little Rock, Arkansas, USA3University Health Sciences Center,

Division of Hematology and Oncology,

Department of Internal Medicine,

Texas Tech Lubbock, Texas, USA

Cannon, Goyne, Stone & Chiriva-Internati


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1. Introduction

2. Cell transplantation for stroke

3. Stroke-induced neurogenesis

4. Conclusion

5. Expert opinion

Review

Stem cells and stroke:opportunities, challenges andstrategiesTerry C Burns & Gary K Steinberg†

Stanford University School of Medicine, Department of Neurosurgery, Stanford, CA, USA

Introduction: Stroke remains the leading cause of disability in the Western

world. Despite decades of work, no clinically effective therapies exist to facil-

itate recovery from stroke. Stem cells may have the potential to minimize

injury and promote recovery after stroke.

Areas covered: Transplanted stem cells have been shown in animal models to

migrate to the injured region, secrete neurotrophic compounds, promote

revascularization, enhance plasticity and regulate the inflammatory response,

thereby minimizing injury. Endogenous neural stem cells also have a remark-

able propensity to respond to injury. Under select conditions, subventricular

zone progenitors may be mobilized to replace lost neurons. In response to focal

infarcts, neuroblasts play important trophic roles to minimize neural injury.

Importantly, these endogenous repair mechanisms may be experimentally aug-

mented, leading to robust improvements in function. Ongoing clinical studies

are now assessing the safety and feasibility of cell-based therapies for stroke.

Expert opinion:We outline the unique challenges and potential pitfalls in the

clinical translation of stem cell research for stroke. We then detail what

we believe to be the specific basic science and clinical strategies needed to

overcome these challenges, fill remaining gaps in knowledge and facilitate

development of clinically viable stem cell-based therapies for stroke.

Keywords: clinical trial, differentiation, ischemic brain injury, migration,

neural progenitor cell, neuroblast, neurogenesis, neuroprotection, neuroregeneration, plasticity,

stem cell, stroke, subventricular zone, translational research


1. Introduction

With an incidence of almost 800,000 new victims per year in theUSA alone, stroke per-sists as the leading cause of disability and the third leading cause of mortality in theWestern world. Stroke leads to rapid destruction of brain tissue over several hours,with an estimated 1.9million neurons, representing approximately 14 billion synapses,dying each minute [1]. It is important to recognize that each lost neuron was born at aspecified time and location during development as a result of complex sequences ofphysical and chemical signals as well as intrinsic timingmechanisms guiding progenitorcell fate. After birth, the immature neurons were precisely guided into appropriatelocations, from which they extended projections along intersecting gradients of diffus-ible, membrane and extracellular matrix-bound molecules. They then competedsuccessfully for neurotrophic signals and established thousands of activity- andexperience-dependant synaptic connections. In the wake of stroke, these intricate net-works are swiftly reduced to an expanding necroticmilieu of dead and dying cells. Adja-cent neurons teeter on the edge of viability with marginal blood supply, wheremounting inflammatory responses may mediate additional cell death. In addition toneuronal cell loss, even greater numbers of glia with probably under-appreciatedlocation-defining and regulatory as well as supportive roles are also destroyed.


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Restoration of blood flow within the first three to four hours ofstroke onset enables measurable improvements in outcome.However, only a small minority of patients arrive early enoughto receive effective therapy. Despite decades of work and prom-ising animal data, neuroprotective strategies aiming to limit fur-ther exacerbation of cell loss within and beyond this timeframehave uniformly failed in human trials [2-5].Stem cells have the potential to generate nearly unlimited

numbers of neural cells. Given the complex fidelity of neuronaldevelopment and integration, however, true cell replacementhas proven an elusive goal. During the past decade, dozens ofcell types have been tested via multiple routes of delivery innumerous animal models of stroke; in many cases, markedlydecreased lesion size and improved functional outcomes havebeen achieved. Though some have claimed ‘replacement’ ofneurons by transplanted cells, others have encounteredpoor survival despite functional benefits, suggesting indirect

mechanisms of recovery. Others have sought to stimulate thebrain’s own stem cells toward regeneration with promising pre-liminary results [6]. Here we evaluate the preclinical and clinicalprogress of stem cell therapy to date. We discuss current evi-dence regarding mechanisms of action, and outline pertinentopportunities, challenges and strategies for safe and effectivetranslation of stem cell therapy into clinical practice.

2. Cell transplantation for stroke

2.1 Exogenous stem cellsPreclinical studies of cell transplantation have identified asurprising variety of cells that promote functional recoveryafter stroke. Work to optimize delivery parameters such asroute, timing, cell dose and immunosuppression is ongoing.To date, demonstrated mechanisms of benefit have includeddirect inhibition of cell death, enhanced regeneration ofvasculature, immunomodulation, induction of neuronalplasticity and promotion of endogenous neurogenesis.

2.1.1 Human fetal brain cellsPioneering cell transplantation work focused initially on replace-ment of dopaminergic neurons for Parkinson’s disease (PD).Studies employing fetal midbrain demonstrated behavioral ben-efits from ‘cell replacement’, prompting several clinical trialswith variable outcomes. In the mid 1980s, Polezhaev and Alex-androva performed transplantations of fetal brain tissue into ratbrains after ischemic injury. Robust engraftment was observedwith evidence of synaptic integration. Grafts also decreased celldeath and promoted the restoration of ‘dysfunctional’ neuronsto their normal state [7]. Grafts, which seemed to survive bestin the penumbra [8] improved local neurotransmitter levels andfacilitated cognitive recovery [9]. Human fetal brain tissue is alimited and ethically challenging resource. As such, no clinicaltrials of fetal cells have been pursued for stroke and significantefforts have sought to develop alternate cell types that may bemore readily amenable to widespread clinical application.

2.1.2 Human teratocarcinoma cellsA teratocarcinoma cell line, NT2, was shown in 1984 to gen-erate pure populations of post-mitotic neural-like cells uponexposure to retinoic acid [10]. In 2000, based on preclinicalevidence for functional improvements in animal models ofstroke [11], these became the first cells reported in a Phase Iclinical trial of a cell-based therapy for stroke (Table 1) [12].Cells were grafted stereotactically into patients with stabledeficits after a basal ganglia infarct; immunosuppression wascontinued for 2 months. Overall, no adverse effects werenoted, and surviving cells were observed post-mortem withno evidence of neoplasm at 27 months [13]. In 2005, thereport of a Phase II randomized controlled trial involving14 treatment and 4 control patients revealed functionalimprovements in some patients. Given the very smallgroup sizes, improvements based on a primary outcomemeasure of European stroke scale at 6 months did not

Article highlights.

. To date, demonstrated mechanisms of stem cell benefithave included direct inhibition of cell death, enhancedregeneration of vasculature, immunomodulation,induction of neuronal plasticity, and promotion ofendogenous neurogenesis.

. Both bone marrow mononuclear cells (BMMCs) andmesenchymal stem cells (MSCs) seem to have quitelimited survival in the brain after either local or systemicdelivery. Thus it is likely that benefits are mediatedpredominantly via trophic signals of variable duration.

. Recent meta-analysis of preclinical studies employingintravenous cell delivery indicated that neural stem cells(NSCs) yielded the greatest behavioral improvements whencompared with bone-marrow-derived or other cell types.

. The recent development of techniques to generateinduced pluripotent stem (iPS) cells, opens the potentialfor autologous neural cell therapy, thereby averting theneed for immunosuppression.

. The optimal timing for cell delivery is unclear, but maydepend upon the predominant. mechanism of action.Therapies aiming for neuroprotection will require earlierdelivery than those targeting neuroplasticity.

. Exogenous cell therapy may act, in part, by augmentingthe endogenous neurogenic response to stroke.

. Increased endogenous progenitor cell proliferation andneuroblast recruitment may persist for at least severalmonths after ischemic injury.

. Multiple cellular and molecular tools now exist toenhance endogenous responses to stroke.

. The failure of hundreds of neuroprotective compoundsin clinical trials illustrates the sobering challenge aheadof translational therapy for stroke.

. In the treatment of stroke, it remains true that ‘time isbrain’. Cell therapies should be developed in conjunctionwith optimized recanalization technologies to targetresidual areas of ischemia, combat reperfusion injuryand provide trophic support in areas ofhemorrhagic conversion.

This box summarizes key points contained in the article.

Stem cells and stroke: opportunities, challenges and strategies


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Table

1.Publish

edtrials

ofcelltherapyforstroke.

Ref.

Sponso

r/location

Cellnumber/

type/route

Age

Infarct

severity/

type

Treatm

ent

window

nPhase

Primary

outcome

Randomized?

Blinding

Follow

up

Resu

lts

Comments

Kondziolka

etal.,2000

[12]

Universityof

Pittsburgh,

PA,USA

2or6million/

hNTcells/IC

44--75

Basalganglia

infarct

6months--

6years

12

ISafety,

feasibility

No

No

6months

Safe.European

StrokeScore

improvedat

6months

(p=0.046)

Adverseevents

intw

opatients,

possibly

unrelated

Kondiolka

etal.,2005

[14]

Universityof

Pittsburgh,PA&

Stanford

University,

CA,USA

5or10million/

hNTcells/IC

40--70

Basalganglia

infarct,

ESS10--45

1--5years

14+

4controls

IIEuropean

StrokeScore

Yes

Observer

only

6months

Feasible;primary

outcomemeasure

notmet

Adverseevents

intw

opatients,

possibly

unrelated.

Somemeasures

improved

Savitz

etal.,

2005

[16]

Harvard

MA

and

Cornell,

NY,USA

Upto

50million/

fetalporcine

cells/IC

25--52

NIHSS5--11

1.5

--10years

5I

Safety

No

No

4years

FDA

term

inated

trialdueto

possible

side

effects

Improvements

intw

opatients

remainedstable

for4years

Bangetal.,

2005

[29]*

YonseiUniversity,

Souel,SKorea

50million�

2/Autologous

MSCs/IV

30--75

NIHSS>6

32--61days

5+

25controls

I/II

Safety

Yes

Observer

only

12months

Safe,feasible;

Barthelindex

higherat

3and6months

only

Decreasedexvacuo

ventriculardilitation

at12monthsin

cellgroup,p=0.019

Suarez-

Monteagudo

etal.,2009

[19]

CentroInternacional

deRestauracion

Neurologica,

Habana,Cuba

14--55million/

Autologous

BMMCs/IC

41--64

NIHSS

10.6

±0.92

1--10years

5I

Safety,

tolerance

No

No

12months

Safe;

neuropsychiatric

improvements

insomepatients

Epileptic-likeactivity

onEEG

at6and

12months;

noclinical

seizures

Barbosa

da

Fonseca

etal.,2010

[18]

UniversidadFederal,

Rio

deJaneiro,Brazil

125--500million/

autologous

BMMCs/IA

24--65

NIHSS

4--17

8--12weeks

6I

Neurologic

deficits

No

No

120days

Noneurologic

worsening

Cells

notseenin

brain

bySPECT

beyond24h

Leeetal.,

2010

[30]*

YonseiUniversity,

Souel,SKorea

50million�

2/Autologous

MSCs/IV

30--75

NIHSS>6

5--7weeks

16+

36controls

I/II

Safety

Yes

Observer

only

5years

Safe,feasible;

mRSim

proved

(p=0.046),

best

ifSVZintact

Enrollm

entsuspended

dueto

concern

regardinganim

al

productsin

culture

media

*[30]Includespatients

previouslyreportedin

[29];mRSnotsignificantlydifferentbetw

eengroupsin

[29];Barthelindexnotreportedin

Lee[30].

BMMC:Bonemarrow

mononuclearcell;

hNT:Humanteratocarcinoma-derivedneuralcellline;IA:Intra-arterial;IC:Intracerebral;IV:Intravenous;

MSC:Mesenchym

alstem

cell;

na:Notavailable;

NIHSS:NationalInstitutesofHealthStrokeScale;SPECT:Single-photonemission-computedtomography.

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reach statistical significance. Reported adverse outcomesincluded one seizure and one subdural hematoma requiringevacuation [14].

2.1.3 Porcine fetal neural cellsA major challenge of adult neural cell therapy is the relativelyinhibitory environment presented by the adult brain toneurite outgrowth. It has been suggested that molecular dif-ferences between species may permit better engraftment ofxenograft than allograft neurons [15]. In 2005, Savitz et al.published results from a trial employing stereotactic deliveryof up to 50 million anti-MHC1 antibody-treated fetal por-cine cells for stable basal ganglia stroke [16]. Of five patientsenrolled, one showed temporary worsening of symptomsand another had a seizure. Both had questionably concerningfindings on MRI, prompting the FDA to terminate enroll-ment. Two of the five patients experienced improvements insymptoms over several months that persisted at four years [16].

2.1.4 Bone marrow mononuclear cells (BMMCs)Endogenous bone-marrow-derived cells are swiftly recruitedto regions of ischemic injury. Administration of supplementalbone marrow mononuclear cells has been under investigationin animal models of stroke since 2000 [17], with benefitsattributed to various trophic mechanisms, in spite of largelypoor long term survival. To date, BMMCs have appearedwell tolerated in multiple small clinical trials, mostly involvingintravascular delivery [18]. However, Suarez-Monteafudo et al.recently reported long-term, asymptomatic EEG abnormali-ties after intraparenchymal BMMC administration [19]. Inter-estingly, meta-analysis of BMMCs in clinical trials for acutemyocardial infarction indicated a 4.77% improvement inleft ventricular ejection fraction after three months [20].G-CSF, which also has direct neurotrophic effects, may inpart replicate the action of BBMCs by promoting mobiliza-tion of bone marrow cells. G-CSF is now under clinical inves-tigation for stroke [21,22], having previously enabled a 3%increase in ejection fraction in meta-analysis of clinical trialsfor acute myocardial infarction [23].

2.1.5 Mesenchymal stem cells (MSCs)By selectively culturing bone-marrow-derived cells that adhereto a culture dish in serum-containing media, cells variablytermed marrow stromal cells or mesenchymal stem cells(MSCs) are generated that have shown benefit in animalmodels of stroke [24]. Recent meta-analysis of intravenously-delivered cells in preclinical studies for stroke showed thebeneficial effect of MSCs on behavioral outcome to beroughly twice that of BMMCs, consistent with the samestudy’s finding that cell lines and cultured or genetically mod-ified cells are significantly more efficacious than primarycells [25]. Much literature has focused on conditions thatmay promote the neuronal differentiation of such cells eitherin vitro or in vivo after transplantation. However, few if anysuch claims withstand current standards of scrutiny [26,27].

MSCs offer a somewhat more homogeneous and wellcharacterized cell population for cell transplantation. Thesecultured cells are also amenable to genetic manipulation,allowing targeted delivery of specific therapeutic compounds.Both BMMCs and MSCs seem to have quite limited survivalin the brain after either local or systemic delivery. Thus it islikely that benefits are mediated predominantly via trophicsignals of variable duration. Though widely regarded assafe, some reports of MSC-derived sarcomas have appeared,suggesting that limits on passage numbers and stringentstandards of cytogenetic quality control will be required forclinical applications [28].

Lee and colleagues recently published five year follow-up data from a previously reported [29] randomized openlabel trial of intravenous administration of two doses of50 million autologous MSCs. Five year outcomes suggestedsignificantly improved modified Rankin Scale scores, asassessed by blinded observers (p = 0.046). It is of interestthat levels of stromal cell-derived factor-1 (SDF-1), whichhave been associated with MSC, as well as neural stemcell (NSC) homing, were found to correlate positivelywith clinical outcomes [30]. MSCs have been suggested tostimulate endogenous neurogenesis after stroke [31]. Thus,it is worth noting that patients in whom the subventricularzone (SVZ) was spared from infarct (n = 5) uniformlyimproved with MSC therapy, though outcomes in MSC-treated patients with infarct extending to the SVZ(n = 11) were more variable. Although no adverse effectswere observed in MSC-treated patients within five years,recruitment was suspended due to the publication ofconcerns regarding use of xenogenic bovine calf serum inculture media for grafted cells [30].

2.1.6 Neural stem cellsTechniques for the in vitro culture of neural stem cells werefirst described in the early 1990s by Reynolds and Weiss [32].With inherent neurogenic potential, demonstrated trophicbenefit and minimal risk of tumorgenicity, NSCs representan excellent cell therapy choice and have been widelyemployed in pre-clinical stroke studies during the past 10 yearswith encouraging results [25,33]. Recent meta-analysis of pre-clinical studies employing intravenous cell delivery indicatedthat NSCs yielded the greatest behavioral improvementswhen compared with bone-marrow-derived or other celltypes [25]. Due in part to regulatory delays, very few clinicaltrials have been initiated employing neural stem cells. NSC-like olfactory ensheathing cells from the olfactory mucosahave been employed in a clinical trial for spinal cord injurywith early establishment of safety and feasibility [34]. Neuralstem cells from Stem Cells, Inc. were employed in a recentlycompleted Phase I trial of six patients for neuronal ceroidlipofuscinosis, also known as Batten’s disease [35]. The resultsof this study remain to be published. An open label trialof the neural stem cell line CTX0E03, from ReNeuron,began in June 2010 and plans to enroll 12 patients for



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intraparenchymal delivery of 2 -- 20 million cells, 6 -- 12months following subcortical stroke (Table 2).

2.1.7 Embryonic stem cells (ESCs) and induced

pluripotent stem (iPS) cellsESCs possess the defining capacity to generate all cell types ofa developing embryo under appropriate conditions. ESCshave received particular attention as a source of cells for whichno reliable tissue-specific progenitor is available, such as cardi-omyocytes, as well as certain neuronal lineages not readilyobtainable from NSCs, including dopaminergic neurons forPD and motor neurons for amyotrophic lateral sclerosis(ALS). The recent development of techniques to generateES-like cells via epigenetic reprogramming, termed inducedpluripotent stem cells, or ‘iPS cells’, opens the potential forautologous neural cell therapy, thereby averting the need forimmunosuppression. Safety concerns regarding the viral con-structs used to reprogram iPS cells are being mitigated by thedevelopment of transient transfection techniques that leavecells genetically unaltered after reprogramming [36]. By defini-tion, however, ESCs do generate teratomas. As such, thedevelopment of differentiation and culture techniques thateliminate any residual undifferentiated ESCs continues tobe a high priority. Neural stem cell lines derived fromhESCs have been generated that promote functional recoveryin animal models of stroke without tumor formation, and areunder development for future clinical applications [37,38].Researchers at Wernig’s lab recently demonstrated thatselective genetic reprogramming may enable direct trans-differentiation of fibroblasts to functional neuronal cellswithout the need for an intermediate ES or iPS cell stage [39].

In 2009, Geron received FDA approval to initiate the firstever clinical trial employing hESC-derived cells. This trial, fortreatment of spinal cord injury, is based on the observationthat pure cultures of hESC-derived oligodendrocyte precur-sors cells (OPCs) promote functional recovery by remyelinat-ing axons in spinal cord-contused rats. A clinical holdimposed shortly after initial approval by the FDA for furthersafety evaluations was lifted on 30 July 2010, allowing thetrial to proceed.

2.2 Delivery variables for exogenous cell therapyFor any given cell type, a number of options are availableregarding when, where and how to implant, and what adjunc-tive treatments should additionally be administered (Table 3).The variety of protocols in use suggests that ‘right’ answers tothese questions are not easily determined; optimal parametersmay vary depending on the model, cell type, extent of injuryand outcome measure being assessed.

2.2.1 Administration routeThough not without risks, stereotactic delivery allows precisetargeting of defined numbers of cells to desired sites, withbest survival seen in the peri-infarct region. The first clinicaltrials of NT2 stem cells for chronic basal ganglia stroke

involved 25 cell deposits along 5 stereotactic tracts throughoutthe infarct area [40]. A recent protocol involving up to 88deposits targeted selectively to the peri-lesion area was recentlydescribed for administration of MSCs [19].

Most studies of bone-marrow-derived cells to date haveemployed intravenous delivery. Meta-analysis of preclinicalresults suggests robust benefit, in spite of limited evidencefor significant numbers of cells reaching the infarct site [25].Intra-arterial or intra-carotid therapy has been advocated byseveral groups to facilitate delivery to the ischemic regionand minimize cell sequestration in systemic tissues such asliver, lung and spleen [41]. With appropriate protocols to reg-ulate cell density and allow continued blood flow duringinjection, risk of microembolic infarcts resulting from adher-ent cell clusters or vessel occlusion can be minimized [42].Brain penetration of NSCs after intra-arterial deliveryappears to be dependent upon upregulation of vascular celladhesion molecule-1 (VCAM-1) following stroke, whichbinds the cell surface integrin CD49 that is expressed on theNSCs [43]. Future studies may assess whether or not geneticmanipulation of receptor expression enhances targeting tothe ischemic region. By comparison with stereotactic implan-tation, intravascular approaches have the advantage of readilyallowing repeated administrations of cells. Combinationsof intraparenchymal and intravascular therapies may alsobe feasible.

2.2.2 Cell dosageRecent meta-analysis of intravenously-delivered cells in ani-mal studies showed a robust effect of cell dosage on lesionsize, behavioral outcome and molecular measures of outcomesuch as apoptosis, neurogenesis and angiogenesis [25]. Darsaliaet al., recently demonstrated that the percentage of survivingcells is decreased with intraparenchymal delivery of largernumbers of cells. However, the total number of surviving cellstrended upwards with incremental increases in cell dose [44].The potential benefits of higher cell dose must be weighedagainst potential risks, including potential mass effects, theo-retical risks of increased tumorgenicity in certain cells andpotential for embolic events with intra-arterial delivery. Assuch, potential toxicity must be evaluated via appropriatedose--response analyses in preclinical studies [2].

2.2.3 ImmunosuppressionThe use of immunosuppression for cell therapy in CNS disor-ders is controversial [45]. Erlandsson et al., recently demon-strated that immunosuppression promotes endogenousprogenitor migration and tissue regeneration with enhancedaccumulation of SVZ-derived cells at the site of corticalinjury [46]. By contrast, in meta-analysis of preclinical studiesof IV-delivered stem cells, immunosuppression had no signif-icant impact on behavioral outcomes, though a trend wasnoted towards more favorable outcomes without immunosup-pression [25]. It should be noted that preclinical studies ofhuman cell lines in animal models will almost always require

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Table

2.Ongoingandpendingtrials

ofcelltherapyforstroke.

Clinicaltrials

ID[Ref.]Sponso

r/location

Cellnumber/

type/route

Age

Infarct

severity/type

Treatm

ent

window

nPhase

Primary

outcome

Randomized?

Blinding

Start

date

Enddate

NCT00473057

[109]

FederalUniversity

ofRio

deJaneiro,

Brazil

500million/

autologous

BMMC/IA

versusIV

18--75

NIHSS4--20

3--90days

15

INeurologic

deficits

No

Openlabel

December

2005

June2011

NCT00535197

[110]

ImperialCollege

London,UK

na/autologous

CD34+cells

from

bone

marrow/IA

30--80

Totalanterior

circulation

syndrome

<7days

10

I/II

Toxicity

No

Openlabel

September

2007

June2010

NCT00593242

[111]

DukeUniversity,

NC,USA

5million

cells/kg/

autologous

cord

blood/IV

<14days

Neonatal

hypoxic/

ischemic

injury

<14days

12

IAdverseevents

No

Openlabel

January

2008

January

2011

NCT01028794

[112]

National

Cardiovascular

Center,Osaka,

Japan

25or50cc

bonemarrow/

autologous

BMMCs/IV

20--75

NIHSS>9

7--10days

12

I/IIA

NIHSS

No

Openlabel

May

2008

March2012

NCT00761982

[113]

HospitalUniversitario

CentraldeAsturias,

Spain

na/autologous

CD34+cells

from

bonemarrow/IA

18--80

NIHSS>7;MCA

5--9days

20

I/II

Adverseevents

No

Single-blind

(assessor)

September

2008

March

2010

NCT00859014

[114]

TheUniversity

ofTexasHealth

Science

Center,

Houston,USA

na/autologous

BMMCs/IV

18--80

R:NIHSS6--15;

LNIHSS6--18

24--72h

10

ISafety,feasibility

No

Openlabel

January

2009

January

2014

NCT00950521

[115]

ChinaMedical

UniversityHospital,

Taichung,Taiwan

2--8million/

autologous

CD34+cells

from

peripheral

blood/IC

35--70

NIHSS9--20

6--60months30

IINIHSS

Yes

Openlabel

June

2009

December

2010

NCT01019733

[116]

HospitalUniversitario,

Nuevo

Leon,Mexico

8--10cc

bone

marrow/CD34+

cells

post

G-CSF/IT

1month

--

18years

Cerebralpalsy

1month

--

18years

10

IBattelle

Devel.

Inventory

No

Openlabel

July

2009

August

2010

CTRI/2008/091/

000046

[117]

IndianCouncilof

MedicalResearch,

New

Delhi

(multi-institution)

30-500million/

BMMCs/IV

18--70

NIHSS>7;

Anteriorcirculation

infarct

7--30d

120

IIModifiedBarthel

Index,

Safety

Yes

Openlabel

January

2009

June2011

NCT01091701

[118]

Stempeutics

Research

Pvt

Ltd,Malaysia

2millioncells/kg/

adultallogenic

MSCsIV

20--80

mRS<5with

motorweakness

<10days

78

I/II

Adverseevents,

NIHSS

Yes

Double

blind

May

2010

December

2011

*TransientNotch1transfection.

BMMC:Bonemarrow

mononuclearcell;

ESS:EuropeanStrokeScale;IA:Intra-arterial;IC:Intracerebral;IV:Intravenous;

MCA:Middle

cerebralartery;MSC:Mesenchym

alstem

cell;

na:Notavailable;

NIHSS:NationalInstitutesofHealthStrokeScale.



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host immunosuppression, which, along with nature of thexenograft model itself, may significantly influence results.To date, autologous cells have been limited to bone marrow,and clinical trials of allogeneic cells for stroke have employedtemporary immunosuppression [13]. Given the difficulty ofaccurately extrapolating immunosuppression results from ani-mal studies, optimal protocols for allogenic cell grafts may bebest derived in the setting of appropriately controlled clinicaltrials. Further development of iPS or nuclear reprogrammingtechnologies may ultimately enable autologous human cells tobe differentiated toward neural or neural stem cell lineagesprior to grafting without need for immunosuppression.

2.2.4 TimingThe optimal timing for cell delivery is unclear, but maydepend upon the predominant mechanism of action. Thera-pies aiming for neuroprotection will require earlier deliverythan those targeting neuroplasticity. Some studies suggestoptimal survival of transplanted cells at early time points(e.g., 48 h) before inflammatory responses are maximal [44],though studies have demonstrated robust benefit even whendelivered over one month after stroke [47]. Meta-analysis ofanimal studies employing IV cell delivery found that thedegree of inhibition of apoptosis was the strongest predictorof functional outcome [25]. This same study demonstrated anon-significant trend towards improved benefit with earliercell delivery [25]. Stem cells have yet to be evaluated as adjunctsto thrombolysis or thrombectomy. However, pre-banked allo-geneic cells may be feasibly delivered at quite early time pointsin this setting. Autologous therapies and intraparenchymaldelivery will predictably be associated with greater delays.

2.2.5 AdjunctsCellular grafts may be supplemented by extracellular matrixproducts to improve survival and neurite outgrowth [48,49] Cellsmay be genetically modified to secrete selected growth factors,with significantly enhanced therapeutic outcomes [25]. Alterna-tively, cells may be grafted after specialized pre-treatmentprotocols ranging from relative hypoxia to cytokine pre-treatment or cellular co-culture. Delivery of cocktails contain-ing multiple cell types, or adjunctive viral constructs is alsofeasible. Finally, cells grafted in clinical trials of stroke have pre-dominantly been identified based on histopathological evalua-tion at autopsy. The modification of cells with appropriatetransgenic or other cellular labels may markedly improve thecapacity to track cells in vivo after transplantation, via MRIand/or positron-emission tomography (PET) [50].

2.3 Proposed therapeutic mechanisms2.3.1 IntegrationGraft-derived neurons can survive and mature, formingsynaptic connections to host brain circuitry after transplan-tation of fetal [51], ESC-derived [52,53], and NSC-derived [54]

cells into stroke-lesioned rodents. However, what role suchintegration, plays, if any, in functional recovery is unclear.T

able

2.Ongoingandpendingtrials

ofcelltherapyforstroke(continued).

Clinicaltrials

ID[Ref.]Sponso

r/location

Cellnumber/

type/route

Age

Infarct

severity/type

Treatm

ent

window

nPhase

Primary

outcome

Randomized?

Blinding

Start

date

Enddate

NCT01151124

[119]

ReNeuron,Ltd,

Glasgow,UK

2,5,10,

20million/

CTX0E03neural

stem

cells/IC

60--85

NHSS>5;

Subcorticalwhite

matterorbasal

ganglia

6--24months12

IAdverseeffects,

MRI,NIIH

SS,

antibodies

No

Openlabel

June

2010

na

NCT00875654

[120]

UniversityHospital,

Grenoble,France

na/autologous

MSCs/IV

18--65

NIHSS>2

<14days

30

IIFeasibility,

tolerance

Yes

Openlabel

September

2010

December

2013

NCT00908856

[121]

Universityof

California,Irvine,

CA,USA

30cc

bonemarrow/

autologousBMMCs/IV

18--85

NIHSS7--24;

supratentorial

2--21days

33

IIMortality

Yes

Double

blind

January

2011

December

2012

na

SanBio,Inc.,

Mountain

View,

CA,USA

2.5,5.0,10million/

SB623cells

--

modified*BMMCs/IC

18--75

mRS3--4;

ESS40--50;

SubcorticalMCA

orstriatum

+/-

cortex

6--12months18

I/II

Adverseeffects,

acute

and

long-term

safety

No

Openlabel

January

2011

na

*TransientNotch1transfection.

BMMC:Bonemarrow

mononuclearcell;

ESS:EuropeanStrokeScale;IA:Intra-arterial;IC:Intracerebral;IV:Intravenous;

MCA:Middle

cerebralartery;MSC:Mesenchym

alstem

cell;

na:Notavailable;

NIHSS:NationalInstitutesofHealthStrokeScale.

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Benefits are frequently seen at early time points, well beforegrafted cells could mature and form synaptic connections.Benefits may also be seen in the presence of few, if any sur-viving cells, and after grafting of cells devoid of neurogenicpotential. As such, popular consensus now favors trophicmechanisms as the predominant basis for functional gainsafter cell transplantation [55]. Selective ablation of graftedcells, for example via administration of diphtheria toxin inrodents after human cell grafting, would be needed to criti-cally assess the requirement for ongoing graft survival formaintenance of functional gains [56]. No such studies instroke-lesioned animals have yet been reported. In lesionsinvolving cell death of select neuronal subpopulations withmaintenance of surrounding cytoarchitecture, integrationof grafted cells may be more feasible than after focal infarctand cavitation [57]. Use of supportive extracellular matriciesmay further maximize integration potential [58].

2.3.2 NeuroprotectionBone-marrow-derived and neural stem cells produce animpressive array of neuroprotective compounds. In a meta-analysis of 60 preclinical studies of intravenously-deliveredcells, Janowski et al. demonstrated that outcomes were moststrongly correlated with inhibition of apoptosis [25]. Less sig-nificant correlations were also found with neurogenesis andangiogenesis. Endogenous NSC-derived neuroblasts intrinsi-cally migrate to the injured region following stroke and pro-mote neuroprotection. This endogenous neuroprotectionmay be significantly bolstered by supplementation with exog-enous cells. Careful attention to cell source may be important.Takahashi et al. found that NSCs derived from embryoswere more effective than those derived from adults in mitigat-ing ischemic damage [59]. Neuroprotection may be directvia secretion of neuroprotective compounds or indirect,

via immunomodulation, angiogenesis or amplifying theendogenous NSC response.

2.3.3 ImmunomodulationAfter acute ischemic injury, secondary injury may occur as aresult of inflammatory mediators. Microglia are among thepredominant regulators of the local inflammatory environ-ment and may be modulated by grafted cells [60]. It shouldbe noted that meta-analysis of preclinical studies employingintravenous cell delivery failed to find a significant correlationbetween immunomodulation and outcome [25]. However, theinteractions between inflammatory signals and stem cells arenotoriously complex and conflicting literature abounds. As ageneral rule, although SVZ and hippocampal neurogenesisincreases after stroke, inflammatory signals following strokeimpair neurogenesis [61]. Anti-inflammatory treatments, suchas indomethacin, can increase neurogenesis following focalstroke [62]. It should be noted, however, that ablation of acti-vated microglia exacerbates infarct size [63], consistent with anadditional role of inflammation in the reparative process [64].The immunosuppressive and anti-inflammatory effects ofmultiple stem cell populations are well documented [65].Exactly when and how these effects impact outcomesfollowing stroke requires further study. Unlike simple anti-inflammatory treatments, it is conceivable that stem cellsrespond dynamically to changing inflammatory signals overtime and may adjust their regulatory activities accordingly.Ideally, future studies should be undertaken in humanizedmouse models to maximize insights relevant to human clinicaltherapies [66].

2.3.4 Vascular repairThe integral relationship between endothelial cells andneural progenitors is well established [67,68]. Neural precursors

Table 3. Overview of exogenous cell types.

Exogenous cell type Tumor risk Cost Survival Autologous Comments Progress of clincal trials

Human fetal brain cells - $$$ + - Limited availablility;ethical challenges

None planned

Teratocarcinoma cells + $$ + - First cell type in trialsfor stroke

Early trials completed.No more planned

Porcine fetal neural cells - $$ + - Complex immuneconsiderations

Prior trial aborted by FDA.None planned

Bone marrowmononuclear cells

- $ +/- + Most readily availablecell type

Trials completed andin progress

MSCs + $$ +/- +/- Trophic effects despitepoor survival

Trials completed andin progress

Neural stem cells + $$$ ++ - Robust efficacy data inanimal studies

Trials now starting

Embyonic-stem-derivedcells

++ $$$$ ++ + (iPS only) iPS cells now available.May be used togenerate NSCs

Still in pre-clinical phase

+: Applicable; ++: Highly applicable; -: Not applicable; +/-: Variable; iPS: Induced pluripotent stem cell; MSC: Mesenchymal stem cell; NSC: Neural stem cell.



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promote endothelial proliferation in the peri-infarct region.Conversely, such proliferation appears to enhance the recruit-ment of SVZ-derived neuroblasts [68]. Bone-marrow-derivedstem cells similarly secrete multiple pro-angiogenic compoundsincluding VEGF, EGF and IGF-1 in response to signalsfrom ischemic brain [69], promoting endothelial proliferationin the peri-infarct region [68]. Blockade of angiogenesis inBMMC-treated cells markedly impedes recovery [70].

2.3.5 PlasticityThe adult brain possesses much greater plasticity than previ-ously appreciated. The spontaneous development of new hostprojections after stroke has been significantly correlated withbehavioral recovery [71]. Moreover, mice devoid of thrombo-spondin 1 and 2, important for synapse formation and plastic-ity, demonstrate poor functional recovery after stroke [72].Recent studies demonstrate that plasticity, as evidenced byincreased synapse formation and new neuronal projections, issignificantly enhanced by treatment with bone marrow-derivedor neural stem cells following stroke [60,73].

2.3.6 Recruitment of endogenous neural

progenitorsNeural stem cells proliferate and give rise to neuroblasts thatmigrate toward the injured region following stroke. Theseneuroblasts exert neuroprotective and pro-angiogenic effectsupon arrival in the peri-infarct region. Bone-marrow-derivedstem cells are known to secrete a variety of compounds thatpromote the proliferation and migration of endogenous neu-ral progenitor cells, suggesting that exogenous cell therapymay act, in part, by augmenting the endogenous neurogenicresponse to stroke [30,74].

3. Stroke-induced neurogenesis

In the adult brain, neural stem cells are located in the hippo-campal dentate gyrus [75] and SVZ [76] that give rise to newfunctional neurons throughout life. Hippocampal NSCsmodulate learning, memory and spatial navigation as well aspsychiatric states [77]. In the SVZ, slowly dividing stem cellsgenerate transit amplifying cells, which in turn generate neu-roblasts [78]. Unlike hippocampal NSC progeny that remainin the dentate gyrus, SVZ neuroblasts migrate along the ros-tral migratory stream (RMS) to generate functional olfactorybulb neurons [79], though they can be redirected towards areasof injury.

3.1 Subventricular zone response to focal infarctsAfter ischemic stroke, hypoxia-induced signals promote theproliferation of neural stem and progenitor cells. SDF-1 andangiopoietin redirect neuroblasts from the SVZ and RMSalong blood vessels toward the infarct region [80]. Rare newneurons are generated, though most recruited cells die orremain undifferentiated in association with blood vesselsnear the infarct boundary zone [80,81]. Increased progenitor

cell proliferation and neuroblast recruitment may persist forat least several months after ischemic injury [82].

Given the very low numbers of new neurons generated, therelevance of stroke-induced neurogenesis to functional recov-ery has been controversial, and has been examined via severalexperiments in the past decade employing irradiation orchemotherapeutic agents to impede neurogenesis. Thesemanipulations worsened stroke outcomes, but conclusionswere tentative, given possible toxicity from the experimentaltreatment. In 2006, Won et al. demonstrated that reelinmice lacking doublecortin (dcx), a protein required for neuro-blast migration, had larger infarcts and worsened behavioraloutcomes following stroke [83]. However, baseline behavioraldeficits in these mice somewhat hampered interpretation. In2009, Jim et al. selectively ablated migrating dcx+ cells andsimilarly observed increased infarct size as well as substantiallyworsened behavioral scores within days following stroke [84].These studies collectively imply that immature endogenousneuroblasts act locally at the peri-infarct region to promoteneuronal survival, well before any new mature neurons couldpossibly be generated.

The exact mechanisms via which endogenous neuroblastsenhance outcomes are incompletely defined. However, NSCsare known to produce neurotrophic factors such as nervegrowth factor (NGF) and glial-cell-derived neurotrophic factor(GDNF), to regulate the inflammatory environment, and toproduce pro-angiogenic complexes including netrin-4, lami-nin and integrins [85]. Notably, NSCs constitutively secrete fac-tors implicated in synaptic plasticity, including those that areconsidered anti-angiogenic, such as thrombospondins [72]. Itis likely that the factors generated by NSC progeny varydynamically as the infarct injury evolves. The specific factorsgenerated by endogenous recruited neuroblasts and theirtemporal patterns of expression after ischemia remain to beevaluated. Such analysis may provide fundamental insightsregarding the endogenous response to ischemic injury.

A plethora of signaling molecules including VEGF, brainderived neurotrophic factor (BDNF), erythropoietin (EPO),fibroblast growth factor 2 (FGF2), noggin, notch, IGF-1,TGF-alpha, stem cell factor (SCF), nitric oxide (NO), EGF,angiopoietin, microphage-interacting protein-1-alpha (MIP1-alpha), stromal cell-derived factor 1 (SDF-1), cell surfacemolecules, including CXCR4, vascular cell adhesion molecule(VCAM), integrins and extracellular matrix molecules, arenow known to regulate NSC proliferation, migration and dif-ferentiation [86,87]. Experimental manipulation of these path-ways in rodents can substantially bolster the neurogenicresponse with subsequently decreased lesion size and improvedfunctional outcomes [88]. The clinical implications of thesefindings may be substantial. Manipulations that increase pro-genitor proliferation and migration with improvements infunctional outcomes may be observed without significantincreases in the number of new post-mitotic neurons generated.However, increased numbers of number of new neurons maybe observed with overexpression of basic fibroblast growth

Burns & Steinberg


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factor (bFGF) [82]. Moreover, ‘filling’ of the infarct cavity withnew neurons may be attained with sequential administration ofEPO and EGF [89]. While robust functional improvementsare observed in these cases, the capacity of the new neuronsto form functional connections with surrounding circuitry orcontribute to functional recovery remains to be assessed.

3.2 Selective neuronal replacement by endogenous

progenitorsIn animal models of selective neuronal loss, endogenous neu-ral progenitors appear more inclined to replace the lost celltype. CA1 neurons in the hippocampus are particularly sus-ceptible to ischemic injury [90]. Spontaneous regeneration ofCA1 cells was observed after hypoxic injury, with cells arisingfrom the periventricular region. This regenerative responsecould be significantly augmented by intraventricular deliveryof EGF and bFGF. Functional recovery occurred in a delayedmanner, which correlated with the appearance, maturationand electrophysiological integration of new neurons. Inanother example, Macklis’ lab developed a selective photo-ablation technique to delete subsets of cortical projection neu-rons without hypoxia, ischemia or other injury. Remarkably,SVZ-derived neurons appeared to migrate along the corpuscallosum to the affected region, and then replace ablated neu-rons with subsequent generation of long distance corticospinaland corticothalamic projections [91,92]. The signals responsiblefor recruitment and directed differentiation of the new corti-cal projection neurons in this case remain unclear, thoughwould be of fundamental importance to any attempts topromote regeneration after cortical injury.

3.3 New neurons from cortical progenitors?Increasingly frequent reports of cortical progenitor cellswith neurogenic potential have appeared in recent years.Ohira et al. recently found that the rat layer 1 cortex containsKi67+/67 kDa glutamic acid decarboxylase (GAD67)+ cellsthat bear few of the usual NSC markers, and generate nonew neurons under baseline conditions. Impressively, inresponse to hypoxic injury, these cells generated new inhibi-tory neurons throughout the cortex that appeared to integrateinto local circuitry and survive for at least eight weeks [93].These cells probably correspond to the mitotically active glialfibrillary acidic protein (GFAP)+ cells in cortical layer 1described recently by Xui et al. [94]. These cells expressedvimentin and nestin in response to a cortical insult, migratinginto deeper cortical layers over subsequent days and assumingan immature neuron-like morphology. The tendency of thesecells to generate GABAergic neurons suggests they may repre-sent residual undifferentiated progeny from the medial gangli-onic eminence where cortical interneurons originate duringdevelopment. The factors regulating the behavior of thisnewly identified population of cells and their response to focalischemic injury remain to be evaluated.Certain astroglial cells from the cortex or subcortical white

matter may also de-differentiate into neural stem cells after

in vitro culture [95,96]. Heinrich et al. recently demonstratedthat reactive adult astrocytes isolated after cortical injury canbe differentiated into glutamatergic or GABAergic neuronsafter transduction with Neurogenein2, or distal-less homeo-box 2 (Dlx2), respectively. These neurons formed maturesynapses with electrophysiological properties of mature neu-rons in vitro. Whether or not a similar strategy may beemployed to redirect glial cells toward neurogenic fatesin vivo after cortical injury remains to be determined [97].Some have argued that small cortical infarcts that spare thestriatum provide a poor stimulus for SVZ neuroblast migra-tion and that neural progenitors surrounding such infarctsmay be locally derived [98]. Lineage tracing studies will beneeded to fully characterize the identity, behavior andfunction of naturally occurring cortical progenitor cells.

3.4 Human implicationsThough distinct in structure from those of rodents, thehuman subventricular zone [99] and rostral migratorystream [100] have been characterized. Moreover, evidence forneuroblasts has been found in multiple studies of patientsafter ischemic and hemorrhagic stroke [101-103]. As such, strat-egies to augment the human neurogenic response may yieldimproved outcomes. As always, rigorous preclinical safetystudies will be needed to ensure factors employed to mobilizeendogenous neural stem cells are safe. For example, EGF andBDNF have both been implicated in the development ofglioma-like growths from SVZ progenitors [104,105], BDNFmay induce spontaneous seizure activity [106], bFGF infusionhas resulted in demyelination [107] and all-causes mortalitywas elevated in a large clinical trial of EPO for treatment ofacute ischemic stroke [108]. Nevertheless, appropriate dosingin animal studies has enabled marked benefits with severalcytokines without clinically untoward effects.

4. Conclusion

Early recanalization remains the most effective treatment foracute stroke by minimizing infarct size. Neural and bone-marrow-derived stem cells appear to function via multiplesynergistic mechanisms to augment natural recovery mecha-nisms. In the acute setting, endogenous endothelial andneural progenitor cells work together to minimize neuronalcell death and both may be bolstered by signals from exoge-nous stem cells. Both neural and bone marrow-derived stemcells directly secrete pro-survival factors in addition to modu-lating the endogenous response to stroke, thereby maximizingthe amount of original neural circuitry that survives the ische-mic insult. Thereafter, stem cells and their progeny functionto promote synaptic plasticity, optimizing functional recov-ery. Multiple cellular and molecular tools now exist toenhance endogenous responses to stroke. While much worklies ahead, ample proof of principle suggests that substantialbenefits may reward an ongoing investment in the scienceand translation of stem cell therapies for stroke.



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5. Expert opinion

5.1 Unique challenges of stem cell therapy for strokeThe failure of hundreds of neuroprotective compoundsin clinical trials illustrates the sobering challenge ahead oftranslational therapy for stroke. Given the inherently hetero-geneous nature of stroke, clinical trials will be prone to inad-equate power, being based on preclinical data from modelsthat imperfectly reflect human disease. They may also sufferfrom being based on a literature that is skewed towards thepublication of only positive results. Collaborative strategieswill be needed to ensure not only that scientifically well-founded studies are initiated, but also that such potentiallybeneficial therapies are not aborted prematurely due toType II errors. Considerations of statistical practicality andtherapeutic potential may be in conflict when therapeuticwindows are selected for trials. Should cells be given earlywhen there is potentially more to gain, or later, when thegains can be most accurately measured from a stable baseline?As a novel technology, stem cells offer new risks, not only topatients, but also to the field as a whole, should early compli-cations undermine public support. Will randomized double-blind studies be acceptable for therapies that may be invasiveand risky, including possible stereotactic intracerebral celldelivery and immunosuppression? Ongoing collaborativediscussions between experts on all sides of the negotiationswill be essential. While stem cell therapies have attracted sig-nificant media hype and venture capital, transparency regard-ing realistic expectations is critical. Regulatory bodies, stakeholders and the public at large should all be prepared for along term investment that is more likely to be marked bysmall steps, than home runs. Refinement of protocols relatedto cell preparation, delivery and detection after the onset ofhuman studies will require diligence and patience.

5.2 Pre-clinical directionsSeveral preclinical questions remain that are pertinent totranslational efforts. How long must cells survive for thera-peutic benefit? Simple timed ablation studies, for example,using diphtheria toxin, are needed and may guide decisionsregarding immunosuppression and cell labeling in clinicaltrials. Selective ablation of specific graft-derived cell typesusing cre--lox technology may help to dissect mechanismsunderlying long-term functional benefits. Knockdown ofputative therapeutic genes in transplanted cells may furtherilluminate molecular mechanisms of benefit. The endogenousneurogenic response remains poorly characterized. Lineagetracing labels based on selective markers such as dcxshould be used for isolation and gene profiling of endogenousneuroblasts at various times post-infarct. Subgroups of

newly born neurons should also be ablated after augmenta-tion studies to assess their functional contributions torecovery. Studies employing humanized mice may yield clini-cally relevant insights regarding immunomodulatory effectsof grafted and mobilized cells, while guiding decisionsregarding immunosuppression.

5.3 Clinical directionsIn the treatment of stroke, it remains true that ‘time is brain’.Cell therapies should be developed in conjunction with opti-mized recanalization technologies to target residual areas ofischemia, combat reperfusion injury and provide trophicsupport in areas of hemorrhagic conversion. Defined ex vivogenetic modifications of grafted cell lines should be consid-ered to introduce transgenic MRI-detectable labels, as wellas inducible suicide genes as insurance against undesired pro-liferation or neoplastic transformation. Transgenes may alsopermit delivery of complementary therapeutic genes. Induc-ible viral constructs should also be prepared for in vivo orcell-based delivery of cytokines for mobilization of endoge-nous NSCs. Use of bicistronic constructs with MRI labelsmay facilitate monitoring for effects upon transduced endog-enous cells, while regulatory elements may improve safety incase of untoward side effects. Therapeutic candidates may beexpanded to include intracerebral hemorrhage, with stereotac-tic cell delivery following stereotactic clot evacuation. Addi-tionally, global ischemic injury after myocardial infarctionmay be amenable to cytokine augmentation of endogenouscell replacement. Upon establishment of safety, stem cell pre-treatment may be indicated for high-risk patients, such as insubarachnoid hemorrhage patients at risk for vasospasm, andpatients undergoing embolizations, complex tumor resectionsor cerebral revascularization procedures.

In sum, although challenges abound, and while vigilanceregarding safety and monitoring will be paramount, maximalefforts are indicated to ensure timely translation of the mostpromising therapies for the treatment of stroke.

Acknowledgements

The authors wish to thank C Samos for editorial assistance.


The authors are supported in part by funding from theWilliam Randolph Hearst Foundation, and Bernard andRonni Lacroute. GK Steinberg has received grants from theNational Institute of Neurological Disorders and Stroke(NINDS) and the California Institute of RegenerativeMedicine. TC Burns declares no conflict of interest.

Burns & Steinberg


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121. Autologous cell therapy after stroke.






AffiliationTerry C Burns MD PhD &

Gary K Steinberg† MD PhD†Author for correspondence

Stanford University School of Medicine,

Department of Neurosurgery,

300 Pasteur Drive, R281,

Stanford, CA 94305-5487, USA

Tel: +1 650 725 5562; Fax: +1 650 723 2815;

E-mail: [email protected]

Burns & Steinberg


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1. Introduction

2. Stem cell trials for

cerebral palsy

3. Potential cell sources

4. Experimental models

5. Possible mechanisms of action

6. Risks of treatment

7. What’s needed next

8. Conclusion

9. Expert opinion

Review

Update on stem cell therapy forcerebral palsyJames E Carroll† & Robert W Mays†Medical College of Georgia, Neurology, Augusta, GA, USA

Introduction: Due to the publicity about stem cell transplantation for the

treatment of cerebral palsy, many families seek information on treatment,

and many travel overseas for cell transplantation. Even so, there is little scien-

tific confirmation of benefit, and therefore existing knowledge in the field

must be summarized.

Areas covered: This paper addresses the clinical protocols examining the

problem, types of stem cells available for transplant, experimental models

used to test the benefit of the cells, possible mechanisms of action, potential

complications of cell treatment and what is needed in the field to help

accelerate cell-based therapies.

Expert opinion: While stem cells may be beneficial in acute injuries of the

CNS the biology of stem cells is not well enough understood in chronic inju-

ries or disorders such as cerebral palsy. More work is required at the basic

level of stem cell biology, in the development of animal models, and finally

in well-conceived clinical trials.

Keywords: animal models, cerebral palsy, embryonic stem cells, induced pluripotent stem cells,

mesenchymal cells, multipotent adult progenitor cells, stem cells, transplantation


1. Introduction

Cerebral palsy is a heterogeneous group of conditions, defined as non-progressive motor disability due to an abnormality of the cerebral hemispheres.While a small proportion of patients with cerebral palsy have as their cause a peri-natal hypoxic-ischemic insult, most have acquired cerebral palsy due to the presenceof one of a wide variety of other illnesses, such as developmental brain abnormali-ties, genetic conditions, traumatic or infectious disorders. Furthermore, insultsmay occur at different times during gestation, resulting in even more variation inpattern and causation. This heterogeneity in cause makes the assessment of anytreatment fraught with considerable difficulty.

Parents, on the other hand, focus on the condition of cerebral palsy and seektreatment based on that terminology. Patoine, in a recent editorial [1], describedthe pressures of a supposed ‘miracle cure’ supplied by stem cells influencing thebehavior of parents of children with cerebral palsy. The United Cerebral PalsyFoundation states that there are 800,000 children and adults in the USAwith cerebral palsy. The Centers for Disease Control estimates that about10,000 babies are born each year with cerebral palsy. Improvements in the careof neonates have done little to alter the percentage of children with cerebral palsy.In fact, the increased survival of very low birth weight infants has contributed tosustaining the present occurrence rate [2]. Thus, the issue of stem cells as a poten-tial treatment for cerebral palsy has assumed a disproportionately elevated posi-tion among parents of children with cerebral palsy. Seven years ago wepresented in this journal the state of stem cell research in cerebral palsy [3]. Whilethere has been definite progress in the scientific study of multiple types of stemcells, particularly the discovery of induced pluripotent stem cells (iPS cells),


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relevant animal models for cerebral palsy are still lacking incritical factors. Consequently, progress with the initiation ofcell based clinical trials for treatment of cerebral palsy hasbeen limited.An additional problem is the timing of treatment. In order

to be effective for most patients with cerebral palsy, the treat-ment will need to address an established or longstanding brainabnormality. But as we accumulate more information aboutthe potential mechanisms of action of stem cells in braininjury, we are led to the conclusion that stem cells are muchmore likely to be effective in the acute situation rather thanlong into the course of a chronic disability. However, it is pos-sible that stem cells could act favorably in a chronic injury byreplacing nerve cells, with even a small replacement being sig-nificant, by making existing connections more effective, or bypromoting blood vessel regeneration.The purpose of this article is to present the current state

of stem cell transplantation for cerebral palsy patients. Wereview the current efforts with patients, the types of cellsthat might be used, the experimental basis for the treatment,animal models for cerebral palsy, the possible mechanismsfor therapeutic success, the need for additional work, andthe potential for harm.

2. Stem cell trials for cerebral palsy

There are two ongoing US trials (Duke University and theMedical College of Georgia) listed in ClinicalTrials.gov [4]

testing the safety and efficacy of autologous umbilical cordblood for cerebral palsy. These trials are obviously depen-dent upon the fact that some parents chose to preserve theirchild’s umbilical cord blood at the time of birth. The factthat the cells are autologous gives a significant safety marginto the trials, which otherwise might not have been allowed toproceed. Given that the parents have a strong commitmentto stem cell therapy and enter the trials only because theyknow their children will receive the cells, both these trials

are double-blinded with a crossover treatment protocol.The crossover allows the children to receive their cells atsome point in the study. The trials attempt to pare downthe long list of causes for cerebral palsy by having extensiveexclusion criteria, such as athetoid cerebral palsy, autism,hypsarrthymia, intractable epilepsy, progressive neurologicaldisorder, HIV infection, extreme microcephaly, knowngenetic disorder, obstructive hydrocephalus, significantdefect of brain development, chromosomal disorder, pres-ence of major congenital anomaly or severe intrauterinegrowth retardation. One of the main justifications for thesetrials is the need to investigate the efficacy of this treatmentin the face of ongoing clinical usage of the treatment. Cur-rently there are no US trials for cerebral palsy dealing withallogeneic cell therapies.

While hypoxic--ischemic injury is a clear cut and easilydefinable cause of cerebral palsy and possibly the mostpotentially open to treatment, this cohort of patients is inthe minority. The current US trials attempt to focus onthis group. Perhaps fewer than 100,000 of the 800,000 indi-viduals with cerebral palsy have hypoxic--ischemic injury astheir cause.

A third trial listed in ClinicalTrails.gov [4] is being con-ducted by the Sung Kwang Medical Foundation in theRepublic of Korea. This study is double-blinded, randomizedwith placebo control using allogeneic umbilical cord blood incombination with erythropoietin. The three arms of the studyare: i) umbilical cord blood, erythropoietin, and rehabilita-tion, ii) erythropoietin and rehabilitation, and iii) rehabilita-tion only. This study employs immunosuppression in orderto allow for the use of allogeneic cells.

A fourth trial listed in ClinicalTrials.gov [4] is active but notrecruiting (Hospital Universitario, Monterrey, Mexico). Inthis trial the patients are given G-CSF in order to stimulatetheir bone marrow to produce stem cells, bone marrow is har-vested, and CD 34+ cells are purified and delivered via theintrathecal route.

Outside the USA, there are a number of facilities thatoffer treatment with various types of stem cell preparationsfor cerebral palsy. These facilities are not conducting formalclinical trials. Stem cells offered from these companies orinstitutions are usually autologous adult stem cells preparedfrom the patient’s own tissue, usually bone marrow. Thespecific details of the preparation methods are generallynot available. The cells are delivered either intravenously orinto the cerebrospinal fluid. Often multiple administrationsare recommended.

3. Potential cell sources

There are many potential cell sources that have been used forexperimental treatment protocols in animal models. The stud-ies employ either direct implantation into brain parenchymaor, more commonly, intravenous injection. We recentlyreviewed the various cell sources [5].

Article highlights.

. Treatment with stem cells is a serious consideration forcerebral palsy parents.

. Several clinical trials are in progress.

. There are numerous types of stem cells that couldbe used.

. While there are many animal models of brain injury,none are completely satisfactory for cerebral palsy.

. The potential mechanism of action of stem cells ispotentially multifaceted.

. Risks of stem cell transplantation are real andprobably understated.

. What is needed includes more knowledge of stem cellbiology, a better chronic injury model and, later on,well-conceived clinical trials.


Update on stem cell therapy for cerebral palsy


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3.1 Mesenchymal stem cells (MSCs)Mesenchymal stem cells (MSCs) are bone marrow stromalcells, comprised of a mixture of cell types, capable of support-ing hematopoiesis along with the capability to differentiateinto multiple cell types. While bone marrow is consideredthe primary source of MSCs, they are also found in humanumbilical cord blood and to a lesser degree in other tissues.MSCs are generally isolated based on their preferential attach-ment to tissue culture plastic. The cells are fibroblast-like andpossess the ability for self renewal. Most of the adult stem cellscurrently studied share some similarities with MSCs.

In all pre-clinical cerebral palsy studies to date testingMSCs, the cells have been administered in the shortterm [6-9], with the longest period being one month afterinjury [10]. The benefit is noted both with intravenous andintracerebral transplantation. The mechanism of cell actionis unknown, but does not appear to be neuronal cell replace-ment. However, the treatment appears to lead to sparing ofintrinsic cells. In a primate model, Li et al. [11] reportedthat the cell transplantation resulted in upregulation ofIL-10 expression. In association they found a decrease inneuronal apoptosis and astroglial activity in the peri-ischemic area. The number of proliferating cells in thesubventricular zone was also increased.

3.2 CD34 cellsCD34 cells are found in umbilical cord blood and bone mar-row. They represent a small subset of MSCs. These cells areisolated based on the presence of a transmembrane glycopro-tein as their surface characteristic. Clinical trials are underwayin stroke patients [4].

3.3 Umbilical cord bloodUmbilical cord blood (UCB) is currently a popular source ofadult stem cells being tested as a therapy for disease andinjury. Numerous private and public banks have arisen inthe USA and other parts of the world. The collection ofumbilical cord blood is somewhat controversial in that variousorganizations, including the American Academy of Pediat-rics [12], have questioned the utility of the collection and pres-ervation in private banks. These concerns are based on thecontention that there are few, if any, proven autologous ther-apies. To date, the main usage of these cells has been treat-ment of childhood diseases of the blood, although theirexperimental use for the treatment of cerebral palsy is cur-rently under investigation. The minimum necessary dosageof cells for cell engraftment is usually considered to be 1 �107 cells per kilogram. This includes the total nucleated cellfraction and not just stem cells. Thus, the child will ‘outgrow’the available dose of autologous cells obtained at birth andavailable for transplant at a later date. Should autologousUCB be found efficacious for the treatment of acquireddisorders, however, its usage would become wide spread.

UCB has been used experimentally in brain injury models.Benefit of the treatment has been shown in a neonatal

hypoxic--ischemic rat model [13], adult rat stroke models [14-16],and a rat traumatic brain injury model [17]. On the otherhand, Makinen et al. [18] did not find benefit with UCB in arat stroke model. These were all acute studies.

3.4 Multipotent adult progenitor cellsMultipotent adult progenitor cells (MAPC) (Athersys) arederived from bone marrow as well as other tissue sources [19,20].The phenotype consists of CD13+, fetal liver kinase1 (Flk1)dim, c-kit-, CD44-, CD45-, MHC class I- and MHCclass II-. These cells differentiate into mesenchymal cells, butalso cells with visceral mesoderm, neuroectoderm and endo-derm characteristics in vitro. They proliferate without senes-cence or loss of differentiation potential. We have used thesecells in a rat model of neonatal hypoxic--ischemic injury,where cell administration results in improvement in behav-ioral outcome and neuronal sparing as determined by histol-ogy. We observed benefit in an acute model via bothintracerebral and intravenous transplantation routes [21].This was an important experiment in that we were able toshow the efficacy of a safe and practical method of administra-tion, that is intravenous. While some of the transplanted cellssurvived, and even displayed neuronal markers, the chiefrestorative feature was enhanced survival of endogenous neu-rons. We speculated this process was mediated by trophic fac-tors, which would be most efficacious in the acute situationand perhaps less so in a chronic injury, as would be the casefor cerebral palsy.

Mays et al. [22] reported recent data from our group in a ratmodel of ischemic stroke. We demonstrated that immuno-suppression was not required for allogeneic or xenogeneiccell mediated benefit. The studies noted that improvementwith MAPC administration persisted at least as long as sixmonths following acute treatment. Based on histologicaldata, it was concluded that MAPC do not exert their benefitvia cell replacement but more probably acted by trophicmechanisms. All of our work with MAPC is in acute studies,and once again we need to show improvement in a chronicinjury model in order to supply pre-clinical evidence thatwould apply to cerebral palsy.

3.5 Induced pluripotent stem cells (IPS cells)Induced pluripotent stem cells (iPS cells) are now consideredto be a substitute for embryonic stem cells [23]. The use of iPScells has not yet been reported in any preclinical model ofbrain injury. It seems that the cells may be an ideal sourcefor tissue repair, as they can be prepared from the patient’sown fibroblasts, eliminating considerations of rejection. How-ever, there are a number of hurdles that will need to be clearedbefore this cell type would be available for clinical usage. First,the safety of the cells will need to be amply demonstrated inanimal models. Do the cells form tumors? Are the viral agentsused in the preparation of the cells a danger to the recipient?Are the cells effective in animal models? Robbins et al. [24]

reviewed the use of these cells for transplantation and

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concluded that reprogramming efficiency and safety consider-ations would need to be addressed before the initiation ofclinical trials. Thus, while iPS cells seem quite promising,much work remains to be done at the basic translationalscience level before they can move into the clinic.

3.6 Oligodendrocyte progenitor cellsOligodendrocyte progenitor cells (OPC) may be derived fromfetal brain tissue [25], embryonic stem cells or iPS cells, the lat-ter two via cell-differentiation protocols. Once again the prob-lem in relation to the chronic nature of cerebral palsy is thatthe models of injury utilized in experimental animals areacute. OPC derived from human embryonic stem cells dem-onstrated some amelioration of function in rats undergoingtraumatic spinal cord injury [26,27]. Keirstead et al. [28] usedhuman embryonic-stem-cell-derived OPC in a rat model ofspinal cord injury and compared the cells in an acute modelversus a chronic model. Animals receiving the transplant sevendays after the injury showed remyelination and improvedmotor ability compared with untreated animals; however theanimals treated 10 months after the injury demonstrated nostatistically significant improvement over control animals.This study underlines the potential difficulty of developingeffective therapeutics in the chronic injury setting of the CNS.Tokumoto et al. [29] evaluated the ability of iPS cells

derived from mouse embryonic fibroblasts to differentiateinto oligodendrocytes and compared this with the differentialability of mouse embryonic stem cells (ESC). They found thatintracellular factors inhibited the differentiation of iPS cellsinto mature oliogodendrocytes.

3.7 Embryonic stem cellsEmbryonic stem cells (ESC) are certainly the most controver-sial type of stem cells. They are derived from embryos and gen-erally require the destruction of that embryo. Consequently,there remain abiding ethical concerns about their use. In addi-tion, the proliferative capacity of the cells and their potential fordifferentiation into many cell types makes the possibility oftumor formation quite real. Given that children receiving thecells would have many years in front of them, there would beample time for tumor formation to occur.The animal models examined with ESCs are all in acute

injuries. Zhang et al. [30] studied transplantation in a rat strokemodel 24 h after the injury and found favorable post-implantation histological changes with survival of the trans-planted cells, their migration and differentiation towardneural cell types. Liu et al. [31] reported that mesenchymal cellsderived from ESCs lessened rat infarction volume, differenti-ated into neuronal and endothelial cells, and improved func-tional outcome when injected intravenously. Ma et al. [32]

showed that embryonic-derived stem cells possessed the abil-ity to migrate into the injury site and improve learning abilityand memory fully eight months after the injury. Even thoughthe benefit of the ESCs was long-lasting, the treatment wasdelivered in the acute phase after injury.

3.8 Fetal stem cellsFinally, stem cells can be collected from fetal tissue. While theutility of these cells has not been widely explored in injurymodels, there are indeed indications of their potential.Aftab et al. [33] demonstrated that retinal progenitor cellsfrom donor tissue of 16 -- 18 weeks gestational age wereable to integrate into host retina and express rhodopsin.In other experiments cells from fetal brain transplantedacutely after hemorrhagic stroke displayed neuroprotectinganti-inflammatory capacity [34].

4. Experimental models

While cerebral palsy is caused by a number of conditions ofwhich brain injury is a minor component, the models forcerebral palsy are generally based on some type of braininjury. The ideas for various therapies, therefore, are predi-cated on the notion that we can reverse the effects of theinjury. Even though this may be the case for an acute injury,this theme does not apply to the many children with cerebralpalsy whose condition arises from abnormalities of braindevelopment. Our discussion in regard to the models of cere-bral palsy is confined to the types of cerebral palsy arisingfrom injury.

Johnston et al. [35] have recently reviewed the availableanimal models and concluded that none are fully adequate.

The Rice-Vannucci model [36] which combines unilateralcarotid artery ligation with hypoxia in 7-day-old rat pupshas been used for numerous studies on the cause and treat-ment of brain injury in the neonatal animal. These are studiesof acute injury.

The use of lipopolysaccaharide as a pretreatment to inducevulnerability to hypoxic--ischemic insult has added the impor-tant aspect of prenatal infection to the examination of theproblem [37]. Girard et al. [38] showed that the combinationof lipopolysaccharide exposure and hypoxic--ischemic injuryin rats mimicked the motor deficits and neuropathologicallesions seen in very premature infants. Their motor deficitswere more persistent making this one of the more promisingmodels for chronic injury.

In view of the frequency of cerebral palsy occurringrelated to prematurity, the importance of white matterinjury is an important consideration. Periventricular leuko-malacia is the most frequent lesion in these patients. Whitematter lesions are not well-seen in rodent models, as therodents have comparatively little white matter. In order tomimic the lesion seen in premature infants, several largeranimal models have been developed which demonstratewhite matter injury [35].

The perinatal rabbit model of cerebral palsy probably bestfits the criterion of an injury producing motor disability.This model is produced by uterine ischemia [39-42] or by intra-uterine administration of endotoxin [43]. However, these mod-els do not appear to supply the chronic or long-lasting deficitwe believe is required for satisfactory assessment.



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Larger animal models, such as the sheep [44] or baboon [45],better reproduce the pathology seen in human infants. Thepre-term baboon mimics the white matter neuropathologyseen in premature human infants [45]. The expense of thesemethods, however, appears to be prohibitive for the numberof animals required for an adequately powered study.

One of the central problems in the development of stem celltherapies for cerebral palsy is still the lack of satisfactory exper-imental models. Ideally the model should include impairmentof movement as a result of a brain injury. Secondly, the modelshould be one of chronic rather than acute injury. The morecritical of these two factors actually is the need for a chronicor long-lasting injury. There have been numerous experimentaltreatments of acute injury models that have demonstrated suc-cess but none that have shown efficacy in a true, chronic modelof injury. We and other investigators have shown that acuteinjuries are subject to repair by cell therapy, while the problemof chronic injury has been more resistant or neglected. Theimportant feature that needs to be demonstrated is the capacityof the cell therapy to repair a chronic injury of any type. Thetype or location of the brain injury is comparatively less impor-tant than the need for a persistent, abnormal behavioralsyndrome of some type in the animal.

5. Possible mechanisms of action

One of the main ideas inherent in stem cell transplantationfor cerebral palsy is that the stem cells would replace the cellsof the damaged nervous system. Most reports dealingwith adult stem cells show only a minimal survival of thetransplanted cells with few, if any, of these cells displayingmarkers/functionality of nervous tissue [21,46,47]. It does notappear that replacement alone would be sufficient to accountfor improvement in the experimental situation. While embry-onic or iPS cells may have somewhat greater potential for suchreplacement and transformation, the number of cells under-going this process is quite limited in vivo. Even though theremay be some replacement by transplanted cells, the cells oftendo not develop normal processes and may not function inneuronal circuitry [48]. Thus, cell replacement as an explana-tion for any improvement in the models is unlikely to bethe case given the current state of our knowledge of the cellbiology of stem cells.

Another possibility is that the transplanted cells differenti-ate into astrocytes [48] or microglia. How this would assist infunctional recovery is unclear.

Bone-marrow-derived cells may participate in blood vesselregeneration by promoting adhesion of CXCR4-positive cellsonto vascular endothelium [49], recruitment of endothelialprogenitor cells [50], and in the formation of periendothelialvascular cells [51]. Borlongan et al. [52] have demonstratedthat crude bone marrow may form endothelial cells in ananimal model of stroke.

A fourth set of ideas related to benefit is that the transplantsinduce a greater survival of intrinsic cells. We reported this

phenomenon in our neonatal hypoxic-ischemic model in ani-mals treated with MAPC [21]. Mahmood et al. [53] used MSCinjection to demonstrate that transplanted cells increased theexpression of nerve growth factor and brain-derived neurotro-phic factor after traumatic injury. This idea, for which the evi-dence seems strong, tends to restrict the benefit of stem celltransplantation to the acute post-injury period.

Another possible mechanism of benefit is the effect of adultstem cells on splenic function during acute brain injury. In astroke model Vendrame et al. showed that UCB lessenedthe splenic release of inflammatory cells and thereby protectedthe brain [54]. In support of this concept Walker et al. [55]

demonstrated that the intravenous injection of MAPC aftertrauma blocked the normal splenic response to injury andimproved outcome. These reports supported the idea thatthe spleen plays a role in adversely increasing the blood--brainbarrier permeability and that the splenic response is blockedby adult stem cell therapy. Once again, this is a benefit onlyfor the acute situation.

6. Risks of treatment

The risks of stem cell therapy occur primarily with allogeneictransplants, which expose the recipient to graft-versus-host dis-ease. Most reports of complications are in children undergoinghematopoietic stem cell transplantation for malignancies.These complications may relate in part to the fact that thesechildren received radiation therapy, chemotherapy, or immu-nosuppressive medications in addition to the stem cell trans-plant. Herpes or cytomegalovirus infections may occur inthese patients [56]. A variety of other medical complicationsare also reported in similar groups of patients [57,58].Woodward et al. [59] reviewed 405 patients who receivedhematopoietic stem cell transplantation for a variety of disor-ders. Of these patients, 26 experienced some type of encepha-lopathy due to infection, organ failure, medication reaction,seizures, acute disseminated encephalomyelitis, thromboticthrombocytopenic purpura or stroke.

Herpes virus-6 encephalitis is also reported as acomplication of unrelated umbilical cord transplant [60].

Clearly, we should not consider stem cell transplantation,particularly allogeneic, to be a benign procedure. Autologoustransplantation may incur some of the same risks, particularlyas the patients may be exposed to chemotherapy or infectiousagents. The complications may relate significantly to the treat-ment accompanying transplantation or the site to which thetransplant is delivered, such as into the cerebrospinal fluid.

While adult stem cell transplants have been carried out inlarge numbers of cerebral palsy patients outside the US, thereis no systematic reporting of complications. One would thinkthat the route of administration, that is intravenous versusdirectly into the CNS, might be a key to the understandingof complications, but the reporting of routes and their com-plications are unavailable. Without question, the long-termcomplications are simply unknown.

Carroll & Mays


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7. What’s needed next

We must have more knowledge of the biology and labora-tory manipulation of the different types of stem cells. Thisarea must include more work in the area of cell differentia-tion strategies. In addition we need to learn more aboutthe effects of the various methods of stimulating intrinsicneural proliferation.A chronic, pre-clinical animal model is required for the

study of the various competing cells types. The different celltypes need to be compared in head-to-head competition.Controlled clinical trials are needed. These should be con-

ducted with very specifically described patient groups, partic-ularly more so than the current, on-going American trials. Wemust recognize that there are considerable differences amongcerebral palsy patients, and therefore the patients need to becarefully matched for each study. This type of trial couldonly be achieved in a coordinated multiple-center paradigm.

8. Conclusion

Current clinical trials in the use of stem cells for cerebral palsyare ongoing and incomplete. While there are a number of dif-ferent cell types that are potential candidates as treatments,none have been shown to be effective in chronic animal mod-els. Furthermore, available animal models do not adequatelymimic cerebral palsy. Risks of the treatment are reported.More work on understanding the underlying beneficial biol-ogy of stem cells and the development and validation ofmore relevant animal models is required.

9. Expert opinion

Stem cell therapy for cerebral palsy remains a frustratingarea. Considering all the publicity about stem cells and

the fact that cell therapy is widely available outside theUSA for a price, parents feel that surely the treatmentmust work. This view tends to be confirmed by preclinicalreports of benefit in animal models of acute injury. Anec-dotal reports of success, of which there are many, contrib-ute little toward clarifying any benefit, but neverthelessencourage parents of cerebral palsy patients to seek theunproven therapy. There is no evidence as yet that stemcell therapy works in a chronic model of injury, as wouldbe relevant to cerebral palsy.

The problem remains difficult for several reasons: cerebralpalsy is not a homogeneous disease, our knowledge of stemcell biology is in its infancy, the pre-clinical models are farfrom ideal, and various preclinical trials show efficacy in acutemodels leading to falsely raised hopes.

We need a safe cell type that is effective in a chronic animalmodel of brain injury. Despite clinical use of stem cell treat-ment for cerebral palsy in many sites outside the USA, evi-dence of efficacy in a chronic animal model will benecessary before a clinical trial will be allowed in the USAusing any type of allogeneic cell. We believe it would be inap-propriate to conduct a clinical trial for cerebral palsy usingallogeneic cells without safety and efficacy data in a chronicanimal model.

For the time being it may better to focus on the treatmentof acute brain injuries with stem cells and thereby theimprovement or prevention of cerebral palsy in this subsetof patients.


JE Carroll has received funding from Associazione AssistenzaFigli Inabili Banca d’Italia, Cord Blood Registry, NINDS5R42NS55606, RW Mays is an employee and stake holderat Athersys, Inc.



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BibliographyPapers of special note have beenhighlightes as


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development of MAPC therapy.

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24. Robbins RD, Prasain N, Maier BF,

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not quite ready for prime time?

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25. Wu C, Chang A, Smith MC, et al.

beta4 tubulin identifies a primitive cell

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mammalian brain. J Neurosci

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Transplanted human embryonic neural

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Growth kinetics and transplantation

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34. Lee ST, Chu K, Jung KH, et al.

Anti-inflammatory mechanism of

travascular neural stme cell

transplantation in haemorrhagic stroke.

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endotoxin leads to motor deficits in a

rabbit model: a link between prenatal

infection and cerebral palsy. Am J

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Human bone marrow stem cells exhibit

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Marrow stromal cells migrate throughout

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2002;99:991-4

AffiliationJames E Carroll†1 & Robert W Mays2

†Author for correspondence1Medical College of Georgia,

Neurology, BG2000H,

1446 Harper Street,

Augusta, GA 30912 USA

E-mail: [email protected], Inc.,

Regenerative Medicine,

3201 Carnegie Avenue,

Cleveland, 44115-2634 USA

Carroll & Mays


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1. Introduction

2. Historical perspective

3. The biology of GvT and GvHD

and potential targets for DLI

4. The role of Tregs

5. The role of MRD in guiding

administration of DLI

6. Efficacy of DLI in specific

disease settings

7. DLI in the pediatric setting

8. General principles of DLI:

effective cell dose, timing,

toxicity and donor issues

9. Strategies to avoid

DLI-associated toxicity

10. Future therapeutic options

and research imperatives in

the field of DLI

11. Conclusions

12. Expert opinion

Review

Donor lymphocyte infusionfollowing allogeneichematopoietic stem celltransplantationClaire Roddie & Karl S Peggs††UCL Cancer Institute, Department of Haematology, Paul O’Gorman Building, 72 Huntley Street,

London, WC1E 6BT, UK

Introduction: Allogeneic hematopoietic stem cell transplantation (SCT) is the

treatment of choice for many malignant hematological disorders. Following

recent improvements in non-relapse-related mortality rates, relapse has

become the commonest cause of treatment failure. Infusion of donor lympho-

cytes can potentially enhance immune-mediated antitumor activity and offers

a salvage option for some patients. This paper reviews the current literature

on the efficacy of this therapeutic strategy.

Areas covered: The biology of adoptive cellular therapy with allogeneic

immune cells to treat relapse across a spectrum of diseases in both the full

intensity and reduced intensity hematopoietic SCT settings is explored.

The review discusses the current limitations of the approach and reviews

several new experimental strategies which aim to segregate the desired

graft-versus-tumor effect from the deleterious effects of more widespread

graft-versus-host reactivity.

Expert opinion: Durable responses to DLI have been noted in chronic myeloid

leukemia and responses have also been described in acute leukemia, multiple

myeloma and chronic lymphoproliferative disorders. The new challenge

in transplantation is to optimize DLI therapy in order to further improve

patient outcomes.

Keywords: allogeneic stem cell transplantation, DLI, graft-versus-host disease, relapse


1. Introduction

The efficacy of allogeneic stem cell transplantation (SCT) as a curative option forhematological malignancy is influenced by three factors: the underlying disease,the pre-transplant conditioning regimen and the graft-versus-tumor (GvT) effectmediated by donor leucocytes within the graft. The last two factors must be bal-anced against transplant related mortality (TRM). For example, despite deliveringa reduction in relapse rates, further intensification of existing myeloablative (MA)conditioning chemo- and radio-therapy beyond current levels increases TRM andmorbidity without improving overall survival (OS) [1,2]. Efforts to minimizetreatment-related morbidity and mortality have focused on modulating condition-ing protocols and improving supportive care. The introduction of reduced intensityconditioning (RIC) and non-MA transplants (the so-called ‘reduced toxicity’ trans-plants), which deliver lower TRM than conventional MA regimens, has revolution-ized clinical practice, permitting allogeneic SCT in a population of previouslyineligible patients [3,4]. The underlying principle of the RIC transplant is to providesufficient immunosuppression to facilitate engraftment without the highly toxic,inflammatory ‘cytokine storm’ induced by conventional MA conditioning. These


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transplants permit tumor eradication through facilitating theGvT effect more slowly post-transplant rather than directlythrough cytoreductive conditioning and for this reason maybe more suited to the management of indolent hematologicdiseases [5-7].Due in part to the lower intensity conditioning of these

procedures, relapse has become the leading cause of treatmentfailure [8-10]. The high uptake of RIC transplantation hasresulted in the prevention and management of relapse becom-ing an increasingly prominent feature of clinical practice. Onekey approach has been the donor lymphocyte infusion (DLI)to enhance GvT responses [11].One of the major problems faced by clinicians is the lack of

explicit published data on the efficacy of DLI. The reasons forthis are manifold. First, there is a reluctance to conduct clini-cal trials in this population due to the small patient numbersand often poor outcomes despite intervention. Second, stud-ies often incorporate heterogeneous patient groups fromwhich it is difficult to identify which factors are relevant toeach disease entity. There is now international agreement onthe need for collaborative multi-center studies and the needfor a central database or sample repository to assess inter-ventions in this area. An initiative was recently outlinedin the National Cancer Institute First International Work-shop on the Biology, Prevention and Treatment of Relapseafter Allogeneic Haematopoietic Stem Cell Transplantation(2010) [10,12].We provide a historical context for the use of DLI, discuss

the biology underlying the GvT effect, review the available evi-dence on the utility of DLI across a range of hematological dis-eases and address the future of DLI along with developmentsin graft engineering. For the purposes of this review, weconcentrate on T-cell rather than NK-cell therapies.

2. Historical perspective

Bidirectional immune reactivity (alloreactivity) between hostand recipient T cells underlies many of the major complica-tions following allogeneic SCT, namely graft failure andgraft-versus-host disease (GvHD), but it is also responsiblefor the advantageous GvT effect [13]. Early evidence for GvTwas based on clinical observation: complete remissions wereobserved in some patients with relapsed disease post-allograftin whom immunosuppression was withdrawn whilst inothers GvHD appeared to protect against relapse [14,15].Higher rates of relapse were observed in patients receivingsyngeneic (i.e., from an identical twin donor) [16] or T-celldepleted transplants [17,18] and it was hypothesized that alloge-neic T lymphocytes could be the active cell in the observedGvT effect.

Preclinical studies revealed how specific donor T cellsprevented growth of leukemia colonies in vitro and preventeddevelopment of acute myeloid leukemia in an in vivoimmune-deficient mouse model of leukemia. Donor T cellsactive against leukemia cells were subsequently demonstratedto target recipient hematopoiesis-restricted minor histo-compatability antigens (mHags) [19] and aberrantly or overex-pressed ligands such as Proteinase 3 [20-22] in myeloidmalignancies. These findings supported the concept ofDLI as a therapeutic intervention, where isolation ofdonor T lymphocytes and their subsequent infusion to therecipient could hasten or intensify the GvT effect in therelapsed patient.

In 1990, Kolb et al. published the first clinical study of DLIin patients with relapsed chronic myeloid leukemia (CML),which showed that infused donor buffy-coats in associationwith IFN-a could induce cytogenetic remissions [23]. Remark-ably, DLI has since been shown to restore durable completeresponses (CR) in up to 80% of these patients [24-33]. Thissuccess in CML prompted others to investigate the use ofDLI across a range of hematological malignancies such asacute myeloid leukemia (AML), acute lymphocytic leukemia(ALL), non-Hodgkin’s lymphoma (NHL), multiple myeloma(MM) and Hodgkin’s lymphoma (HL) with variablesuccess [34-38]. The role of DLI in these disorders remainspoorly defined and response rates, optimal approaches andlong-term outcomes are still unclear (refer to Table 1 fordetails of several of the larger trials of DLI therapy in a rangeof hematological diseases).

3. The biology of GvT and GvHD andpotential targets for DLI

The efficacy of allogeneic SCT derives largely from theallorecognition which permits donor cell engraftment andfacilitates the GvT response. GvHD is an undesirable sideeffect of therapy and is thought to be initiated by tissue injuryleading to activation and proliferation of alloreactive T cells,which then home to sites of inflammation and potentiate

Article highlights.

. Response to donor lymphocyte infusion (DLI) isdisease-dependent, with the best evidence in chronicphase chronic myeloid leukemia.

. Encouraging results have been obtained in indolentlymphoproliferative disorders, mantle cell lymphoma andHodgkin’s lymphoma, but less so in acute lymphocyticleukemia, more aggressive non-Hodgkin’s lymphomaand multiple myeloma, which may benefit more fromnon-cellular therapies in the setting of relapse.

. Graft-versus-host disease (GvHD) and to a lesser degreemarrow aplasia are the major side effects of DLItherapy. The escalating dose regimen administrationschedule is one approach to minimizing GvHD.

. Promising advances in graft engineering (e.g. selectionof T-cell subsets and selection of T cells directed againstrecipient minor histocompatability antigens) andgene therapy (e.g. TCR and chimeric antigen receptorconstructs) may also help to tip the balance of immunitytowards graft-versus-tumor effect and away fromGvHD and improve clinical outcomes.


Donor lymphocyte infusion following allogeneic hematopoietic stem cell transplantation


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tissue injury further. A major focus of transplant biology is todevise strategies to dissociate GvT from GvHD, with focus onboth the antigen presenting cells (APCs) and the effectorT cells.

The mechanism(s) by which allorecognition occurs duringthe GvT response is not fully understood. Murine models ofantigen presentation have shown that recipient APCs are cru-cial to initiation of GvT, whereas donor APCs appear not tobe so critical [39,40]. Dendritic cells (DCs) are professionalAPCs which present antigen via both Class I and II MHCpathways to the adaptive immune system. Research is ongoinginto methods of loading DCs with tumor antigen to enhanceGvT activity and to try to develop methods to selectivelyeliminate DCs responsible for GvHD whilst promoting theexpansion of those involved in GvT responses [10].

The GvT response is dependent on the ability of the graftto either induce immunity against tumor-specific neo-antigens (probably a less common event) or to break toleranceand induce antigen-specific autoimmunity to overly- oraberrantly-expressed tumor-associated antigens such asWilm’s tumor 1, Proteinase-3 and several mHags (HA-1,HA-2, HB-1 and BCL2A1) which are differentially expressedon hematopoietic cells [41-43]. Dissociation of GvT fromGvHD might be achieved through the adoptive transfer ofT cells directed against these antigens.

Alternative approaches to dissociate GvT from GvHD havefocused on potential differences in effector pathways. GvTand GvHD related tissue damage is thought to occur viadifferent mechanisms: GvHD effectors generally utilize the

Fas:Fas-ligand cytolytic pathway, whilst GvT effectors useperforin-mediated cytotoxicity and possibly TNF-relatedapoptosis-inducing ligand with selective activity for malignanttargets. Manipulation of these different mechanisms of cyto-toxicity may facilitate enhancement of GvT and at the sametime suppress GvHD [44].

4. The role of Tregs

Maintenance of transplant tolerance is dependent on thesuppression of alloreactive donor T-cell clones by means ofcentral deletion, clonal anergy and the inhibitory effects ofregulatory T cells (Tregs). Tregs are naturally occurring clus-ters of differentiation (CD)4+ CD25+ forkhead box P3+

T cells that constitute ~ 1 -- 2% of the circulating CD4+

T-cell population. In the autologous setting, they helpto prevent autoimmunity by dominantly suppressing the acti-vity of autoreactive lymphocytes using a variety of mecha-nisms including the secretion of inhibitory cytokinessuch as IL-10 and TGF-b, and direct cell--cell contactinhibition [45,46].

In murine models, Tregs have been shown to preventGvHD when co-infused with effector T cells, albeit withthe potential to also suppress GvT [47,48]. It is, therefore,possible that infusions of ex vivo expanded Tregs given topatients with GvHD could ameliorate their symptoms andthat depletion of Tregs from the stem cell (or DLI) productcould enhance alloreactivity by ‘releasing the brake’ on theGvT effect.

Table 1. Efficacy and toxicity of DLIs in hematological malignancy (selected series).

Disease/study author Patient

number

Cell dose

(� 108/kg)

CR post

DLI (%)

GvHD (acute > grade II/

chronic, extensive) (%)

CML/Kolb et al. [23] 3 4.40 -- 7.40 100 66.6CML/van Rhee et al. [30] 14 0.70 -- 5.30 57 28a/28cCML/Collins et al. [33] 56 0.50 -- 3.62 100 (Cy)/73 (H)/33 (A) 46a/21c*CML/Porter et al. [75] 25 0.005 -- 5.21z 46 28a/12cAML/Kolb et al. [32] 23 0.10 -- 7.83z 29 41*AML/Shiobara et al. [35] 32 0.01 -- 7.40z 25 34a/33c*AML/Schmid et al. [72] 171 0.001 -- 2.250 35 43a/46cAML/Porter et al. [75] 23 0.001 -- 31.8 35 35a/17cALL/Kolb et al. [32] 22 0.30 -- 11 0 41*ALL/Collins et al. [33] 15 0.50 -- 6.20z 18 46a/32c*ALL/Shiobara et al. [35] 30 0.01 -- 11.3z 20 34a/33c*NHL/Russell et al. [76] 17 0.05 -- 1 10 44a/89cNHL/Bloor et al. [78] 17 0.01 -- 1 76 15a/31cNHL/Bishop et al. [80] 5 0.1 -- 1 50 50HL/Peggs et al. [85] 14 0.01 -- 1 57 -MM/Collins et al. [33] 5 1.63 -- 5.53z 50 46a/32c*MM/van de Donk et al. [95] 63 0.01 -- 3 12 22a/43cMM/Lokhorst et al. [96] 13 0.01 -- 3.30 31 62a/15c

*The studies marked encompass different disease entities and the incidence of GvHD reported relates to the whole study cohort rather than to individual diseases.zIndicates the mononuclear cell dose rather than the T-cell dose.

a: Acute GvHD; A: Accelerated phase/blast crisis; ALL: Acute lymphocytic leukemia; AML: Acute myeloid leukemia; c: Chronic GvHD;

CML: Chronic myeloid leukemia; CR: Complete response; Cy: Cytogenteic relapse; DLI: Donor lymphocyte infusion; GvHD: Graft-versus-host disease;

H: Hematologic relapse; HL: Hodgkin’s lymphoma; MM: Multiple myeloma; NHL: Non-Hodgkin’s lymphoma.

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5. The role of MRD in guiding administrationof DLI

The detection of minimal residual disease (MRD) maypredict the likelihood of overt disease relapse in somediseases [49-52]. Monitoring for mixed chimerism (MC), thatis, the return of recipient-derived hematopoietic cells is onepotential approach. In classical MA conditioning, host hema-topoiesis is replaced entirely by donor cells to create full donorchimerism (FDC). In this setting, the return of host cells(MC) is generally indicative of relapse with few exceptions.The potential importance of early intervention in terms ofmaximizing therapeutic efficacy is well established in CMLand increasingly recognized in other disorders. For example,it was demonstrated in a retrospective analysis of patientswith acute leukemia and myelodysplastic syndrome (MDS)who received DLI for relapse based on leukemia lineage-specific chimerism analysis following transplantation. The3-year survival in those treated for molecular relapse was42% compared to 16% in those treated for hematologicrelapse, further suggesting that early intervention withDLI during molecular relapse rather than waiting for hemato-logic relapse has the potential to improve responses andsurvival [53].In the RIC transplant setting, early MC is more common

(particularly with T-cell depletion) with more gradual con-version to FDC over many months. Whether persistentMC, particularly when it is present only within the T cell line-age, is associated with a higher incidence of relapse and canthus be used as a basis for early intervention with DLI toachieve FDC and prevent relapse, remains controversial [54].It is likely that the significance of MC differs accordingto whether T-cell depletion is incorporated as part of theconditioning regimen and remains stable or is increasingover time.Other methods of MRD detection, such as multiparamet-

ric flow cytometry (MFC) and PCR amplification of fusiontranscripts and antigen receptor genes are used across a rangeof diseases. In many cases, they offer a much greater sensitivitythan routine chimerism assays. However, except in the case ofCML, these strategies have not yet been fully validated. Fur-ther detail is beyond the scope of this review, but the readeris referred to several studies of interest in specific diseasesettings [55-62].

6. Efficacy of DLI in specific disease settings

6.1 CMLTo date, most experience with DLI has been gained in CML.Post-transplant relapse rates are markedly higher for patientswith advanced CML (accelerated phase (AP) or blast crisis(BC) compared with CML in first chronic phase (CML-CP)). Responses to DLI are often durable and are bestin those with molecular relapse (90 -- 100%) followed bycytogenetic relapse (90%), hematological relapse in

CP (75%), relapse in AP/BC (36%) and worst in resistantdisease (0%) [63].

Relapse following DLI represents a varied spectrum. Theoutlook for patients with extramedullary relapse is oftenpoor and optimal treatments are yet to be defined [64]. Alter-natively, patients who achieve hematological remission butremain molecularly or cytogenetically positive for bcr-abl(oncogenic fusion protein) can be successfully re-treatedwith DLI [65]. Disease persistence may be due to CML stemcells which do not express the maturation-associated antigenstargeted by CD8+ mHag-specific cytotoxic T lymphocytes(CTLs) [66]. Work is ongoing to identify and eradicate CMLstem cells.

The role of additional agents in combination with DLI isunclear. There is evidence to suggest that IFN-a may poten-tiate the therapeutic efficacy of DLI such that lower totalcell doses are required to achieve remission or that patientsresistant to conventional DLI achieve responses [67,68]. Studiesof cytoreductive chemotherapy plus DLI in patients withadvanced CML have also been conducted, but outcomeshave been disappointing [32].

The combination of DLI with a tyrosine kinase inhibitor(TKI) has been explored, although results have been con-flicting. One study suggested more rapid remissions andimproved OS and disease-free survival, particularly in thecontext of accelerated disease [69]. Other groups have obtainedcompelling preclinical data to suggest that the anti-proliferative effect of TKI therapy affects both residual leuke-mia cells and tumor-responsive CTLs [70,71]. TKI therapymay, therefore, adversely affect the potentially curativeimmune effects of allogeneic transplantation and DLI. Thiswould be an ideal area for a randomized controlled study.

6.2 AMLThe probability of post-transplant relapse in AML has beenvariably reported as 20 -- 60% and the prognosis is generallypoor. As a single agent, DLI is generally unable to induceremission in florid relapse. One study of DLI in combinationwith induction chemotherapy has revealed poor remissionrates (15 -- 20%) and a low 2-year OS (15 -- 20%). Sub-group analysis revealed that chemo-responsive patients withfavorable cytogenetics and low bulk disease at relapse didsignificantly better, with an OS of 56% at 2 years [72].

Combination regimens incorporating newer agents such asdecitabine and azacitadine into DLI protocols are the subjectof current clinical interest, but data are limited. Bearing inmind the poor outcomes in relapsed AML with current sal-vage modalities, a clinical study of prophylactic DLI in acohort of AML patients was conducted and showed improvedOS compared with case-matched controls [73]. Interventionbased on MRD monitoring may reduce the exposure ofpatients to the risk of GvHD inherent in prophylactic strate-gies as previously discussed [53], and the results of combinationof DLI with novel hypomethylating agents in this setting areeagerly awaited.



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These data require further maturation and validation,but are particularly relevant to a growing populationof patients with AML or high risk MDS undergoingRIC transplantation.

6.3 ALLRelapsed ALL has a poor prognosis, with only 7% of adultpatients surviving 5 years [74]. DLI is rarely effective in floridrelapse and one small study reports a CR rate of 10 -- 20%of limited duration in patients receiving matched siblingDLI [75].

The current research emphasis in ALL is on the preventionof relapse through optimization of up-front therapy andthe development of new targeted therapies. Post-transplantcellular therapies have not yet realized their potential.

6.4 NHLNHL comprises a heterogeneous group of histological diag-noses which can be divided clinically into indolent andaggressive groups. The curative potential of MA allogeneictransplantation is established in NHL, but is precluded inthe majority of patients due to an unacceptably high TRM.The lower toxicity RIC strategies have facilitated the applica-tion of allogeneic transplantation and DLI in this groupof patients. A significant GvT effect has been reported inindolent NHL following stem cell and DLI therapy, withless evidence supporting a strong effect in more aggressivehistologies [76-80].

In our recent study of T cell depleted RIC transplantationin multiple relapsed follicular lymphoma, 13/82 patientsrelapsed, 10/13 received DLI and 9/10 experienced sustainedremission. Overall, the incidence of GvHD was low,affecting < 20% of patients [77]. An overlapping study ofDLI in ‘indolent NHL’ comprising 28 patients with eitherMC (n = 11) or relapsed disease ± MC (n = 17) demonstratedcumulative response rates of 91% in the MC cohort and 76%in the relapsed disease cohort, complicated by GvHD in15 -- 31% [78].

Aggressive NHL is thought to be relatively poorly respon-sive to allogeneic SCT and DLI [76], but a recent study of48 patients with relapsed diffuse large B-cell lymphomaundergoing RIC transplantation demonstrated progression-free survival (PFS) and OS rates at 4 years of 48 and 47%,which improved further to 55 and 54% in those withchemo-sensitive disease before transplant. Overall, 12/48patients received DLIs ± chemoimmunotherapy for relapseand 3/12 obtained durable remissions although the role ofother therapies administered in close temporal approximationis difficult to evaluate [79,80].

Mantle cell lymphoma (MCL) is an aggressive NHL whichgenerally responds poorly to treatment. In a study of RICallogeneic transplantation in 70 patients with MCL, 15relapsed post-transplant and 11/15 achieved CR withDLI [81]. This demonstrates that DLI is an effective salvagetherapy in MCL, confirming the importance of GvT inferred

from the encouraging PFS survival rates delivered in T-cellreplete RIC programs.

Rituximab, a chimeric anti-CD20 mAb, is purported toaugment the GvT effects of DLI by promoting antigen prim-ing. Preclinical studies of rituximab-treated tumor cell linesdemonstrated more effective alloantigen presentation [82].Clinical experience remains largely anecdotal at present,though encouraging responses have been claimed and this isanother area that would benefit greatly from a consolidatedclinical study.

6.5 HLHistorically, poor risk HL patients rarely underwent alloge-neic transplantation due to prohibitively high TRM [83].RIC conditioning protocols have effectively reduced TRMand post-transplant survival outcomes have improved, butthis has been complicated by high relapse rates (44 -- 81%at 2 -- 3 years) [84-86]. Published experience with DLI is rela-tively scarce. In many cases, response rates have been disap-pointing (around 30 -- 40%), and perhaps more significantlyof limited duration. The experience following T-cell depletedtransplantation seems qualitatively different. Our single-institution experience of 24 patients treated with DLI eitheralone (n = 14) or combined with debulking or consolidatingchemo-radiotherapy (n = 10) demonstrated CR in 14 andpartial responses (PR) in five patients (overall response79%) [87]. The majority of responders developed GvHD.Responses were maintained in 11 patients at a median of2.2 years from last DLI, and a further three died in CR ofcomplications of GvHD. It seems likely that experience withDLI in relapsed HL will grow rapidly over coming years.

6.6 CLLRelapse following allogeneic SCT for CLL is reported in20 -- 48% of patients and responds poorly to standard salvagechemotherapy. Reports on the use of DLI are limited, butshow highly variable response rates (0 -- 60% CR) which aredurable only in a minority [88-91].

The reasons why DLI often fails in CLL giving low durabil-ity responses is unclear. One group attempted to correlateclinical responses post-allograft with MRD kinetics by MFCand/or real time PCR and identified a pattern of an initialclinical GvT effect accompanied by a failure to completelyeradicate MRD followed by subsequent frank relapse despiteextensive chronic GvHD [92]. Possible mechanisms to explainthis disease kinetic include clonal evolution, sanctuary sites,the development of tolerance and the presence of CLLstem cells which fail to be targeted by DLI. Unraveling thispicture may elucidate further the pathophysiology underlyingrefractory and relapsing CLL.

Novel immunological strategies to target CLL include theuse of activated DLI (aDLI) which is addressed later inthe article [93]. Combined therapy with DLI and Bi20(FBTA05), a trifunctional, bispecific antibody targetingCD20 and CD3 which is thought to direct T cells towards

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CLL cells has been used in patients with refractory, tumorprotein 53-mutated CLL. Transient clinical responses wereobserved, but disease recurrence was identified soon aftercessation of therapy, despite further doses of DLI [94].

6.7 MMRelapse post-transplant is a significant problem in MM,affecting 50% of patients who achieve CR and 80% of thosewho do not achieve CR. Therapy with DLI gives overallresponse rates of 38% (CR = 19%, PR = 19%) complicatedby high levels of acute GvHD (38% of patients) and chronicGvHD (42% of patients). A strong correlation exists betweenthe likelihood of a favorable response to DLI and the deve-lopment of GvHD [95]. Long-term survival is possible in60% of patients who achieve CR with DLI whereas survivalat 22 months is < 20% in those who achieve only PRpost-DLI [96].In one study of RIC transplantation in chemo-sensitive

MM, only 2/20 patients achieved CR. In all, 14/20 receivedescalating-dose DLI for residual/progressive disease and5/14 developed GvHD (which appeared to correlate withdisease responses). Response durations were short (five were< 12 months) and progression often occurred despite persistingFDC, giving 2-year PFS rates of 30%. Dose escalation did notpermit dissociation of the GvT from the GvHD effect [97].Attempts are ongoing to define strategies to augment the

efficacy of DLI using novel chemotherapy agents. The proteo-some inhibitor bortezomib has been combined with DLI inanimal models and found to reduce the incidence of GvHDwhilst preserving the crucial GvT effect [98]. Lenalidomideand thalidomide can activate T cells and NK cells and mayaugment GvT activity [99].

7. DLI in the pediatric setting

Compared to adults, children with hematologic malignancyshow superior survival post-allogeneic SCT, but post-transplant relapse represents a significant problem [100]. Therole of DLI in managing post-SCT relapse in pediatric hema-tologic malignancy is unclear, as most published data on thesubject comprise small case series [101-103].One important comparative analysis of outcomes in

49 children who received DLI for post-transplant relapse(18 ALL, 17 AML, 8 CML, 4 MDS, 2 juvenile myelomono-cytic leukemia) and 1229 historical controls (no DLI)reported to the Centre for International Blood and MarrowTransplant Research demonstrated that DLI did not resultin survival benefit for the majority of children treated [100],perhaps primarily reflecting the disease histologies representedrather than the age group of the patients.Several other studies have focused on strategies to try to

prevent relapse. Bader et al. have defined how increasingMC after allogeneic SCT is an important prognostic factorfor unfavorable outcome in children with ALL. They showthat the probability of 3-year event-free survival (EFS) in

patients with FDC or low level MC is 66% compared with23% for patients with increasing MC. They go on to showthat of the increasing MC cohort, those who received immu-notherapy had a 3-year EFS rate of 37% compared to 0% inthose who did not receive immunotherapy and they proposethat overt relapse could be prevented in a considerablegroup of patients through chimerism monitoring and earlyintervention with DLI in cases of increasing MC [104,105].

As in adults, post-transplant cellular therapies for ALL havenot yet been fully optimized, although clinical studies ofchimeric antigen receptors (CARs) targeting CD19 on leuke-mia cells have been established in pediatric ALL and resultsare awaited with interest.

It is important to note that DLI is also an accepted treat-ment option for some non-malignant hematologic conditionssuch as severe aplastic anemia (SAA) and thalassemia major.Treatment failure is rare in SAA, but is mostly caused by graftrejection. Pediatric studies have suggested that early, low-doseDLI for increasing MC can prevent graft rejection whilstincreasing the risk of GvHD [106].

It is recognized that MC and secondary graft failure arefrequent complications of allogeneic SCT for thalassemia.Escalating doses of DLI in this setting have been shown tobe safe but are only efficacious in restoring FDC in patientswith low-risk relapse rather than in patients with high-riskrelapse. For this reason, the authors suggest that DLI becommenced on detection of level 2 -- 3 chimerism (donor< 90%) [107].

8. General principles of DLI: effective celldose, timing, toxicity and donor issues

The best evidence addressing the optimal cell dose for DLItherapy has been established in CML. In one study,68 patients received DLI in an escalating dose regimen(EDR) and the proportion of clinical responses increasedwith each increment in cell dose. Subgroup analysis showedthat the effective cell dose in CML correlates with diseasestage and donor type such that patients with cytogenetic ormolecular relapse and those with unrelated donors respondto lower doses (< 107 CD3+ cells/kg) compared to patientswith advanced CML or those with fully-matched siblingdonors [108]. The optimal cell dose for most hematologicalmalignancies is yet to be defined, but in some diseases suchas MM there appears to be no clear correlation betweenCD3 cell dose and clinical response [109].

GvHD complicates DLI therapy in ~ 30% of patients andcan be life threatening [110], although it is important to recog-nize that GvHD developing either following transplantationor DLI is a manifestation of alloreactivity which often actsas a marker for GvT activity. Therefore, although severeGvHD may compromise overall outcomes, less severe acuteGvHD or limited chronic GvHD has been associated withsuperior outcomes in terms of reduced relapse risk. Thisimpact was first recognized in CML, but has been described



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across many other pathologies now including acute leukemias,myeloma and a variety of lymphoma subtypes. GvHD riskdoes not appear to correlate with the underlying hematologi-cal malignancy being treated, but is positively correlated withthe administered dose of DLI. In CML, a dose of 1 � 107/kgcan induce complete donor chimerism and a potent graft-versus-leukemia (GVL) effect, in some cases in the absenceof clinical GvHD particularly if given at later time pointsfollowing transplantation [31].

The time interval between SCT and DLI therapy appearsto influence the likelihood of developing GvHD. A smalldose of 1 � 105 T cells/kg can induce GvHD if administeredon the day of transplant [111], yet a dose of 1 � 107 T cells/kgcan be given at 12 months post-transplant without GvHDdeveloping [31]. It is thought that the inflammatory cytokinesproduced in the immediate post-transplant period activatealloreactive donor T cells and lower the threshold for theemergence of GvHD. Changes in the frequency of recipient-derived APCs over time may also have relevance to GvHDrisk. Furthermore, the development of extrinsic mechanismsof peripheral T-cell tolerance (e.g., Tregs) at later time pointsmay also modulate this risk.

Other factors which make GvHD more likely to occurinclude donor sex mismatch (female donor to male recipient),advanced patient age and mismatch at the mHag level [112].

Aplasia is now a relatively infrequent complication of DLI.It is often transient, but in some cases may require hemato-poietic stem cell rescue. It was reported historically in15 -- 20% of treated CML patients with an associated mortal-ity rate of ~ 5%. Aplasia is more common in hematologicalrelapse of CML, possibly due to poor donor myeloid reserve,and is rarely reported in patients with exclusively cytogeneticor molecular relapse [33,113] or in those treated for low levelsof recipient MC.

Another important issue to consider when planning DLI isdonor availability and willingness to undergo further leukophe-resis. This process can bring significant delays in treatment andmay compromise patient outcomes. Some groups advocateDLI collection at the outset to avoid delays, but the inherentdifficulties with this approach include the need for additionaldonor leukopheresis, the financial burden of collecting DLIfor all transplant patients and the handling and storage issuessurrounding the DLI product once it is collected when thereis a strong possibility that it will never be needed or used. Itis the practice in our institution to prepare and store aliquotsof DLI from the primary harvests of matched unrelated donorswhere the yield is in excess of the target dose of 4� 106 CD34+

cells/kg necessary for transplantation.

9. Strategies to avoid DLI-associated toxicity

9.1 The escalating dose regimenAdministration of DLI as a single bolus of cells collected froma single leukopheresis and containing variable numbers ofCD3+ T cells is referred to as a bulk dose regimen (BDR)

and this approach is associated with a high incidence ofGvHD [33,75,114].

The EDR approach is fundamentally different in that theDLI product is quantitated for CD3+, CD4+ and CD8+

T-cell numbers and is then administered in multiple smallaliquots with a dose escalation over time. In this way, the min-imum cell dose needed to achieve disease remission is admin-istered and with more modest cell doses, the likelihood ofGvHD may be reduced [31]. One study in CML comparingBDR and EDR approaches demonstrated equivalent remis-sion rates with both schedules, but a significantly lowerincidence of GvHD in the EDR cohort [113].

It is critical when using the EDR schedule to allow an ade-quate interval between DLI doses to allow for assessment ofresponse and toxicity. The optimum interval between dosesis yet to be defined, but Dazzi et al. report that shorter inter-vals (rather than total cell dose) leads to a higher incidence ofGvHD [113]. Mackinnon et al. recommend a minimum periodof 3 months between escalating doses in clinically stablepatients [31].

9.2 Suicide gene transfected donor T cellsA potential solution to the undesirable alloreactivity of unma-nipulated T cells is the transfection of donor T cells with a‘suicide gene’ to permit their elimination in the event ofGvHD. The ideal suicide gene is non-immunogenic, non-toxic in the quiescent state, but can efficiently trigger celldeath once activated.

Transfection of donor T cells with the herpes simplex virus1-thymidine kinase (HSV-TK) gene has been described inseveral clinical trials. This strategy is purported to be safeand effective in controlling GvHD via ganciclovir-inducedelimination of HSV-TK transfected T cells [115,116]. Problemswith this approach include concerns over the possible adverseimpact of this approach on GvT activity, the possible immu-nogenicity of the transfected cells which could promote theirrapid clearance from the blood and alterations in the TKgene which lead to the expression of a non-functional pro-tein [117,118]. These issues need to be addressed prior to thisstrategy being consolidated in clinical practice.

Research is ongoing into other suicide genes such as chime-ric Fas and caspase-9. Timed induction of these genes canlead to T-cell apoptosis and this may represent a promisingnon-viral alternative to HSV-TK [119,120].

9.3 Cell selection/subsetsWith advances in available graft engineering techniques, DLIcan now be tailored in attempts to tip the balance of immu-nity away from GvHD and towards GvT. Strategies to definethe optimal cell type/combination to use, the cell activationstatus and the antigenic specificity are outlined below.

9.3.1 Selective depletion of alloreactive cellsAlloreactive T cells, defined by their expression ofactivation-induced antigens such as CD25, CD69,

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CD137 or CD134 in response to exposure to host antigens,can be depleted from the DLI product with a view of reduc-ing the incidence of GvHD. Co-incubation of donor lympho-cytes with allogenic recipient stimulator cells followed bytargeting with immunotoxin-conjugated antibodies specificfor cell-surface activation markers or antibodies which canbe sorted via immunomagnetic beads, permits separationof activated cells from DLI prior to infusion. Whetherthis approach reduces alloreactivity towards hematopoeisis-restricted mHags and reduces potential GvT activity remainsto be clarified [121,122].

9.3.2 Manipulated DLI (CD8 T-cell depletion)CD8+ T cells are thought to be the primary mediators ofGvHD in humans whilst CD4+ T cells are reported to con-tribute more to the GvT effect. A number of recent papershave focused on demonstrating how CD4 T lymphocytesare crucial to the development of the DLI-associated GvTeffect. In one study, in vivo CD4 T-cell depletion abolishedthe antitumor effect of DLI which, by contrast, was notimpacted by depletion of CD8 T cells. CD4 T cells clearlyplay an essential role in mediating early antitumor effects [123].For this reason, a number of groups have explored CD8+

T-cell depletion as a strategy to reduce the incidence ofGvHD. CD8+ T-cell depletion of the stem cell graft hasbeen reported to reduce the risk of GvHD without a parallelincrease in relapse rates [124]. This has also been confirmedin the DLI setting in CML [125,126].A Phase I study of high-stringency immunomagnetic CD8

T-cell depletion of DLI was reported in which escalatingdoses of CD8 T-cell depleted DLI were given at 3-monthlyintervals to patients with persistent disease or MC or disease.Responses were documented in 8/16 of the former and5/11 of the latter. Five developed acute grade II -- IVGvHD and two died of GvHD-related complications.Clearly, GvHD remains a major problem despite CD8+

T-cell depletion and further studies are warranted to definethe potential benefits and risks more clearly [127].

10. Future therapeutic options and researchimperatives in the field of DLI

10.1 T-cell engineeringThe main clinical imperative driving research in DLI biologyremains how to shift the immunological balance of cellularimmunotherapy away from GvHD towards GvT. Ideally,one would aim to select (and possibly expand) a high-avidity tumor-reactive T-cell clone in vitro and then infuseit to the patient as DLI. Difficulties with this approachinclude identifying a high-avidity tumor-reactive clone inthe first place (many have undergone central deletion due tothe risk of autoimmunity of any high-avidity self-specificT cell) and also the deleterious impact of prolonged cell cul-ture on subsequent T-cell persistence and function (terminaldifferentiation and exhaustion). Optimal cell culture

conditions are beyond the scope of this review, but arediscussed in a review by Aqui and June [128].

T-cell engineering can potentially overcome the limitationsof adoptive cellular therapies by introducing antigen receptorsinto T cells to re-direct their specificity. This potentiallyallows rapid generation of tumor-reactive T cells expressingeither HLA-restricted, heterodimeric TCRs or CARs thatrecognize native cell-surface antigens.

Initial clinical studies of TCR gene transfer have beendescribed in metastatic melanoma using a high affinityMART (melanoma antigen recognized by T cells)-1 specificTCR. Tumor regression was reported in 30% of patients.Treatment was complicated by off-target toxicity associatedwith the destruction of melanocytes elsewhere in the body,but this responded to steroid therapy [129].

Clinical studies of first-generation CARs have been con-ducted in ovarian and renal cancers, lymphoma and neuro-blastoma, although results to date have been somewhatdisappointing and have highlighted potential issues withtoxicity [130-133]. Second generation CARs often comprise anantibody binding motif and a CD28--CD3z dual signalingreceptor which facilitates T-cell activation and expansion fol-lowing stimulation. Studies of refined second generationCARs directed against CD19, CD20, CD23, CD33 andCD74 are awaited with interest.

It is important to note that the development and safetymonitoring of these new immunotherapies as advancedmedicine therapy products are the subject of EU regulations,but this is beyond the scope of this review.

10.2 aDLIResistance to the therapeutic effects of DLI may occur due tofailure of ‘in vivo’ activation of donor T cells. Several studieshave reported that IL-2 stimulation of donor T cells (bothin vivo and ex vivo) can induce clinical responses in patientswho are resistant to DLI alone. Porter et al. showed thatinfusion of ‘ex vivo’ activated donor lymphocytes (usinganti-CD3 and anti-CD28 coated beads) in patients with arange of hematological malignancies led to responses whereconventional DLI had been disappointing. A total of17 patients were evaluated and 8 achieved CR. Of those,4/8 relapsed. The incidence of GvHD in this cohort com-pared favorably with that of conventional DLI [93]. Presently,Phase I and II studies of aDLI in CLL are underway at theUniversity of Pennsylvania.

11. Conclusions

Current experience provides evidence that DLI is the mostconsolidated approach to the management of disease relapsefollowing allogeneic SCT. DLI is particularly effective inmanaging relapsed CML-CP, but encouraging clinicalresponses have also been reported in other hematologicalmalignancies. The main drawback of DLI is GvHD whichaffects ~ 30% of all treated patients and can be life



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threatening. Rapid advances in our understanding of trans-plant biology have led to the development of strategies whichaim to preserve the GvL effect whilst inhibiting the GvHDeffect. The EDR approach, cell selection and genetic engi-neering strategies all offer the potential for refining DLI, butthere is still a lack of definitive published data to guide ourclinical practice. Ultimately, large, collaborative clinical trialsare warranted.

12. Expert opinion

DLI will continue to have an important role in the manage-ment of disease relapse following allogeneic transplantation.Its potential has been demonstrated in a number of diseases,but attempts to standardize approaches within the context ofprospective studies have been limited. There are a numberof reasons for this, but growing international recognition ofthe importance of collaborative programs may help to definethe most important outstanding questions and define appro-priate therapeutic strategies either across disease subtypes(e.g., timing, dosing) or within specific disease histologies(e.g., the role of particular cytoreductive combinatorial appro-aches). Toxicity due to GvHD remains common andattempts to reduce this should be further evaluated in pro-spective studies. DLI administration via the EDR hasbeen shown to minimize the GvHD risk in studies of CMLwith no apparently adverse impact on GvT function, andconfirmation of similar efficacy in other diseases should beforthcoming within the next few years.

Improvements in defining the risk of relapse followingallogeneic transplantation are crucial for future studies and

may help to establish which patients are of sufficiently highrisk to merit consideration for prophylactic DLI studies. Dis-ease type and status at transplant taken together with MRDmonitoring will be important factors in directing interven-tion. Elucidation of the impact of MC, potentially differingaccording to the transplantation platform used, will alsorequire more concerted attention.

It is likely that the role of graft manipulations will be fur-ther defined within the next 5 -- 10 years, including aDLI,T-cell subset selection, allodepletion and genetic modifica-tion. It will be critical to define the impact on GvT activityof any manipulation designed to reduce GvHD (e.g., Treginfusion). Parallel studies aimed at manipulating APCs maybe equally important.

The durability of DLI responses in many diseases and thefactors which might influence this also merit further investiga-tion, and perhaps there is a need to explore the concept ofmaintenance therapy (either in terms of DLI or combinationsof newer pharmacological agents).

One thing that becomes increasingly apparent whenreviewing the literature on DLI is that there are many morequestions than answers. The remarkable ability of thedonor immune system to eradicate hematological tumorsis undoubted, but taming this potential will require con-siderably more research within the context of largercollaborative networks.


The authors declare no conflict of interest and have receivedno payment in preparation of this manuscript.

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AffiliationClaire Roddie1 FRCPath MRCP MBChB BSc

(Hons) &

Karl S Peggs†2 MD FRCPath MRCP MBChB†Author for correspondence1Clinical Research Fellow,

UCL Cancer Institute,

Department of Haematology,

Paul O’Gorman Building,

72 Huntley Street,

London, WC1E 6BT, UK2Senior Lecturer,

UCL Cancer Institute,

Department of Haematology,

Paul O’Gorman Building,

72 Huntley Street,

London, WC1E 6BT, UK

Tel: +0207 679 6236; Fax: +0207 679 6222;


Roddie & Peggs


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1. Introduction

2. Clinical trials with adult

autologous cell therapy

3. Enhanced cell strategies

4. Host effects

5. Conclusions

6. Expert opinion

Review

Autologous cell therapy forcardiac repairDarryl R Davis* & Duncan J Stewart†

*University of Ottawa Heart Institute, Ottawa, Ontario, Canada; and †Ottawa Hospital Research

Institute, Ottawa, Ontario, Canada

Introduction: While new therapies have improved the prognosis of patients

post acute myocardial infarction, many patients still suffer from irreversible

damage and live with the debilitating consequences. However, with the

advent of stem cell-based therapies, future treatments may enable us to har-

ness the potential of autologous stem cells to prevent and even reverse

heart damage.

Areas covered: We outline the results of the early clinical trials using autolo-

gous cell therapy and highlight the hurdles and limitations that still need to

be addressed. We also discuss new approaches that hold promise for develop-

ing the next generations of autologous cell therapy by exploring strategies to

enhance their regenerative activity using biomaterials, genetic modification,

optimal cell types and small molecule preconditioning.

Expert opinion: Autologous cell therapy may be on the cusp of being widely

adopted for the treatment of patients with large areas of myocardial damage.

Techniques to enhance the activity and retention of autologous cell products

may represent the next generation of this therapy.

Keywords: cardiac stem cells, cell therapy, endothelial progenitor cells, mesenchymal stem cells,

myocardial infarction, small molecules, somatic gene transfer


1. Introduction

Although there has been remarkable progress in the last several decades in the devel-opment of new pharmaceutical and interventional therapies for cardiac and vasculardiseases, many patients still suffer from irreversible damage and live with the debilitat-ing consequences, in particular heart failure (HF), which is emerging as a major chal-lenge for health care systems worldwide. As the contractile function of the heartdeclines, so too does its ability to respond to medical therapies, leading eventuallyto the need for cardiac replacement with all the attendant costs and serious health con-sequences of living with a transplanted heart or mechanical device. With the explosionof new knowledge about the role of stem cells in tissue repair and regeneration, we areon the cusp of a new era in medicine, one in which we will be able to harness thepotential of endogenous regenerative mechanisms to prevent and even reverse organdamage. The discovery of stem and progenitor cells, and the increasing understandingof their role not only in early embryonic development, but also organ homeostasis andrepair throughout adult life, has led to the burgeoning field of cell therapy, which isbeing explored for a wide variety of medical problems, from spinal injury to diabetes.

This review outlines the progress of first generation autologous stem cells and thehurdles that confront their ready translation to the clinical setting. The influence ofthe host factors that influence engraftment and regenerative potency will be exam-ined. Techniques to enhance regeneration using biomaterials, genetic modification,optimal cell types and small-molecule preconditioning are being evaluated whichoffer tremendous promise to enhance the healing of injured myocardium. Thisreview also looks forward at the future of autologous cell therapy for cardiac repair,


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and assesses attempt to predict what will be required in orderfor this innovative strategy to be adopted widely in the man-agement of patients that sustain large myocardial infarctionsdespite all modern reperfusion therapies.

2. Clinical trials with adult autologous celltherapy

Despite our incomplete understanding of the biology of stemand progenitor cells, a number of clinical trials have alreadybeen performed; many of which have rigorous design includ-ing randomization and blinding. The results of stem celladministration for the treatment of cardiac disease appear toshow real promise. Even quite simple approaches have alreadyyielded positive results for enhancing cardiac repair in the postmyocardial infarction (MI) setting (Table 1) [1-30]. In mostcases these trials have used unselected autologous bonemarrow mononuclear cells (BMC), since these are routinelyused for bone marrow transplantation in all large tertiarycare centers.Moreover, several recent systematic reviews and meta-

analyses have been performed of all randomized studies, total-ing over 900 patients, and these support a highly significant,albeit modest, improvement in global left ventricular ejectionfraction (LVEF), infarct area and end systolic volumes post-MI [31-33]. Although it was initially thought that transdiffe-rentiation of stem and progenitor cells into new vascular orcardiac tissue would be the predominant mechanism of resto-ration of structure and function, the results from both clinicaland preclinical studies using BMCs indicate that the mecha-nism of this benefit is probably not due to direct cardio-myocyte replacement but rather to a variety of othereffects which modulate cardiac repair [34,35]. These includeenhancing neovascularization of the ischemic zone [36],

paracrine stimulation of endogenous cellular repair mecha-nisms [37,38] and modulation of immune responses withreduced fibrosis and scarring [39]. In the early post-MI setting,the delivery of BMCs may be effective in attenuating theinitial inflammatory response, reducing scar formation andpromoting more adaptive healing. However, in the settingof extensive damage and chronic organ failure (i.e., HF), itis likely that regeneration of new contractile myocardial ele-ments either directly or indirectly will be required to improvecontractility. In this case, it may be necessary to harness cellsthat have the capacity to transdifferentiate to cardiomyocytes,be they resident within the myocardium or harvested fromother tissues, and both strategies are discussed below.

3. Enhanced cell strategies

3.1 Optimal autologous cell typesAlthough truly totipotent stem cells, such as embryonic stemcells or inducible pluripotent cells, demonstrate the greatestcapacity for organ regeneration; inherent concerns aboutsafety will probably impede their use in clinical cell therapiesfor the foreseeable future. Another class of bone marrow‘stem’ cell is represented by the mesenchymal stromal/stem cells (MSCs). These cells have been studied extensivelyover the last several decades and in addition to some capacityto differentiate to cardiac and vascular cell lineages [40], haveimportant modulatory effects upon the host myocardium.Recently, tissue-resident stem cells have been identified in anumber of adult organs including the brain [41] and heart [42].As outlined below, these cells appear to have specific abilitiesto regenerate the cells of these tissues.

3.1.1 Autologous blood and bone marrow cellsBone marrow contains a variety of stem and progenitor cellsincluding mesenchymal stem cells (MSCs) and endothelialprogenitor cells (EPCs) [31,43]. However, progenitor or stemcells represent a very small proportion of bone marrow orcirculating mononuclear cells (MNCs) (i.e., < 0.05%). None-theless, unselected MNCs appear to be able to improvecardiac function after delivery into the infarct-related artery,albeit modestly as reviewed above. While these benefits stillneed to be validated in a large, pivotal Phase III trial, it isalso apparent that there may be tremendous opportunity torefine current cell therapy strategies to enhance efficacy bythe selection or enrichment of cell populations with greatertherapeutic activities, and the enhancement of regenerativeactivity of a given cell population.

Most of the published clinical studies for cardiac cell ther-apy have used bone marrow or blood derived MNCs isolatedby Ficoll gradient centrifugation, drawing on the establishedclinical expertise and infrastructure developed to supportbone marrow transplantation as a routine clinical procedurein most tertiary hospital settings [1,2,21,22]. While expeditingthe road for translation to clinical application, it is highlyimprobable that unselected MNCs will ultimately prove to

Article highlights.

. Clinical trials demonstrate that even quite simpleapproaches to administer autologous cells yieldpositive results.

. While truly totipotent cells, such as embryonic stem cellsand induced pluripotent stem cells, may have thegreatest capacity to regenerate organs; safety concernswill significantly delay translation to clinical use.

. A host of bone-marrow-derived cell products have beendeveloped for clinical use; however the main mechanismof benefit appears to be paracrine-mediated.

. Resident cardiac stem cells show great promise forautologous cardiac repair strategies as they posses bothparacrine-mediated myocardial salvage and the capacityto differentiate into working myocardium.

. The next generation of autologous therapy will beengineered using combination cell therapy, improvedmechanical retention, modified culture environmentsand somatic gene transfer.


Autologous cell therapy for cardiac repair


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be the most effective cell therapy product for cardiac andvascular repair. Although it is attractive to use antigenic cellsurface markers to select subpopulations of cells with greaterregenerative capacity, at this time there is no agreement asto what markers can be used to enrich the true ‘EPC’fraction [5,44-48]. In addition, cell selection based on surfacemarkers introduces major challenges in scaling up to manu-facture sufficient cell numbers for effective clinical treatmentsgiven the very small proportion of MNCs which meet themost common ‘EPC’ definitions [44-46]. Indeed, the use ofselected MNCs subsets (i.e., CD133+, CD34+) has notyielded substantially greater benefit compared with unselectedMNCs in the limited number of clinical studies that haveused this strategy [5,47,48].

An alternate strategy is to select regenerative cells fromthe MNC fraction based on their functional characteristics,in a manner analogous to the commonly used selectionprocedure for deriving mesenchymal stem cells or dendritic

cells. Specifically, the ability of subpopulations of MNCs todifferentiate in attached culture in the presence of specificextracellular matrix components and growth and differentia-tion factors. There has been a wealth of experience in charac-terizing the regenerative activities of cultured-derived ‘EPCs’in a variety of in vitro and in vivo models, which is brieflysummarized below [49,50].

3.1.1.1 Culture modified endothelial progenitor cells (EPCs)So-called ‘early growth EPCs’ or ‘circulating angiogenic cells’appear within 2 -- 3 days of MNC selection culture in thepresence of endothelial growth factors (i.e., VEGF, IGF,EGF, etc.) [49,51]. While they express a number of endothelialcharacteristics (CD31, VEGFR2, tunica interna endothelialcell kinase (Tie2), eNOS, lectin binding and dioctadecyl-3,3,3¢,3¢-tetramethylindo-carbocynanine perchlorate-labelledlow-density lipoprotein (DI-LDL) uptake), they still retainmonocyte (CD14) and leukocyte (CD45) markers. However,

Table 1. Clinical trials of autologous bone marrow stem cells in the post myocardial infarction setting.

Study Design N* Cell type Delivery Days post MI Outcome

Non-randomized trialsStrauer et al. [1] Primarily safety 10 BM-MNCs IC 8 ± 2 Stroke volume"; infarct size#;

wall motion"; perfusion"TOPCARE-AMI [2,3] Primarily safety 59 BM-MNCs,

PB-EPCsIC 4.9 ± 1.5 EF"; remodelling#; infarct size#;

perfusion"Fernandez-Aviles et al. [4] Primarily safety 20 BM-MNCs IC 13.5 ± 15.5 EF"; ESV#; wall motion"Bartunek et al. [5] Primarily safety 19 CD133+,

BM-MNCsIC 11.6 ± 1.4 EF"; perfusion"; myocardial

viability"BALANCE [6] Efficacy 62 BM-MNCs IC 7 ± 2 EF"; stroke volume index;

mortality benefitSTAR-heart [7] Efficacy 191 BM-MNCs IC 3102 ± 1168 EF"; stroke volume index;

mortality benefitRandomized controlled trials (RCTs)Chen et al. [8] Randomized 34 BM-MSCs IC 18.8 ± 0.5 EF"Ge et al. [9] Randomized 10 BM-MNCs IC < 1 EF"; perfusion"Ruan et al. [10] Randomized 9 BM-MNCs IC < 1 EF"; wall motion"Huang et al. [11] Randomized 20 BM-MNCs IC < 1 EF"; infarct size#Yao et al. [14] Randomized 24 BM-MNCs IC 13 ± 8 No change in systolic function;

improved diastolic functionPenicka et al. [15] Randomized 17 BM-MNCs IC 9 No change in EFBOOST [13,17,18] Randomized 30 BM-MNCs IC 4.8 ± 1.3 EF"(6 months only)Janssen et al. [19,20] Randomized,

double blind33 BM-MNCs IC < 1 No change in EF; infarct size#;

regional systolic function"REPAIR-AMI [21-23] Randomized 102 BM-MNCs IC 4.3 ± 1.3 EF"; greater benefit in < 49%

EF and > 5 days after MIMeluzin et al. [16,25] Randomized 44 BM-MNCs IC 6.8 ± 0.3 EF" in a dose-dependent

mannerASTAMI [24,26] Randomized 50 BM-MNCs IC 6.0 ± 1.3 No benefitSuarez de Lezo et al. [27] Randomized 10 BM-MNCs IC 7 ± 2 EF"FINCELL [28] Randomized 39 BM-MNCs IC < 4 EF"REGENT [29] Randomized 80 BM-MNCs IC 7 No benefit

80 CD34+ BM-MNcsTraverse et al. [30] Randomized 40 BM-MNCs IC 4.5 No change in EF; Improved

LV end diastolic volume

*N: Number of patients receiving cells.

BM-MNCs: Bone marrow-mononuclear cells; EF: Ejection fraction; ESV: End-systolic volume; IC: Intracoronary; LV: Left ventricle; MI: Myocardial infarction;

PB: Peripheral blood.

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there is abundant in vitro and in vivo evidence that this cellpopulation is highly angiogenic with the ability of these ‘earlygrowth’ EPCs to restore perfusion in the ischemic hindlimbor improve cardiac function post MI having been soundlyestablished [2,31,49,52]. Therapeutically, transplanted EPCscan provide stimulatory cytokines [53,54] and induce humoraleffects that are sustained by the host tissues [55]. Therefore,these culture-modified MNCs may result in one or more ofthe following: i) the cells may modify the local milieu in orderto increase recruitment of other circulating progenitor cells;ii) the injected cells may themselves be involved in producinggrowth factors and recruiting additional cellular and paracel-lular elements; or iii) the cells may differentiate and directlypartake in new blood vessel growth. All these processes havebeen observed experimentally, however, it is believed that vas-cularization from paracrine/humoral factors and secondaryrecruitment of host stem/progenitor cells is probably themain mechanism leading to functional improvement [3,55-60].Thus these cells have a number of properties that are quitefavorable for their use in clinical therapy with extensive pre-clinical evidence demonstrating potent angiogenic activity ina number of relevant models [61-63] as well as ease of scalabilityof manufacturing for human studies.

3.1.1.2 Late outgrowth EPCsMore prolonged culture of blood or bone marrow-derivedMNCs (i.e., > 1 -- 2 weeks) under the conditions describedabove gives rise to colony-like clusters or aggregates of highlyproliferative cells, which then rapidly overgrow the dishes toproduce uniform ‘cobblestone’ cultures of so-called ‘late out-growth EPCs’ [31,64]. Unlike the early-growth EPCs describedabove, these cells have lost all leukocyte determinants andexhibit a robust endothelial phenotype. Indeed, apart fromtheir markedly increased proliferative potential and possiblyan increase in the expression of some markers of EC activation(i.e., E-selectin), the late growth cells are nearly indistinguish-able from mature EC cultures. Thus, this population may beof great interest for regenerative medicine applications;however, to date they have been far less studied in relevantpreclinical models. It has been suggested that they may actsynergistically with early outgrowth EPCs, acting mainly todirectly repair the vascular endothelium [65], however, theydo not appear to share the same paracrine abilities of the‘early growth’ cells, and in our experience, have not beeneffective in a head to head comparison of in vivo efficacy inthe prevention of experimental pulmonary hypertension [66].

3.1.1.3 Mesenchymal stromal (stem) cells (MSCs)MSCs represent an important population of bone marrow-derived cells which have been studied extensively over severaldecades, and may offer certain advantages for specific regener-ative medicine applications. MSCs are a type of non-hematopoietic, adult somatic stem cells that can be isolatedfrom bone marrow [67] and extensively expanded in vitro[68]. In contrast to embryonic stem cells, which are capable

of differentiating into all cells of the body (i.e., are totipotent),MSCs have only limited in vivo differentiation and prolifera-tion potential (i.e., are multipotent) [69]. However, they alsoexhibit important ‘immunomodulatory’ properties whichprobably contribute to their ability to reduce tissue damageand enhance repair [70]. Additionally, several recent studieshave also shown that MSCs also stimulate the proliferationof other progenitor cell populations within target organs topromote endogenous repair [71-73]. Clinical trials utilizingMSCs for acute MI [8] or for graft-versus-host disease [74]

have shown various levels of success, even though these resultsneed to be confirmed in large and rigorously designed studies.Like EPCs, MSCs can be isolated directly from a patient’sown bone marrow, thereby avoiding complications involvingthe immune rejection of allogeneic tissue [75]; however,there is considerable evidence that they may be ‘immune-privileged’, potentially permitting their use in allo-transplan-tation (i.e., cells from another individual) without the needfor immunosuppressive therapy [76-80]. The use of allogeneiccells would make it feasible for MSCs to be delivered as an‘off-the-shelf’ product for the treatment of acute (such as inthe case of acute lung injury/acute respiratory distress syn-drome (ALI/ARDS) patients) and chronic disorders. Thisunique feature makes MSC-based therapies very attractivefor the treatment of acute organ injuries.

While the clinical experience to date has demonstrated thatautologous bone marrow or circulating stem/progenitor celldelivery is generally well tolerated, some preclinical reportshave shown that intramyocardial injection of MSCs may dif-ferentiate into encapsulated structures with calcifications andossifications [81,82]. This in vivo plasticity replicates in vitroobservations that this highly proliferative cell source maypermit phenotypic drift and the posibility of cancerous trans-formation [83]. Despite these findings, preclinical data hasgenerally been encouraging with no signal reported of adverseevents. But these findings may be a harbinger of the challengesthat will confront the use of more pluripotent stem cells.

3.1.2 Resident cardiac stem cells (CSCs)Ten years ago, prevailing dogma posited that the adult mam-malian heart was incapable of self-regeneration after injury [84].Adult cardiomyocytes were considered terminally-differentiatedcells that have exited the cell cycle. This dogma was challengedby the discovery that the heart contains a reservoir of small cellsthat stain for stem cell markers, propagate in vitro and developphenotypic features of heart cells after differentiation [85-92].Retrospective studies using 14C birth dating of cells or chemo-therapeutically labeled adult cells have since confirmed that car-diomyocyte turnover occurs in the adult human heart [93,94].Given that CSCs are autologous and capable of formingall cardiac lineages, they represent logical candidates forcell therapy.

Numerous markers have been proposed to identify residentCSCs for isolation and ex vivo amplification (Table2) [85-88,95-100].The first paper describing isolation of resident cardiac stem cells



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built upon previous work using isolated skeletal myoblasts [42].In this work, the capacity of discreet subpopulations withinmyocardium to actively transport Hoechst dye defines a sidepopulation of cells on flow cytometry proved to be capable ofmultipotentiality [42,95] and myocardial repair [101]. This sidepopulation phenotype was later ascribed to the surface expres-sion of the ATP-binding cassette protein G2 (ABCG2, amembrane transport protein mediating multi-drug resistance;CDw338) [95]. In humans, ABCG2 expression co-localizeswith CD31+ cells, suggestive of a potential endothelial fate [102].

The most widely studied CSC marker, c-Kit, was firstidentified in histology sections from sex-mismatched trans-planted adult human hearts [103]. This marker co-expresswith other stem cell markers (multidrug resistance protein 1(MDR1) and surface marker antigen (Sca-1)) and has beenshown to improve post MI function with differentiationinto numerous cardiac lineages (cardiomyocytes, endothelialcells and smooth muscle cells) [104-107]. A Phase I clinical trialusing antigenically purified sub-populations of c-Kit hasbegun to assess the feasibility and safety of intra-coronary-delivered CSC in chronic HF patients undergoing cardiacsurgery [108].

Of note, studies using Sca-1 in mice have isolated asmall CSC population capable of improving post-MIcardiac function [86]. These mouse cells partially expressedmarkers of endothelial/BMC origin (ABCG2, CD31,CD38) while not expressing markers of other stem cell(c-Kit, fetal liver kinase 1 (Flk-1)) or hematological (CD34,CD45, fms-like tyrosine kinase (Flt-1)) lineages. Severalstudies have explored the capacity of Sca-1+ cells isolatedfrom human samples to improve cardiac function [98-100].While the human epitope recognized by Sca-1 is not known,these studies found a homogenous cell population can be

isolated that partially expresses c-Kit and is capable ofin vitro cardiac differentiation.

Recently it has been shown that distinct subpopulations ofCSCs can be isolated directly from cardiac tissue [109]. Thistechnique simplifies culture methods by focusing on theprimary cellular outgrowth product from cardiac samples,without recourse to antigenic sub-selection [87,97,110]. Whensamples of minced cardiac tissue are cultured, a lawn of flatcells emigrates spontaneously from the plated cardiac tissue.Within that lawn, clusters of CSCs emerge and proliferate.Using mild enzymatic dissociation, loosely-adherent cellssurrounding the explant (termed cardiac outgrowth) can beserially harvested. This outgrowth contains complimentarysub-populations of cells expressing embryonic (stage-specificembryonic antigen 1 (SSEA-1)), stem cell-related (c-Kit,ABCG2), endothelial (CD34, CD31) and mesenchymal(CD90) antigens. Transitioning this direct outgrowth thoughthree dimensional sphere culture has been shown to enhanceboth CSC content and potency [111]. Further expansionthrough monolayer culture provides a single cell producttermed Cardiosphere Derived Cells (CDCs) that have beendemonstrated to be clonogenic, multipotent and capable ofself-renewal [112-114]. Recent studies of CDCs have demon-strated that in vivo these cells secrete VEGF, hepatocytegrowth factor (HGF) and IGF-1 [115]. It has been estimatedthat direct cardiomyocyte and vascular transdifferentiationrepresents 20 -- 50% of the overall increase in cardiac tissue,suggesting that, like EPCs, indirect effects of CDCs on tissuepreservation and/or recruitment of endogenous progenitorssignificantly contribute to therapeutic outcomes [114,115] Stud-ies have focused on validating culture techniques [116-118] forbroad clinical translation beginning with a recently startedPhase I clinical trial [112,119,120].

Table 2. Cell surface marker expression and stem cell characteristics of cardiac progenitor cells.

Marker Species Source Surface expression Clonogenicity Differentiation Function

benefit

c-kit [85] Mouse/rat Whole heart MDR1+, Sca-1+, CD45-,CD34-, VWF-, CD31-,vimentin-, lin-

Yes IHC/coupling Yes

sca-1 [86] Mouse Whole heart ABCG2±, CD31±, CD38±,c-Kit-, Flk-1-, CD34-,CD45-, Flt-1-

Yes IHC Yes

abcg2 [88,95,96] Mouse Whole heart Sca-1+, CD31+ c-Kit±,CD34±, CD45-

-- IHC/coupling --

SSEA-1 [87] Rat Ventricular ABCG2±, c-Kit±, Sca-1±,Flk-1±, CD31-, CD45-,VE-cadhedrin-, SSEA4-

-- IHC Yes

c-kit [97] Human Biopsy KDR-, CD45-, lin-, CD34-,CD133-

Yes IHC/coupling Yes

Sca- [98-100] Human Atrial appendage CD105+, CD31+ c-Kit±,CD45-, CD14-, CD34-,CD133-

Yes IHC Yes

ABCG2: ATP-binding cassette protein G2; Flk-1: Fetal liver kinase-1; Flt: Fms-like transcript; IHC: Immunochistochemistry; KDR: Kinase insert domain receptor;

MDR-1: Multidrug resistance protein 1; Sca-1: Surface marker antigen-1; SSEA: Stage-specific embryonic antigen; VWF: Von Willebrand factor.

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3.1.3 Other autologous cell types for cellular

cardiomyoplastyA host of other autologous cell types have been cultured fromdiverse extra-cardiac progenitor sources for cellular cardio-myoplasty. Early experience with skeletal myoblasts demon-strated the challenging nature of this approach as myoblasttransplantation was plagued by ventricular tachyarrhythmiasand sudden cardiac death [121,122]. The pathogenesis of thesearrhythmias is poorly understood, but may be related to thefact that skeletal muscle cells, unlike heart cells, are electricallyisolated by the absence of appropriate gap junctions [123].Adipose stem cells represent a promising extra-cardiac

stem cell source that can be retrieved in high number fromeither liposuction aspirates or subcutaneous adipose tissuefragments [124,125]. Although adipose tissue represents aheterologous cell population, culture selects for a relativelyhomogenous cell population, enriched for cells expressinga stromal phenotype (CD13, CD29, CD73, CD90,CD133) [126-129]. In vitro, these cells can be easily be expandedand, like MSCs, can modestly differentiate into cardio-myocyte [130-133] and endothelial [134-137] lineages. Consistentwith the MSC experience, adipose stem cells improve cardiacfunction [133,138-140] with limited evidence for in vivo differen-tiation [138,140,141], suggesting that paracrine-mediated effectsmediate the majority of the beneficial effects seen with thiscell type [142,143]. Based on their ease of culture and benignprofile, adipose stem cells are currently under investigationfor patients with ischemic heart disease [144] and acuteMI [145].

3.1.4 Combination cell therapyDespite evidence that stem cell injection improves myocardialfunction, studies consistently demonstrate modest overallbenefit, possibly related to very limited long-term engraft-ment and retention after intra-myocardial injection [146-150].This finding is not surprising given that cells are injectedinto a hostile inflammatory milieu with poor mechanicaland vascular support. Several studies have investigated theeffect of transplanting complimentary cell types on myocar-dial function [99,151-153], based on the premise that cellssupporting the surrounding host tissue through paracrinesecretion may increase the retention and proliferative capacityof cells capable of forming true contractile cells. Earlyattempts focused upon angiogenic progenitor cells and skele-tal myoblasts [151,152]. These studies demonstrated that whileboth cell types reduce scar size and improve ventricular func-tion, combination therapy was superior. While intriguing,this data is not translatable into clinical trials given theproblems of association of myoblasts with ventriculartachy-arrhythmias and sudden cardiac death [121,122].Recently, a group from the Netherlands has taken this

concept forward using combination therapy derived fromthe adult heart (i.e., epicardium-derived cells (EPDCs) andcardiac progenitor cells (CPCs)) [99]. These investigatorsdemonstrated that co-transplantation improved post infarct

function compared with single-cell transplantation whichitself was superior to vehicle controls. These findings usingmixed cardiac derived cell populations (CPCs and EPDCs)mirror studies showing that purified human CPCs (c-Kit+)and mesenchymal cells (CD90+) are capable of independentlyimproving post myocardial infarct cardiac function in animmunodeficient mouse model [154]. These promising resultssuggest synergies exist between multiple cell types, providingnew directions in cardiac cell therapy using existing firstgeneration cell types.

3.2 Pre-conditioning using small molecules or

enhanced culture techniquesThe concept of introducing exogenous factors to promoteendogenous repair is well established with many studiesdemonstrating benefit through intra-myocardial injection ofcytokines (i.e., G-CSF, HGF), growth factors (IGF-1) orsignaling proteins (Akt, proto-oncogene serine/threonine-protein kinase Pim-1, cardiotrophin 1 (CT-1)) in cardiacdamage models [155-160]. However the potential clinical utilityof these strategies is limited by their reliance on extra-cardiac cell types, lack of specificity, transient retentionand requisite transduction with potentially oncogenic fac-tors. Moreover, clinical trials of bone marrow mobilizationusing G-CSF and related factors have been disappointingbecause of lack of specificity and harmful pro-inflammatoryeffects [161].

Importantly, this work has provided tantalizing clues aboutthe triggers underlying stem cell cycling. Recent work by ahost of labs indicates that targeted manipulation of the verysame pathways underlying cardiogenesis or pluripotencymay significantly increase the overall number and potency ofresident stem cells [158,162-167]. These insights have been usedto enhance cells ex vivo prior to injection by the addition ofprotein factors or small molecules, such as AVE-9488 [168],PPARa agonists [169,170], statins [171,172] and TGF-b [116,167].

Alternatively, variations in culture techniques have beenshown to alter the phenotype of cells without recourse todirect stimulation of potentially oncogenic pathways orgenetic reprogramming. Conceptually, this approach hasmerit as the environments to which stem cells are injectedfor myocardial repair are considerably harsher than traditionalculture conditions or natural stem cell niches [173-176]. Briefexposure to hypoxic conditions (2% O2) has been successfullyused in several studies to enhance the retention and survival ofcells within the low oxygen tension of infarct regions (aslow as 0.2% O2) [177-180]. Interestingly, ‘normal’ cell cultureconditions may be toxic to progenitor and stem cells asnon-physiologic hyperoxic conditions (ambient atmosphericconditions, 20% O2) can promote oxidative DNA damage,genomic instability and premature cell senescence [181-184]. Itfollows that that culture of stem cells within physiologicalnormal oxygen conditions (5% O2) improves cellular yieldand potency [185-187]. A recent study has taken this observationto its natural conclusion by demonstrating that cell culture



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conditions recapitulating the natural three dimensional stemcell niche/embroid state enhance adhesion molecule expres-sion, cardiac progenitor cell content, in vivo cellular retentionand regenerative potency of ex vivo-cultured cardiac stemcells [188].

These findings highlight the capacity of altered culture con-ditions to provide a superior cell product with the potentialfor ready translation to clinical use. Such ‘preconditioning’has the advantage of simplicity but potentially suffers fromlack of durability of effects once the cells are transplantedin vivo. An alternative approach involves direct geneticmanipulation (i.e., gene transfer) or incorporation of cellsinto scaffolding matrices. As outlined below, the suitabilityof the specific strategy will depend on the specific pathwaythat is being targeted and other considerations that effect theefficacy of the approach (i.e., duration of effect), as well assafety, regulatory and ethical considerations.

3.3 Genetic modification of stem cellsDirect genetic engineering of stem cells has also been usedto improve transplanted cell survival (b-Akt [189,190], colonystimulating factoer 1 (CSF-1) [191], cellular repressor ofE1A stimulated genes (CREA1) [192], heme oxygenase 1(HO-1) [193,194], survivin [195], IGF-1 [196,197], heat shock pro-tein 20 (HSP20) [198], B cell leukaemia/lymphoma relatedprotein2 (Bcl-2) [199], Pim-1 [200]), electrical integration (con-nexin 43 (Cx43)) [123], differentiation (TNF-a), homing/migration (CXCR4 [201], monocyte chemotactic protein-3(MCP-3) [202], eNOS [203]) and vasculogenesis (HGF [204],hypoxia-inducibel factor 1 (HIF-1) [205], VGEF [206], stromalcell-derived factor 1 (SDF-1) [207], bFGF [208]). Comparingthese different approaches is problematic given variationsin cell type (fetal myocytes, embryonic stem cells, skeletalmyoblasts, mesenchymal stem cells and several cell typesderived from the bone marrow), models of therapeuticbenefit (ischemic hind limb, carotid injury, MI and pressureoverload HF) and strategies for gene transfer to cells(viral, plasmid). Additionally as outlined in Table 3, theseapproaches have been broadly applied to increase retentionand survival of cells whose primary mechanism of actioninvolves paracrine-mediated survival rather than true cardio-myogenesis [123,189-200,203,207-230]. Notably, a promising studyincorporates overexpression of the pro-survival factor Pim-1into cardiogenic CSCs [200]. This approach is based on theobservation that the benefits of CSC therapy occur even atgenerally low levels of cell engraftment [150,231,232]; whichis presumably due to transplanted cell loss mediated byapoptosis. Pim-1, by enhancing the pro-survival pathways,resulted in better engraftment and long-term retention ofCSCs [158,233].

Another approach targets the paracine profile of earlyoutgrowth EPCs [234]. Given that reduced eNOS expressionand NO production have been strongly implicated notonly in endothelial dysfunction [235,236] but also in EPCdysfunction [237,238], measures to enhance eNOS production

represent a logical target for cellular therapy. Preliminarystudies have demonstrated that overexpression of eNOSenhances the regenerative capacity of EPCs isolated frompatients with ischemic cardiomyopathy [168], suggesting a syn-ergistic effect between cell and gene therapy. Accordingly, thefirst clinical trial of genetically modified autologous stem cells(EPCs) targets has been initiated to target patients with sig-nificant LV dysfunction (left ventricuar ejection fraction(LVEF) £ 45%) early after a reperfused ST elevation MI(< 30 days) [239]. In this study, consenting patients willundergo apheresis to collect peripheral blood MNCs andplasma before randomization to one of three arms, receivingeither Plasma-Lyte A (placebo), autologous endothelial-like, culture modified MNCs (E-CMMs), or autologousE-CMMs transfected with eNOS, by coronary injection tothe infarct-related artery. After 2 -- 3 days, the early outgrowthEPCs will be transfected with a human eNOS-pVAX plasmidcomplexed with linear polyethylenimine. The final cell prod-uct (20 million eNOS-transfected or non-transfected EPCs)will be injected directly into the infarct-related artery distalto an inflated angioplasty balloon. The primary outcomemeasures is a change in global LVEF from baseline to 6 monthfollow-up as determined by cardiac MRI. Secondary efficacymeasures include changes from baseline to 6 month follow-up in i) regional wall motion, wall thickening, and infarct vol-ume as determined by cardiac MRI; ii) echocardiographicassessment of LVEF and ventricular volumes; iii) time toclinical worsening (death, hospitalization for angina, reinfarc-tion); and iv) quality of life (the SF-36 and the Duke ActivityStatus Index). This study represents the first clinical trial ofnext generation of cell products and promises to provide aneffective adjunctive to revascularization therapies in patientswith large myocardial infarcts.

3.4 Scaffolds, matricellular materials and mechanical

retentionRemarkable progress has been made in engineering bio-materials with the aim of creating multiscale 3D architecturesto promote tissue repair and regeneration [240-247]. Throughthe application of nanotechnology in medicine, it has becomepossible to design smart, multi-functional structures com-posed of polymeric materials incorporating suitable architec-tures and bioactive signaling factors, which can interact withthe surrounding tissue environment and facilitate tissue regen-eration [248,249]. Combining bioactive materials with stem cellsincreases both the differentiation potential and the secretionof ‘trophic’ factors involved in tissue repair and survival.Disrupting the normal matrix interactions and placingattached cells in suspension for injection into tissue or bloodis a profoundly disruptive process which triggers rapid pro-grammed cell death, or anoikis [250]. Tissue engineering offersthe possibility of developing biomaterials to provide appro-priate surfaces for integrin binding to improve cell survival,resulting in better retention and function of transplantedcells [251,252]. The design of synthetic materials that mimic

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Table

3.Geneticmodificationofautologousstem

cells.

Celltype

Geneticmodification

Genetransfer

method

Mech

anism/rationale

fortargeting

Resu

lt

Enhancedintegration

SkM

Cx-43overexpression

[123,209]

Plasm

idPerm

itseffectiveextracellularcouplingwith

surroundingmyocytes

Enhancedpost-M

Iengraftmentand

functionwithoutarrhythmiasnoted

EPCs

HIF-1

overexpression

[210,211]

Adenovirus

Protectscells

from

hypoxiaandpromotes

dangiogenesis

Enhancedvascularizationin

ischemic

hindlim

bandcornealmodels

EPCs

VEGFoverexpression

[212]

Adenovirus

Promotesangiogenesis

Enhancedvascularizationin

ischemic

hindlim

bmodel

MSCs

SDF-1overexpression

[213,214]

Plasm

idIncreasesengraftmentandenhancesMSC

survival

Improvedpost-M

Imyocardialfunction

bypreservingreversibly

damaged

myocytesandrecruitingCSC-likecells

MSCs

VGEFoverexpression

[215-219]

Adenovirus,

plasm

idPromotesangiogenesis

Improvesneo-vascularizationandfunction

inmodelsofmyocardialinfarction,ischemic

hindlim

bandpressure

overloadHF

MSCs

HGFoverexpression

[220]

Adenovirus


Improvedpost-M

Ifunction

MSCs

Angiopoetinoverexpression

[221,222]

Adenovirus


Co-expressionwithAktreducesapoptosis

andprovesgreaterfunctionalim

provement

thankeitherfactoralone

MSCs

GATA-4

overexpression

[223]

Retrovirus

GATA-4

enhancestheMSC

secretome-therebyincreasingcell

survivalandpromotingpost-infarction

cardiacangiogenesis

Enhancedhypoxiaresistance,angiogenesis

andfunctionalrecovery

aftermyocardial

infarction

SkM

CSF-1overexpression

[191]

Plasm

idPromotesangiogenesisandmetalloproteinase

expressionpotentially

leadingto

enhancedextracellularremodeling

Enhancedpost-M

Iengraftmentandfunction

withoutarrhythmiasnoted

SkM

SDF-1overexpression

[196]

Plasm

idIm

provedpost-M

Imyocardialfunctionin

rats

Effect

mediatedbyenhancedrecruitment

ofprogenitorcells

Enhancedstem

andprogenitorcell

migrationto

theheartprovidingim

proved

post-M

Iangiogenesisandfunctionrecovery

Enhancedretention

MSCs

CCR1overexpression

[224]

Retrovirus

Mediatesrecruitmentofinflammatory

cells

toareasofinflammation

MSCsover-expressingCCR1accumulate

inareasofdamagedmyocardium

andim

prove

myocardialfunction

MSCs

CXCR4overexpression

[225,230]

Retrovirus,

adenovirus

CXCR4mediatesthehomingofMSCsto

damagedregionsofdamagedtissue

ExpressionofCXCR4increaseshoming

ofgenetically

modifiedcells

toinfarctregions

BAD:BCL2-associatedagonistofcelldeath;Bax:

Bcl2-associatedXprotein;Bcl-2:B-cellleukamia/lym

phoma-associatedprotein

2;CPCs:

Cardiacprogenitorcells;CREG1:CellularrepressorofE1A-stimulatedgenes;

CSCs:

Cardiacstem

cells;Cx43:Connexin43;EPCs:

Endothelialprogenitorcells;FG

F:Fibroblast

growth

factor;GATA-4:Gata

bindingproten4;HF:

Heart

failure;HGF:

Hepatocyte

growth

factor;

HIF-1:Hypoxia-inducible

factor;HO-1:Hemeoxygenase

1;HSP20:Heatshock

protein

20kDA;hTERT:Humantelomerase

reversetranscriptase;MDR1:Multidrugresistance

protein-1;MI:Myocardialinfarction;

MSCs:

Mesenchym

alstem

cells;pim

-1:Proto-oncogeneserine/threonine-protein

kinase;SDF-1:Stromal-cell-derivedfactor-1;SkM:Skeletalmyo

blasts.



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Table

3.Geneticmodificationofautologousstem

cells(continued).

Celltype

Geneticmodification

Genetransfer

method

Mech

anism/rationale

fortargeting

Resu

lt

Enhancedsurvival

EPCs

hTERToverexpression

[226]

Adenovirus

Maintainstelomerase

activityandreplication

oftelomericDNA;perm

ittingtheindefinite

divisionofcells

Enhancedmitogenic

activity,

migratory

activity,

andcellsurvivalusingischemic

hindlim

bmodel

EPCs

eNOSandHO-1

overexpression

[203]

Retrovirus

Regulatesendothelialfunctionandprotects

against

apoptosis

eNOSenhancedendothelialreconstitution

inamodelofcarotidinjury

while

HO-1

had

noeffect

Cardiomyoblasts

(H9c2)

Bcl-2

overexpression

[199]

Adenovirus

Inhibitsoligomerizationofpro-apoptotic

proteinsBax/Bakandsubsequentmembrane

perm

ealization

Increasedgraftsurvivalin

ischemic

ratmyocardium

CPCs

PIM

-1overexpression

[200]

Lentivirus

Downstream

mediatorofAKTactivation

preventingapoptosis

Enhancedpost-M

Icellularengraftment,persistence

andfunctionalim

provementin

mice

MSCs

Aktoverexpression

[189]

Retrovirus

Inhibitsapoptosisbyinactivatingthe

pro-apoptoticprotein

BADandbyincreasing

transcriptionofpro-survivalgenes(via

activatedNF-kB

)

Improvedpost-M

Imyocardialfunctionin

rats

Effect

mediatedbyparacrineeffects[227]

MSCs

CREG1overexpression

[192]

Adenovirus

InhibitsofapoptosisbyactivatingAktand

degradingp53

Protectsagainst

apoptosis,

enhancescellsurvival

andupregulatesVEGFsecretion

MSCs

HO-1

overexpression

[193,194]

Adenovirus

Involvedin

theoxidative

cleavageofheme

andprotectsagainst

apoptosis

MSCs

HSP20overexpression

[198]

Adenovirus

Restoresfunctionofpro-survival/angiogenic

cytokines(Akt,FG

F-2,IGF-1andVEGF)

IncreasedMSCsurvivalandenhancedpost

MI

functionthroughincreasedsecretionofgrowth

factors

(VEGF,

FGF-2andIGF-1)

MSCs

IGF-1overexpression

[197]

Adenovirus

ActivatesAKTpathwayandinhibitsapoptosis

Enhancescellsurvivalandrecruitmentofckit+,

MDR1+,CD31+andCD34+cells

into

the

infarctedratheart

MSCs

Survivin

overexpression

[195]

Lentivirus

InhibitsapoptosisbyinhibitingBax/Fasactions

andpossibly

byinhibitingcaspase

activation

Enhancedcellsurvival,paracrinesecretion(VEGF)

andpost-M

Ifunction

BAD:BCL2-associatedagonistofcelldeath;Bax:

Bcl2-associatedXprotein;Bcl-2:B-cellleukamia/lym

phoma-associatedprotein

2;CPCs:

Cardiacprogenitorcells;CREG1:CellularrepressorofE1A-stimulatedgenes;

CSCs:

Cardiacstem

cells;Cx43:Connexin43;EPCs:

Endothelialprogenitorcells;FG

F:Fibroblast

growth

factor;GATA-4:Gata

bindingproten4;HF:

Heart

failure;HGF:

Hepatocyte

growth

factor;

HIF-1:Hypoxia-inducible

factor;HO-1:Hemeoxygenase

1;HSP20:Heatshock

protein

20kDA;hTERT:Humantelomerase

reversetranscriptase;MDR1:Multidrugresistance

protein-1;MI:Myocardialinfarction;

MSCs:

Mesenchym

alstem

cells;pim

-1:Proto-oncogeneserine/threonine-protein

kinase;SDF-1:Stromal-cell-derivedfactor-1;SkM:Skeletalmyo

blasts.

Davis & Stewart


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natural stem cell micro- and macroenvironments environ-ments (i.e., niches) may potentially be a powerful tool bothto understand and control stem cell function. Research inthis field is still very limited, and artificial niches for stem cellshave been developed mainly using synthetic proteins withapplications mainly focused on neural stem cells [253]. Usingpatterned substrates, it can be shown that stem cells can ‘sense’substrate features or topography at the micro- or nano-scalelevel, and react by changing cell shape (i.e., spreading) andorganizing into multicellular physical patterns [254]. Recently,it has been demonstrated that a single cell encapsulationstrategy can markedly increase the survival of MSCs byre-introducing cell--matrix interactions via integrin clusteringand activation of the MAPK(ERK) signaling cascade [255].These promising results demonstrate that bioengineered‘micro-niches’ incorporating adhesion molecules provides aversatile tool for the enhancement of viability and targetedcellular engraftment.Mechanical approaches to enhance acute retention represent

a complimentary means of increasing the potential of stemcell therapy for myocardial regeneration. Low initial engraft-ment of cell products has been attributed to back leak ofinjected cells due to myocardial contraction, clearance throughvenous or lymphatic drainage and/or washout by injectiontrauma [232,256-259]. Sealing the injection site with fibrin glueand reducing the ventricular rate have been shown to increaseacute retention with resultant salutary benefits on myocardialcontractility [232]. Similarly, labelling cells with superparamag-netic microspheres followed by intra-cardiac injection with asuperimposed magnet [260] or intra-coronary implantation ofmetal stents [261-265] has been shown to enhance the acuteretention and functional benefit of cell therapy.

4. Host effects

The translation of cell-based approaches developed in other-wise healthy animals to therapeutic strategies is not straightfor-ward as most patients suffering from acute MI have a historyof multiple coronary artery disease (CAD) risk factors (RF)(diabetes, advanced age, smoking, hypertension and hypercho-lesterolemia) which have been shown to affect the number andfunction of circulating EPCs [31]. Clinical reports have alsoshown that patients with CAD and/or various RFs also showreduced numbers and function of circulating EPCs [266-271].A clinical report by Vasa et al. showed that in patients withischemic heart disease, there is an inverse correlation betweenthe number of cardiovascular RFs and the number and migra-tory activity of EPCs [270]. More recently it has been shownthat EPCs derived from older individuals [269], and type IIdiabetics without diagnosed heart disease [267], have impairedsurvival, proliferation and migration and reduced incorpo-ration into vascular structures. In addition, BM-MNCsharvested from patients with ischemic cardiomyopathy havea profoundly reduced potential for neovascularization [268],suggesting that the dysfunction is not restricted to circulating

cells. This impaired ability of EPCs to contribute to neovascu-larization may reduce the efficacy of autologous cell deliveryfor therapeutic applications.

5. Conclusions

Autologous cell therapy holds the hope of mending the bro-ken heart. Cell therapy with multiple cell types (includingthose that do not differentiate into new muscle) appearsto be beneficial. Also, significant functional improvementsoccur despite low levels of cell engraftment. New strategies(including more cardiogenic cell types, genetic modification,culture/small-molecule preconditioning and biomechanicalengineering) promise to enhance these benefits and improvextgeneration of autologous cell therapies.

6. Expert opinion

Over the last decade, a variety of promising autologous celltherapies (CDCs, CPCs, EPCs and MSCs) have been devel-oped from a variety of tissue sources (adipose, bone marrow,blood and heart). The mechanisms of benefit underlying themajority of these cell types appears to be rescue of reversiblydamaged myocardium, vascularization resulting from theaction of paracrine/humoral factors and secondary recruit-ment of host stem/progenitor cells [3,55-60]. To date, only themore cardiogenic resident cardiac cell sources appear capableof efficient cardiomyocyte and vascular transdifferentia-tion [85,97,111,112,114,115]. Interestingly, only the aggregates ofthe distinct subpopulations differentiated from myocardialtissue appear capable of indirect effects on tissue preservationand/or recruitment of endogenous progenitors [115]. Auto-logous cell candidates from first-generation therapies areundergoing clinical trials, with second-generation cell prod-ucts using modified/refined products just beginning clinicalassesment [239].

The next fundamental advance in autologous cell candi-dates promises to be based on recent advances in cellularreprogramming, whereby it has become possible to generateembryonic-like cells from virtually any cell of the body [272].These induced pluripotent stem (iPS) cells are capable ofindefinite self-renewal while maintaining the ability to differ-entiate into all cell types [273-277]. This research has lead to agreater understanding of the cellular pathways that regulatethe balance between pluripotency and differentiation. Notsurprisingly, the key nuclear factors (i.e., octamer-bindingprotein 4 (Oct4), sex determining region Y-box 2 (Sox2),nanog) that drive this equilibrium towards pluripotencyand away from differentiation are those that can be exploitedto reprogram somatic cells [276,278]. Additional epigenicprocess significantly contribute to the complex balancebetween pluripotency and differentiation as pluripotent cellchromatin is transcriptionally more permissive while differen-tiation is accompanied by a transition to chromatin that istranscriptionally less active [279].



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The rapid pace of this field has lead to the generation of iPScells from a host of different cell lineages including humanamniotic fluid, bone marrow, dermal fibroblasts, hair follicles,hematopoietic cells, neural stem cells and testis [274,280-287].These cells have been shown to be almost identical to embry-onic stem cells in terms of global gene expression, DNA meth-ylation and histone modification [277,288,289]. Furthermore, ithas been shown that cell types endogenously expressing lowlevels of the key reprogramming factors (i.e., neural stem cellsand Sox2) require less genetic manipulation to become iPScells [290]. This approach holds great promise in the generationof functional cell types that are reliable, scalable and capable ofdifferentiating into an adult phenotype [272,291].

Intriguingly, an abbreviated protocol has recently beendeveloped to generate cardiomyocyte-like cells from somaticcells though the introduction of factors that define

cardiogenesis (GATA binding protein 4 (Gata4), myocyteenhancer factor 2C (Mef2c), and T-box transcription factor5 (Tbx5)) [292]. This data hints that the future of cardiac celltherapy may lie in the generation of autologous cardio-myocytes precursors from easily attainable and expandablesources (e.g., dermal fibroblasts). However, more study isneeded to characterize and understand these approachesbefore they can be effectively (and safely) translated to theclinical setting.


This paper has been sponsored by the Canadian Institute ofHealth Research, Heart and Stroke Foundation of Canada.The authors declare no conflict of interest and have receivedno payment in preparation of this manuscript.

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AffiliationDarryl R Davis*1 MD &

Duncan J Stewart†2 MD†,*Author for correspondence1University of Ottawa Heart Institute,

Ottawa, Ontario,

K1Y 4W7, Canada2Ottawa Hospital Research Institute,

501 Smyth Road,

Ottawa, Ontario,

K1H 8L6, Canada

Tel: +1 613 739 6686; Fax: +1 613 739 6294;




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1. Introduction

2. Methods

3. Results

4. Discussion

5. Conclusion

6. Expert opinion

Review

Platelet rich plasma therapies forsports muscle injuries: anyevidence behind clinical practice?Isabel Andia†, Mikel Sanchez & Nicola Maffulli†Research Department, Osakidetza Basque Health Service, 48170 Zamudio, Spain

Introduction: At present, no drugs are available to hasten restoration of

muscle function after injury. Platelet-rich plasma (PRP) therapies may help

athletes by promoting muscle regeneration.

Areas covered: This is a systematic review assessing the evidence base for PRP

therapies in the management of muscle injuries. A computerized literature

search, citation tracking and hand searching for original studies assessing

the effect of PRP therapies on skeletal muscle cell biology, skeletal muscle

repair, or regeneration in animals or humans was performed. No randomized

trials have studied the merits of PRP injections for muscle healing. Clinical

studies indicated that PRP therapies may enhance muscle repair after strain

or contusion, and laboratory data indicated that they can enhance diverse

aspects of myogenesis. However muscle injuries present a complicated picture

that includes many components other than muscle cells, such as blood vessels,

connective tissue and neural components.

Expert opinion: The field is relevant but under-researched. No PRP for-

mulation has yet displayed proven solid evidence for the stimulation of

healing and recovery after sports muscle injuries. Therefore, major issues,

including standardization of formulations and application procedures, need

to be addressed to inform clinical studies before recommending best

practice guidelines.

Keywords: platelet-rich plasma, regeneration, skeletal muscle, sport injuries


1. Introduction

Muscle injuries resulting from extrinsic or intrinsic mechanisms are extremely com-mon in sports, accounting for about 35 -- 45% of all injuries [1], with contact sportsand sports that require the production of large eccentric forces presenting the high-est risk [2,3]. The vulnerability of athletes [4] to strains and contusions represents asubstantial problem for professional players and their clubs. Such injuries involvesignificant time lost from training and competition. Given the increasing demandsof training and competitions, treatment modalities able to accelerate recoveryfrom muscle injuries without adversely affecting recurrence rate whilst minimizingscarring are of paramount consequence.

At present, no drugs have been proven to hasten the restoration of muscle func-tion after injury. Therefore, in the absence of any available evidence-based treat-ments, injection therapies may be an important option to help professionalathletes [5]. Among the injected agents are Traumeel� (a homeopathic formulation),Actovegin� (an amino acid mixture) [6-8] and autologous serum [9] or platelet-rich plasma (PRP) [10]. PRP involves the use of the patients’ own proteins to restoretissue integrity and function. Initially, PRP therapies were developed to treatcutaneous ulcers [11], but an increased understanding of the biological properties


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of platelets [12,13] and the realization of their healing potentialextended the applications to other medical problems [14,15].Moreover, given the biocompatibility of using the patient’sown proteins, safety is guaranteed, simplifying translationfrom the laboratory to the patient.PRP therapies may influence muscle regeneration by acting

on the satellite cells [16], whose activities are controlled bygrowth factors and other cytokines, including IGFs, hepato-cyte growth factor (HGF), VEGF, basic fibroblastic growthfactor (bFGF) or angiopoietin type I (ANGPT-1), plasminand urokinase plasminogen (uPA) [17-24]. In clinical manage-ment of muscle injuries, the current hypothesis is thatintramuscular injections of PRPs deliver supraphysiologicalconcentrations of the above-mentioned factors [25-27] at theinjured site, influencing cell migration, proliferation, differen-tiation or fusion and ultimately enhancing muscle regenera-tion [28]. Assuming this knowledge, PRP therapies holdpromise for accelerating muscle healing and returning the eliteathlete to competition earlier.However, the rapidity of translation has sparked debate

regarding the level of evidence of clinical benefit neededto introduce PRP technologies in the sports medicine set-ting. We performed an electronic systematic search usingcomprehensive sources and focusing on the use of PRP in

the management of muscle injuries. To gain a more completeunderstanding from a scientific and medical point of view, wehave covered the entire health research spectrum, and, usingpre-specified criteria, we have included all potentially relevantarticles from laboratory and clinical research. The field is rel-evant to orthopaedic sports medicine, but under-researched:we aim to define the current status of our knowledge concern-ing PRP and muscle healing, a necessary task to guide futureresearch efforts and to identify potential implications.

2. Methods

2.1 Search strategyThe search strategy had two main components. First, in termsof treatment, we searched using all current names thatdescribe this therapy modality, that is, platelet rich plasma(PRP), platelet-rich fibrin matrix (PRFM), autologous fibrin,autologous conditioned serum (ACS), platelet concentrate(PC), platelet gel (PG), autologous growth factors (AGF),plasma or preparation rich in growth factors (PRGF) andplatelet releasate or lysate (PL). The applied search strategy(Table 1) covers all variants of the treatment in review, includ-ing materials containing leukocytes such as leukocyte-plateletrich plasma (L-PRP), platelet-leukocyte-rich plasma (P-LRP)or platelet-leukocyte gels (PLG). Secondly, we searched forthe target, combining the following terms: skeletal muscleinjury, strain or contusion and skeletal muscle healing, repairor regeneration.

The applied search strategy in Medline and EMBASE usingthe OVID platform is displayed in Table 1. Via the Webof Science, searches combining the above key words wereperformed in the Science Citation Index Expanded (SCI-EXPANDED) from 1899-present and in the ConferenceProceedings Citation Index - Science (CPCI-S) from 1990to the present (the first week of October, 2010). GoogleScholar was also searched. All seemingly relevant articlesand reviews were screened for meaningful references andthe retrieved article references were further examined foradditional publications.

2.2 Criteria for study consideration and data

extractionStudies were eligible if they provided specific informationrelated to the effects of PRP therapies (including ACS) inskeletal muscle and if they were original studies assessing theeffect of PRP-therapies on skeletal muscle cell biology, skeletalmuscle repair or regeneration in animals or humans. Studiesfocusing on the repair of non-skeletal muscle were not con-sidered. There were no language or data restrictions. Studieswere identified by two authors independently. From theincluded studies, the following data were extracted: studydesign (descriptive or controlled, laboratory studies, in vitroor in vivo or clinical experimentation), sample type (cellline, primary culture, animal species, number of animals,target population, number of patients), type of PRP product,

Article highlights.

. In clinical management of muscle injuries, the currenthypothesis is that intramuscular injections of platelet-richplasma (PRP) deliver supraphysiological concentrations ofgrowth factors and cytokines to the injured site,influencing cell migration, proliferation, differentiation orfusion and ultimately enhancing muscle regeneration.

. Given the biocompatibility of using the patient’s ownproteins, safety is guaranteed, simplifying translationfrom the laboratory to the patient. However the rapidityof translation has sparked debate regarding the level ofevidence of clinical benefit needed to introduce PRPtechnologies in the sports medicine setting.

. No randomized trials have tested PRP injections inmuscle healing, and our systematic search identifiedonly four clinical reports, all of them level 3 or 4observational studies. Moreover, although laboratoryresearch typically informs clinical studies in this area, inthis field basic science experiments were performedsimultaneously or even after clinical applications ratherthan the other way round. The field is relevant butunder-researched.

. Muscle injuries present a complicated picture thatincludes many components other than muscle cells,such as blood vessels, connective tissue and neuralcomponents. PRP therapies are exceptional in that theylargely target multiple regenerative processes because oftheir ability to secrete high levels of the chemokines,cytokines and growth factors, which are required tocontrol activities of different cell types.


PRP therapies for sports muscle injuries


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anatomical location of the injured muscle, outcome measuresand principal conclusions. Articles that focused on satellitecells treated with PRP for tissue engineering were excluded.

3. Results

3.1 Systematic search for PRP and muscle: identified

articlesEligibility of the studies based on titles, abstracts and full-text articles was assessed as shown in Figure 1. Numerousreviews and opinion papers highlight the relevance of PRPtreatments in orthopedics and sports medicine [28-37]. How-ever, no randomized trials have tested PRP injections inmuscle healing, and our search identified only four clinicalreports [9,10,38,39], all of them level 3 or 4 observational studies.Three of these were published reports [9,38,39], and the otherwas an oral presentation [10]. Other relevant articles were threelaboratory experimental reports, two in vivo [40,41] and onein vitro [42] (Tables 2 and 3). All papers were publishedin English.

3.2 Description of the studies3.2.1 Clinical studiesWright-Carpenter [9] assessed the effects of ACS injections in anon-blinded, non-randomized case control study. ACS is anautologous liquid serum conditioned by incubation of wholeblood with glass beads; it contains signaling proteins thatinclude IL-1b, TNF-a, IL-7, fibroblast growth factor-2(FGF-2), IL-1 receptor antagonist (IL-1Ra), HGF, plateletderived growth factor (PDGF-AB), TGFb-1 and IGF-1. Theexperimental group treated with ACS included 17 patients,while the control group, which was analyzed retrospectively,included 11 patients who had received Traumeel�/Actovegin�(3:2). Traumeel is a homeopathic formulationcontaining both botanical and mineral ingredients in

homeopathic concentrations. It is purported to suppress therelease of inflammatory mediators and stimulate the releaseof anti-inflammatory cytokines. Actovegin is a deproteinizedcalf blood hemodialysate consisting of a physiological mix ofamino acids. The rest ice compression elevation protocol wasemployed for initial care in both groups. The severity of thetear, which was scored as grade 2 with detection of bleeding onMRI, was similar for all control and experimental groups [43].Most tears were located in the hamstring and adductor muscles(12 in the experimental group and 9 in the control group).The injected volumes (5 ml) were identical in both groups.The injection technique and post-injury treatment aredescribed well. The mean number of treatments per patientwas 5.4 in the ACS group and 8.3 in the reference group.The main outcome measured was the time needed to resumefull sporting activities. Return to competition was decidedafter isokinetic strength assessment. The experimental groupreturned to competition after 16.6 days, while the controlgroup took 22.3 days; in addition, MRI scans taken at16 days in both groups confirmed that regression of theedema/bleeding was faster in the ACS group. Both treatmentswere safe.

At the 2nd World Congress of Regenerative Medicine,Sanchez et al. [10] reported the application of leukocyte-freePRP [44] in 21 muscle injuries of different severities and atdifferent anatomical locations; small tears progressed wellwith a single application, while more severe tears requiredtwo or three ultrasound-guided injections. The injected vol-ume depended on tear severity. These athletes, who playedin first division teams of the Spanish Soccer League, resumednormal training activities in half the time needed by matchedhistorical controls. Using the same leukocyte-free PRP prepa-ration, Wee et al. [38] reported good outcomes (1 week toreturn to pre-injury activities) after three weekly ultrasound-guided injections to treat adductor longus strain in a profes-sional bodybuilder. Objective measurements, such as swellingor manual muscle testing, were not reported. Pain is alwaysmentioned, but the visual analogue scale or analgesic con-sumption were not reported as outcome measures. Recently,Hamilton et al. [39] reported buffered L-PRP injection in agrade II hamstring strain injury and daily physiotherapy pro-gram. Seventeen days after injury, the patient had full range ofmotion and was pain free in maximal contraction consistentwith MRI demonstrating complete resolution.

3.2.2 In vivo controlled laboratory studiesMyogenesis relies upon satellite cell activation, proliferation,migration to the site of injury, differentiation, fusion withexisting damaged muscle or other satellite-cell-derived myo-cytes and maturation (increased myofiber diameter). Thus,to assess the progress of muscle regeneration from a biologicalperspective, researchers measure the number of activated satel-lite cells, molecular markers of cell differentiation (i.e., RNAor proteins) or the diameter of regenerating myofibers. Usingthis strategy, two separate research teams [40,41] used syngeneic

Table 1. Search strategy in EMBASE 1980 to 2010

Week 41, Ovid MEDLINE� 1959 to October Week 1

2010, Ovid MEDLINE Daily Update October 15, Ovid

MEDLINE in process & other non-indexed citations.

No Search strategy

1 (Plasma adj3 (growth factor* or relasate)).mp2 ((thrombocyte* or platelet*) adj3 (plasma or

concentrate* or gel or fibrin* or lysate*)).mp3 ((Autologous or endogenous or autogenous)

adj3 (serum or blood)).mp4 OR/1 -- 3 (note: combination of terms related

to product)5 (Musc* adj5 (heal* or injur* or strain* or contus*

or regener* or repair*)).mp6 AND/4 -- 57 Remove duplicates from 6

*Truncation, adj3: words in either order between 3 words, mp: title, original

title, abstract, subject heading word, name of substance word.

Andia, Sanchez & Maffulli


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animals (mice or rats) to test the therapeutic effects of ACSand L-PRP, respectively.In 2004, Wright-Carpenter et al. [40] applied ACS in a con-

tusion injury model by injecting 10 µl at 2, 24 and 48 h afterinjury. The control group was treated with the same volumeof saline. The number of activated cells was assessed at30 and 48 h after contusion, and the size of regeneratingmyofibers was measured in histological samples on days0, 2, 4, 6, 7, 8, 14, 21, 28 and 35, assessing the progress ofregeneration. The number of activated satellite cells washigher in ACS-treated contusions at 30 and 48 h. Moreover,larger regenerating myofibers were observed in the ACS groupby days 7 -- 8; however, by day 14, there were no differencesbetween groups. These results indicated that ACS hastensmuscle regeneration after contusions by promoting earlier ini-tiation of the activation and/or recruitment of satellite cellsand by achieving earlier fusion.Hammond et al. [41] described the effects of leukocyte-

platelet-rich plasma (L-PRP) in the treatment of strains.The authors induced either weak or severe strains (bysingle or multiple repetitions) in the tibialis anteriorior ofsyngeneic rats and injected 100 µl of L-PRP at days 0, 3,

5 and 7. The control group was treated identically butwith PPP. Muscle regeneration was assessed by molecularmeasurements of myogenin and MyoD, measuring bothmRNA and protein levels. In addition, myogenesis wasassessed by quantification of centrally-nucleated fibers (widelyaccepted as a marker for muscle regeneration) peaking 2 weeksafter injury in the PRP-treated group. Functional recoverywas evaluated by torque measurements at days 3, 5, 7,14 and 21. In weak strains, PRP ameliorated the forceloss at day 3, while in more severe strains PRP improved thecontractile function at days 7 and 14, and shortened therecovery time from 21 days to 14 days. The authors con-cluded that L-PRP injections hasten functional recovery andthat myogenesis was probably the mechanism underlyingthis acceleration.

In a poster communication [45], muscle lacerations trea-ted with leukocyte-depleted PRP in a sheep model showedenhanced regeneration when compared to platelet-poorplasma. Thus, three independent in vivo studies of differentmethodological values have assessed the effects of three differ-ent autologous preparations injected in three different injurymodels: contusions, strains and lacerations (Table 3).

Potentially relevant articles based on search terms: n = 158Search strategy in EMBASE 1980 to 2010 week 41, Ovid MEDLINE®

1959 to October week 1 2010, Ovid MEDLINE Daily Update October 15, Ovid MEDLINE in process and other non-indexed citations

Articles excluded after screening titles(n = 125)

Potentially relevant articles retrieved forevaluation (n = 33)

Excluded after evaluation of abstract:narrative review, opinion papers(n = 25); tendon/ligament (n = 3)

Included studies:Clinical studies (n = 4)Controlled laboratory studies (n = 3)Descriptive laboratory study (n = 1)

Search via Web of ScienceClinical reports (n = 1)

Search in Scholar GoogleCongress communications:Clinical studies (n = 1)Controlled laboratory study (n = 1)

Figure 1. Flow diagram of the systematic literature research. A total of 125 articles were excluded as the title or the abstract

clearly indicated that they were not relevant, and 28 articles were further excluded because they were narrative reviews or

opinion papers or evaluate tendon/ligament.



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3.2.3 In vivo controlled laboratory studiesRanzato et al. [42] evaluated proliferation and motility inC2C12 mouse myoblasts treated with platelet lysates. Dueto technical difficulties associated with isolating and main-taining cultures of primary satellite cells, immortalized celllines are frequently used as satellite cell models. C2C12 is acommonly used cell line, isolated from clonal cultures derivedfrom the thigh muscles of 2-month-old C3H mice 70 h aftercrush injury. To obtain a platelet lysate, platelet pellets arewashed, repeatedly frozen and thawed and finally centrifugedto eliminate debris. A 20% platelet lysate was used in theseexperiments. The authors used a scratch wound model andchemotaxis assays. In scratch models, the wound healing spaceis reduced by both migration and proliferation of cells. In thechemotaxis model, on the other hand, the effect of migrationdoes not overlap with proliferation. The results showedincreased proliferation and motility, and the latter effect wasmore evident [42]. This is relevant given the isolation and rel-atively sparse distribution of satellite cells in uninjured tissues;proliferation and directional motility are both required toreach large populations of activated myoblasts at a site of focalinjury. This study also provided a mechanistic explanation bydemonstrating that activation of p38 and PI3K was involvedin the myogenic program (differentiation) in cell motility.

Taken together, these studies contained too many vari-ables regarding the product (ACS, L-PRP, pure PRP andplatelet lysates), the method of application (variable numberof injections, volume, frequency), the type of injury (contu-sion, strain or laceration), anatomical location and severity.Moreover, although laboratory research typically informsclinical studies in this area, in this field basic science experi-ments were performed simultaneously or even after clinicalapplications rather than the other way round.

4. Discussion

Although the management of sports injuries with PRP injec-tions has been advocated since 2003 [46], this strategy hasnot yet been tested in clinical trials dealing with muscle inju-ries. In reviewing the published work on PRP therapies formuscle injuries, we found only one peer-reviewed clinicalstudy [9] in recreational athletes, and it contained importantmethodological limitations, such as a lack of blinding, retro-spective controls, incomplete reporting and a lack of objectivemeasurements. The absence of studies may impress clinicalresearchers. This is not so extraordinary for muscle sport inju-ries, as their management is based largely on experimentalstudies or empirical evidence. Even when considering theclinical evidence base for the universally-accepted early man-agement of soft tissue injuries, that is ice (also known ascooling or cryotherapy), after meta-analyses [47], conclusionsand recommendations were greatly limited and guidelinescontinue to be formulated on an empirical basis. Thispresumably reflects not only the importance of key details ofthe application procedures, such as the interaction of theT

able

2.Plateletrich

plasm

atherapiesto

treatmuscle

injuries:

clinicalstudies.

Clinicalstudies

(typeofarticle)

Studydesign/target

population

Treatm

ent

Mech

anism/

location

Outcomemeasu

res

Principalresu

lts

Levelof

evidence

Wright-Carpenter

etal.2004[9]

(Originalarticle)

Case-control

n=16(experimental)n=11

(controlgroup)

/recreationalathletes

ACSversus

Traumeel/Actovegin

ACS:5.4

injections/patient

Controlgroup:8.3

injections/patient

Strain/

Hamstringand

adductor(most)

Regressionoftheoedema

Strength

(isokinetictest)

Return

tocompetition

Fasterregressionofoedema

Fasterreturn

tocompetition

Safety

III

Sanchezetal.2005

(Oralpresentation)[10]

Case-seriesn=20,historical

controls/professionalathletes,

Oneto

threeinjections

Pure-PRP

Strain

orcontusion

/Differentlocations

Return

tocompetition

Nore-injuries

Fasterreturn

tocompetition

Safety

IV

Weeetal.2009

(Letter)

[38]

Case

report

/professionalathlete

Threeinjections

Pure-PRP

Strain/Adductor

longus

Return

tocompetition

Pain

Safety

Accelerationofhealing

Reducedpain

Safety

IV

Hamiltonetal.

2010

[39]

Case

report

/recreationalathlete

Oneinjection

BufferedL-PRP

Strain/

hamstring

Return

topre-injury

activities

MRIevaluation

Pain

Safety

Return

topre-injury

activities

at3weeks

FullrangeofmotionandMRI

resolutionatday17

IV

ACS:Autologousconditionedserum;L-PRP:Leukocyte

plateletrich

plasm

a;PRP:Plateletrich

plasm

a.



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cooling surface with the tissue, but also the major hurdles fordeveloping adequate clinical trials, which include large varia-tions with regard to injury severity and affected musclegroups, non-specificity of reported symptoms, concomitanttreatments, allocation of elite athletes into randomizedcontrolled trials and outcome measurements independent ofpatient motivation.

PRP injection is a form of management of muscle injuriesthat can be considered for clinical practice. However, it ishard to recommend it as best practice, first because it is basedon scarce level III -- IV studies and the recommendations ofexpert opinion; second because PRP therapies are unclearregarding the best formulation for muscle injuries. Essentially,there are two different liquid formulations gathered under thesame PRP terminology. To differentiate and define those, twodescriptive terms have been proposed [48]: L-PRP, which con-tains fivefold to -- eightfold more platelets and more leuko-cytes than peripheral blood; in contrast, P-PRP avoidsleukocytes, and has a moderate increase in platelet count(1.5 -- 2.5-fold above baseline). It is not known whether mus-cle injuries treated with L-PRP or P-PRP progress in differentways. Theoretically, L-PRP may mimic the initial phase ofinflammation in which a high number of neutrophils infil-trate the injured site; the interactions of neutrophils withplatelets can induce a hyperactive leukotactic response of cir-culating neutrophils toward the injury site. Neutrophils mayexacerbate tissue damage via several different mechanisms(i.e., secreting pro-inflammatory cytokines such as TNF-a,IFN-g , IL-6 or IL-1b) that cause matrix destruction throughthe production of MMP-1, -3 and -13. Moreover, neutrophilssecrete high concentrations of a number of cytolytic and cyto-toxic chemicals, such as oxygen radicals and hydrochlorousacid [49]. Consequently, interaction of activated neutrophilswith the damaged tissue can and does intensify muscle dam-age, which is known to be the secondary injury related tothe inflammatory response [50]. Indeed, research shows thathindering neutrophil infiltration can result in reduced overallmuscle damage [51]. Hence, assuming their probable dis-similarities in neutrophil chemotaxis and activation, L-PRPand P-PRP might be critically different in regulating thecomplex innate immune response and subsequent healingoutcome. To gain further information about those criticaldifferences in the early healing phase, both formulationsshould be compared, preferably using large-animal modelsand adequate outcome measurements.

Proponents of PRP therapies in muscle applications mayoffer several arguments in their defense. First, medicine isdynamic, and it is worthwhile to exploit the therapeutic valueof an otherwise safe technology that has the potential to ben-efit patients, as shown in other clinical applications [52,53],even if it will probably be refined as laboratory and clinicalresearch are conducted. Second, while in recreational athletesmuscle injuries may recover uneventfully in a matter of weeks,professional athletes need urgent solutions because they mustreturn to higher levels of performance and activities in aT

able

3.Plateletrich

plasm

aandmuscle

repair:laboratory

studies.

Anim

alstudies

Studydesign/anim

al

Treatm

ent

Injury/anatomical

location

Outcomemeasu

res

Principalco

nclusions

Hammondetal.

2009

[41]

Controlledlaboratory/

syngenic

rats,n=72

L-PRPversusPPP

Fourinjections

Strain

Tibialis

anterior

Percentageofmaximaltorque

mRNA:MyoD

andmyogenin

Histology:

centrally

nucleatedfibers

Enhancedfunctionalrecovery

Stimulationofmyogenesis

Carda,etal.

2005

[45]

Controlledlaboratory

Sheep,n=4

Pure-PRPversusPPP

Oneapplication

Lacerationsover

theback

Qualitative

histology

Enhancedstructuraloutcome

withPRP

Wright-Carpenter

etal.2004[40]

Controlledlaboratory

syngenic

mice,n=108

ACSversussaline

Threeinjections

Contusion

gastrocnemius

Histology:

numberofactivated

satellite

cell,

fiberdiameter

Enhancedsatellite

cellactivation

andlargerfiberdiameterwithACS

Cellcu

ltures

Design/Celltype

Treatm

ent

Assaytype

Effect

measu

res

Principalco

nclusions

Ranzato

etal.

2009

[42]

Controlledlaboratory/

mouse

myoblastsC2C12

Plateletlysate

Inhibitors

of

MAPKsignallingERK,

p38,PI3K

Scratchwoundclosure

Antibodyblockade

Percentagewoundclosure

rate

Proliferation

Chemotaxis

Enhancedproliferationandmotility

Motilitymore

centralthanproliferation

P38andPI3Kdrive

cellmigration

ACS:Autologousconditionedserum;L-PRP:Leukocyte

plateletrich

plasm

a;myoD:Myoblast

determ

inationprotein;PPP:Platelet-poorplasm

a;PRP:Platelet-rich

plasm

a.



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shorter time. Third, knowledge of repair mechanisms sup-ports the biochemical basis of adding supraphysiologicalconcentrations of growth factors to injured tissues [54]. Thereare important insights arising from research that may helpin understanding the clinical potential of PRPs and theirsuitability as a therapeutic tool in muscle injuries.

Muscle injuries certainly present a complicated picture thatincludes many components other than muscle cells, such asblood vessels (endothelial cells and pericytes), connective tis-sue (fibroblasts) and neural components (motor neuron,Schwann cells) [55-57]. In addition, PRP therapies are excep-tional in that they largely target multiple regenerative pro-cesses because of their ability to secrete high levels of thechemokines, cytokines and growth factors, which are requiredto control activities of different cell types. It is not knownwhich are the key factors, but several growth factors abundantin PRPs have been extensively studied in muscle regenera-tion [24]. For example, HGF is the primary component ofcrushed muscle extract [58], and it is currently the most prob-able candidate for initiating regeneration by satellite cellactivation via c-met receptors. HGF promotes activation, pro-liferation, differentiation and chemotaxis. IGF-I and -II eachincrease following muscle injury and promote myoblast pro-liferation and myofiber differentiation as well as enhancingmuscle cell survival and hypertrophy under tissue-specific cir-cumstances. While not as intensively studied as HGF andIGFs, VEGF, bFGF and ANGPT-1 appear to have potentialin regeneration by inducing muscle angiogenesis [55]. Also lesswell-recognized, brain derived neurotrophic factor (BDNF) isa relevant component of PRPs with an important role in reg-ulating satellite cell function and regeneration, as shownin vivo [18]. BDNF has been known since the early 1990s insports research because, of all neurotrophins, it is the mostsusceptible to systemic regulation by exercise and physicalactivity; it is also known because of its metabotropic activ-ity [59]. When axonal communication with the muscle cellbody is interrupted by injury, Schwann cells produce neuro-trophic factors, such as nerve growth factor (NGF) andBDNF [60]. Thus, additional increases in BDNF in the con-text of PRPs may help in the progressive recovery of neuralcommunication [61-64].

On the other hand, the presence of relatively high concen-trations of TGF-b1 prompts the question of whether PRPsmay favor healing by fibrosis instead of regeneration. Bothplatelets and leukocytes secrete TGF-b1, and there is goodreason to think that boosting TGF-b1 levels might induceexcessive accumulation of fibrotic tissue [16,65]. However,molecular combinations may be antagonistic or even suppres-sive, and the combined effect of TGF-b1 with other PRP-secreted molecules on collagen synthesis was weaker thanthat of TGF-b1 in isolation [66]. Then again, with the releaseof proteases, such as plasmin or thrombin, PRPs may fuel thefibrinolytic activity required for myogenesis [23]. For example,IGF-binding proteins (IGFBPs) still need to be cleaved todeliver bioactive IGF to its receptor and stimulate cell

activities. Unraveling the protease-induced activation ofIGFs, HGF or TGF-b1 may be the key to understandingsome PRP actions needed to optimize PRP formulations.Thus, in complex systems such as PRPs, the major challengeis to disentangle the relative effects of the components andto understand how they influence given cell activities. Indeed,the PRP story has turned out to be immensely more complexthat it seemed at first.

5. Conclusion

According to the findings of this review, no PRP formulationhas yet displayed proven solid evidence for the stimulation ofhealing and recovery after sports muscle injuries. Pilot clinicalstudies along with empirical experience indicate that PRPtherapies may enhance muscle repair after strain or contusion.Laboratory data indicate that such treatments can enhancemyogenesis. However, the fundamental principles governingwhen and how PRP therapies can be usefully employed inmuscle injuries are emerging at a slow pace. The key to attainstandardization and improved formulations will be the identi-fication of crucial elements in these preparations. Given ourrudimentary knowledge of the mechanism of action of thePRPs, it remains uncertain how best to use this technologyto affect early healing, and produce improved and acceleratedfunctional recovery.

6. Expert opinion

Currently, the use of PRPs in elite athletes and ensuing dis-cussion in the media has fueled clinical demand outpacingbasic and clinical research and hindering progress on suchtherapies. The ease of use and lack of fear of side and adverseeffects involved with PRPs is detrimental, as this allows prac-titioners to use it frequently without guidelines such as timingof treatment, number and technique of injections or volume;frequently, the personal experience of the practitioner is theonly source of evidence to substantiate practice. Failure tounderstand the mechanism of action of PRPs frustrates effortsto develop best formulations regarding the optimal plateletconcentration. Earlier, in oral and maxillofacial surgery, aminimum four-fold to fivefold increase in the number ofplatelets was considered necessary to produce a therapeuticeffect [67]. In retrospect, it is obvious that such an assertionwas inappropriate, and not supported by basic science [68,69].The best PRP formulation for muscle injuries will beclearer after research efforts have provided a comprehensivedescription of the relations between PRP components, healingmechanisms and functional outcome.

In particular, several critical questions about how to opti-mize PRP therapies should be a high priority for researchers.First, to standardize PRP formulations, research must identifykey elements in these preparations. For example, it is relevantto establish differences between pure platelet-rich plasma andleukocyte-platelet concentrates regarding tissue damage



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exacerbation [50-57]. In addition, the optimal balance betweenplasma myogenic factors, such as IGFs and HGF, andplatelet-secreted angiogenic or chemotactic factors needs clar-ification. In fact, platelets are the major source of chemotacticfactors such as platelet factor 4 (PF-4) which, in cooperationwith PDGF and CXCL7, activates fibroblast migration.Second, to identify the best timing for application, the impli-cations of physicochemical temporal conditions of the tissue(i.e., pH, NO and oxygen) should be evaluated. Indeed,most injured tissues, which are under hypoxic conditions,shift to normoxia after angiogenesis; thus, the biological andclinical effects of PRPs under these circumstances may differ.Moreover, which cells or biological events PRPs target in eachtemporal phase of repair is unknown [54]. Furthermore, if

reduction of scarring is a plausible goal, it would involveidentifying the actions of TGFb-1 in this context.

These questions need to be addressed to standardize theformulations and procedures for application. Because of thesafety of these products, basic science, clinical discovery andpatient-oriented research should be interdependent ratherthan successive steps. The substantial challenges of incorpo-rating such research into clinical care must be pursued if thepotential of PRPs is to be realized.


The authors state no conflict of interest and have received nopayment for the preparation of this manuscript.

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jss.2010.10.001

AffiliationIsabel Andia†1 PhD, Mikel Sanchez2 MD &

Nicola Maffulli3 MD PhD†Author for correspondence1Research Department,

Osakidetza Basque Health Service,

B Arteaga 107,

48170 Zamudio, Spain2Unidad de Cirugıa Artroscopica,

UCA ‘Mikel Sanchez’,

Clınica USP-La Esperanza,

c/La Esperanza 3,

01002 Vitoria-Gasteiz, Spain3Queen Mary University of London,

Barts and the London School of Medicine

and Dentistry,

Center for Sports and Exercise Medicine,

Mile End Hospital,

275 Bancroft Road,

London E1 4 DG, UK



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1. Introduction

2. First-line medical treatment of

metastatic colorectal cancer

3. Conclusion

4. Expert opinion

Review

Cytotoxic triplets plus a biologic:state-of-the-art in maximizing thepotential of up-front medicaltreatment of metastatic colorectalcancerFotios Loupakis†, Chiara Cremolini, Marta Schirripa, Gianluca Masi &Alfredo Falcone†University of Pisa, Department of Oncology, Transplants and New Technologies in Medicine,

Pisa, Italy

Introduction: Up-front treatment of metastatic colorectal cancer (mCRC)

has progressively become more complex during last few years. Nowadays,

treatment options range from monotherapies with biologics or traditional

chemotherapeutic agents to intensive combinations of chemotherapy plus

targeted drugs.

Areas covered: This review deals with the results of the most recent first-

line trials in the medical treatment of mCRC, with a special focus on recently

closed or ongoing trials of intensive concomitant combinations of all active

cytotoxics plus a biologic agent.

Expert opinion: Combinations of three cytotoxic drugs plus a biologic are under

clinical investigation and therefore should not be recommended for routine

use, nevertheless preliminary results are promising. The main challenges for

the future are not only to demonstrate the real clinical usefulness of intensive

approaches, but also to gain the ability of defining prior to treatment which

patients will benefit most on the basis of clinical and molecular elements.

Keywords: bevacizumab, cetuximab, chemotherapy, colorectal cancer, panitumumab


1. Introduction

In the last 20 years the median overall survival (OS) of patients with metastatic colo-rectal cancer (mCRC) has increased from 8 -- 12 months to 18 -- 24 months thanksto the introduction of irinotecan, oxaliplatin and monoclonal antibodies, such as theanti-EGFR cetuximab and panitumumab and the anti-VEGF bevacizumab, and thedevelopment of integrated treatment strategies to achieve metastases resection [1].

The availability of this wide variability of therapeutic options offers new possibil-ities of up-front treatment, but a subsequent question arises: which is the best ther-apeutic strategy for each patient according to the aim of the treatment, the biologyand the clinical characteristics of both tumor and patient?

Both sequential and combination chemotherapy can be employed in the treat-ment of mCRC. The commonly accepted decisional algorythm suggests reservingmost active up-front regimens to patients with potentially resectable metastases, inorder to achieve secondary resection and long-term disease control. On the otherhand, patients with never resectable, widespread disease and no options of furtherresectability are candidates to receive less intensive regimens and even single agentfluropyrimidine, with the objective of prolonging survival without affecting quality


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of life [2]. Nevertheless, in our opinion, the use of combinedfirst-line regimens might apply to a wider range of clinical sce-narios, so that, for example, an intensive up-front treatmentmight be the preferred option also for patients with symptom-atic and widespread, aggressive disease, while the sequentialapproach should be actually reserved for patients unfit forcombinations, due to age or relevant comorbidities.However, this way of modeling doesn’t fit the complexity

of clinical practice, so that the choice of the intensity of theup-front chemotherapy might be, nowadays, influenced bymultiple clinical and biological considerations.Moreover, such increasing complexity is further compli-

cated by both the availability of biological drugs and thesoaring insights into molecular determinants.In fact, in the last few years, clinical trials have shown that

treatment with two cytotoxics in association with the anti-EGFR [3-5] or the anti-VEGF [6,7] monoclonal antibodies(mAbs) is safe, active and effective, but at the moment, wedon’t have a scientific demonstration concerning the bestchemoterapeutic regimen to be associated with a biologic,nor the best biologic to be associated with chemotherapy.The biomolecular characterization of mCRC has gained

a determinant role: the presence of KRAS mutation isa well-ascertained predictive factor of resistance to cetuxi-mab [3,4,8,9] and panitumumab [5,10] and the presence ofBRAF mutation defines a subgroup of patients with extremelybad prognosis [9,11,12].

Hence, in the era of target therapies and of molecular selec-tion, one wonders which settings might be the most suitablefor an intensive first-line therapy as a three-drug regimenand ongoing clinical trials are evaluating the opportunity foradding biological agents to the triplet.

The objective of the present review is to rapidly summarizeevidence from literature about the efficacy of up-frontcombined regimens, with particular regard to the triplet,and to secondly explore the potential role of the associationof a biologic to such an active regimen, in an attempt todefine the clinically and molecularly selected population,that might benefit as more as possible from an intensiveup-front strategy.

2. First-line medical treatment of metastaticcolorectal cancer

2.1 The choice of up-front chemotherapy: two is

(nearly always) better than oneA nodal issue in the choice of the up-front treatment formCRC patients concerns how intensive the chemotherapyregimen should be. It has been firstly demonstrated [13] andthen confirmed [14] by Grothey et al., that the exposure toall cytotoxics during the course of the disease determines a sig-nificant improvement in OS. The striking positive correlationof the percentage of patients treated with 5-fluorouracil,oxaliplatin and irinotecan at some point of their disease withOS strongly supports the strategy of making all active agentsavailable to patients, in order to maximize their survival.

All Phase III randomized trials, conducted with the aim tocompare sequential with combined approaches, achievedanalogous results. The capecitabine, irinotecan, and oxalipla-tin in advanced colorectal cancer (CAIRO) trial [15] random-ized 820 patients to receive first-line capecitabine, followedby second-line irinotecan and third-line capecitabine plusoxaliplatin (sequential strategy) or first-line capecitabine plusirinotecan, followed by second-line capecitabine plus oxalipla-tin (combination strategy). The Fluorouracil, Oxaliplatin, andCPT-11 (irinotecan): Use and Sequencing (FOCUS) trial [16]randomized 2135 mCRC patients, not amenable to curativestrategy, to first-line single-agent 5-fluorouracil (FU) untilfailure followed by single-agent irinotecan (strategy A)or 5-FU until failure followed by combination therapy (strat-egy B), or up-front combination chemotherapy (strategy C).In both strategy B and C, patients were further divided in a1:1 ratio to receive 5-FU plus oxaliplatin or 5-FU plus irino-tecan as combination treatment. The study, initially launchedto establish the superiority of one of the strategies in terms of2 year-OS, was then amended as a consequence of positiveresults of trials of 5-FU-based irinotecan [17-19] or oxaliplatindoublets [20-22] versus 5-FU monotherapy and, before comple-tion of accrual, a supplementary analysis was planned toexamine the non-inferiority of strategy B in comparisonwith strategy C. The main results of the CAIRO and FOCUStrials are summarized in Table 1.

Article highlights.

. A fluoropyrimidine-based doublet as up-frontchemotherapy backbone for the addition of a biologic isa standard-of-care for the vast majority of metastaticcolorectal cancer patients.

. Three-cytotoxics regimen 5-fluorouracil/folinic acid,oxaliplatin, and irinotecan (FOLFOXIRI) might represent apreferrable option when aiming to induce relevanttumor shrinkage, such as for potentially resectabledisease or for never resectable, widespread, biologicallyaggressive, symptomatic disease.

. The efficacy of biologics in the up-front setting iswell-established. The indication of anti-EGFR monoclonalantibodies is restricted to KRAS wild-type patients.Molecular predictors, able to drive the therapeuticdecision in KRAS wild-type patients, are currentlyunder investigation.

. The safety and activity of four-drug regimens,combining the three cytotoxics with a biologic agent,have been recently evaluated in early clinical trials withpromising results. Other Phase II and III studies arecurrently ongoing.

. Future studies might explore the strategy ofconcentrating the greatest cytoreductive activity byadopting an intensive four-drug regimen in a short initialphase of the up-front treatment, followed by amaintenance period until progression.


Cytotoxic triplets plus a biologic in mCRC


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In both studies the up-front combined approach did notproduce an advantage in OS, except for patients treatedwith first-line 5-FU and irinotecan in strategy C of theFOCUS trial, compared with those randomly assigned tostrategy A (hazard ratio (HR): 0.84, 95% CI 0.73 -- 0.96,p = 0.01). On the other hand, the up-front combinedapproach warranted significantly higher objective responserate (RR) (41 versus 20%, p < 0.0001 in the CAIRO trial;49 -- 57% versus 28%, p < 0.001 in the FOCUS trial) andprogression free survival (PFS) (7.8 versus 5.8 months, HR:0.77, p = 0.0002 in the CAIRO trial; 8.7 versus 6.3 monthsfor first-line 5-FU plus oxaliplatin versus 5-FU monotherapy,p < 0.001 and 8.5 versus 6.3 months for first-line 5-FU plusirinotecan versus 5-FU monotherapy, p < 0.001 in theFOCUS trial). Although such trials did not demonstrate abetter OS for patients receiving up-front combined chemo-therapy, thus leading authors to suggest the staged approachas a valid choice for patients with extensive disease, somecrucial points probably need deeper analysis.

First of all it should be noted that both trials reportedmedian survivals in the range between 13.9 and 17.4 months,that is relevantly shorter than expected even for combinationarms, if compared with other contemporaneous experienceswith first-line doublets.

Secondly, one of the most attractive facets of a stagedapproach certainly lies in the possibility of limitingtreatment-related toxicities, thus allowing guarantee of thepreservation of a better quality of life. However, in both trialswhile oxaliplatin- and irinotecan-related adverse events wereobviously more frequent among patients in the combinationarms, no significant differences in the occurrence of grade

3 -- 4 toxicities over all lines, or in the incidence of deathsdue to toxicity was evidenced between staged and combinedstrategies. Also in terms of quality of life, no significantdifferences in the perception of health status were detected.

Thirdly, the percentage of patients exposed to all threecytotoxics was considerably lower in sequential arms inboth trials (36 versus 53% in CAIRO and 16 and 19%versus 33% in the FOCUS trial). Such a finding is easilyexplained by considering that approximately only 50 -- 70%of patients starting a line of therapy will be suitable to receivea next-line treatment.

Last, but not least, the item of patients’ selection deserves tobe emphasized. As underlined by authors themselves [23], bothCAIRO and FOCUS trials included poor prognosis patients’populations. Patients with liver-only metastases, with a chanceto achieve secondary resection, were excluded from FOCUSand underrepresented in CAIRO. Such consideration explainsthe short survivals registered in both trials and widens the wayto the crucial importance of the choice of the best strategywhen planning mCRC patients’ treatment from the verybeginning. In the light of data from the CAIRO and FOCUStrials, it appears, therefore, mandatory to choose a combinedup-front treatment for patients with marginally or potentiallyresectable metastases. However, taking into account both thelow percentage of patients exposed to all three drugs, whichis linearly related to OS, and the absence of a clear advantagein terms of safety and quality of life for the sequential strategy,combination therapy might be considered as a reasonablestandard of care also for the majority of never-resectablepatients, thus reserving first-line monotherapy to patientsclearly unfit for combination therapy, due to age and/or

Table 1. Comparisons of staged versus combination approaches in FOCUS and CAIRO studies: main measures

of outcome.

Strategy FOCUS (2135 patients) CAIRO (820 patients)

First line 5FU

A = 710 patients;

B = 712 patients

First line combination

C-ir = 356 patients;

C-ox = 357 patients

Sequential Strategy

410 patients

Combination

strategy

410 patients

Response rate 28% (A, B) 49% (C-ir); 57% (C-ox) 20% 41%p < 0.001z p < 0.0001

PFS (months) 6.3 (A, B) 8.5 (C-ir); 8.7 (C-ox) 5.8 7.8p < 0.001z HR = 0.77; p = 0.0002

OS (months) 13.8 (A); 15.1 (B) 16.7 (C-ir); 15.4 (C-ox) 16.3 17.4C-ir versus A HR = 0.84; p = 0.01§ HR = 0.92; p = 0.328

Exposure to 3cytotoxic agents

16% (A); 19% (B) 33% 36% 53%

Safety No relevant differences No relevant differencesQoL overall No difference No difference

*A: single-agent 5-fluorouracil (5-FU) until failure followed by single-agent irinotecan; B: 5-FU until failure followed by combination therapy (5-FU with oxiplatin or

irinotecan); C: up-front combination chemotherapy (5-FU with oxiplatin (C-ox)or irinotecan (C-ir).zfor both C-ir versus A + B and C-ox versus A + B.§mOS for C (C-ir + C-ox) = 15.9; p = 0.02 for A versus C (level of significance for multiple comparisons = p < 0.01.

Loupakis, Cremolini, Schirripa, Masi & Falcone


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comorbidities or with a reluctant attitude to accepting anincreased risk of toxicity in a first-line treatment.

2.2 The choice of up-front chemotherapy: when

three is better than twoA diametrically opposite approach to mCRC consists of thecombined administration of all active cytotoxics as up-fronttreatment. The schedule developed by the Gruppo Oncolo-gico Nord Ovest (GONO) [24,25] allowed to achieve, in aPhase III randomized trial, remarkable results in terms ofboth activity and efficacy, with an acceptable increase inadverse events, that did not compromise treatment’s feasibil-ity and safety [26]. Compared with first-line Folinic Acid,Fluorouracil & Irinotecan (FOLFIRI), up-front GON-O-5-FU/folinic acid, oxaliplatin, and irinotecan (FOLFOX-IRI) resulted in higher RR (60 versus 34%, p < 0.0001),PFS (median PFS: 9.5 versus 6.6 months, HR: 0.59,p < 0.001) and OS (median OS: 23.4 versus 16.7 months,HR: 0.74, p = 0.026) in a population of 244 untreatedmCRC patients, deemed initially unresectable. The superioractivity for the experimental arm resulted in a significantlyhigher rate of secondary resections. Of patients treated withGONO-FOLFOXIRI 15% underwent radical surgery onmetastases, compared with 6% of patients treated withFOLFIRI (p = 0.033). Such percentages rise to 36 and 12%respectively, considering patients with liver-only metastases.Another Phase III randomized trial, coordinated by the

Hellenic Oncology Research Group (HORG) [27], comparedthe three-drug regimen HORG-FOLFOXIRI to FOLFIRIin a population of 285 untreated mCRC patients. Althoughthe trial failed to demonstrate any superiority for the experi-mental arm, some improvements for the triplet were reportedin terms of RR (43 versus 33.6%, p = 0.168), time to progres-sion (TTP) (median TTP: 8.4 versus 6.9 months, HR: 0.83,95% CI 0.64 -- 1.08, p = 0.17) and OS (median OS: 21.5versus 19.5 months, HR: 0.86, 95% CI 0.64 -- 1.16,p = 0.337). The secondary resection rate was higher amongpatients treated with HORG-FOLFOXIRI (10 versus 4%,p = 0.08).However, at least two main facets should be taken into

account when globally interpreting results. Firstly, differentschedules of triple-drug regimens were adopted in these stud-ies: while in GONO-FOLFOXIRI, 5-FU was administeredat a dosage of 3200 mg/m2 as a continuous 48-h infusionand 5-FU bolus was abolished, in HORG-FOLFOXIRI,5-FU was administered both on days 2 and 3 as continuous22-h infusion at a dosage of 600 mg/m2/day and as bolus ata dosage of 400 mg/m2/day. Irinotecan and oxaliplatinplanned doses were considerably higher in the GONO-FOLFOXIRI versus HORG-FOLFOXIRI (irinotecan: 165versus 150 mg/m2; oxaliplatin: 85 versus 65 mg/m2). Sec-ondly, study populations were selected according to slightlybut relevantly different criteria. While patients aged > 75years, as well as patients aged 70 -- 75 and with a performancestatus (PS) ‡ 1, were not included in GONO trial, all

patients aged ‡ 18 and with PS £ 2 were eligible for theHORG trial.

As a potential consequence of such discrepancies, in theHORG trial, the compliance to FOLFOXIRI was signifi-cantly worse compared with FOLFIRI, with a higher percent-age of courses being delayed (8.3 versus 14%, p = 0.04) anddose reductions (7 versus 3%, p = 0.001). Both dose reduc-tions and treatment delays were more frequent in the groupof elderly patients and, among them, in those treated withHORG-FOLFOXIRI [28].

Despite these major criticisms, in the HORG trial thethree-drug combination showed a trend toward better TTPand RR and a more than doubled secondary resection rate.In the metanalysis by Golfinopoulos et al. [29], GONO andHORG trials have been comprehensively analyzed, demon-strating that the addition of oxaliplatin to first-line 5-FUand irinotecan provided a significant advantage in terms ofboth PFS (HR: 0.73, 95% CI 0.55 -- 0.95) and OS (HR:0.79, 95% CI 0.63 -- 0.98).

With particular regard to secondary surgery, the pooledanalysis of patients treated with GONO-FOLFOXIRI intwo Phase II and in the Phase III trial revealed that 37 outof 196 initially unresectable patients achieved radical resectionof metastases, with a ‘rescue rate’ of 19% [30]. Notably, after amedian follow up of 67 months, in the group of radicallyresected patients, 5-year and 8-year survivals were 42 and33% respectively. At 5 years, 29% patients were free of dis-ease. For patients undergoing secondary hepatic resection,an increasing amount of evidence supports the relevance ofpathologic complete response (pCR) as a meaningful end-point, significantly related to longer survival [31,32]. In partic-ular, in the series presented by Adam et al. [33], 29 out of767 (4%) patients with liver metastases, who underwent rad-ical liver resection after systemic chemotherapy, achievedpCR. Three- and five-year survivals for patients with pCRwere 91 and 76%, respectively, and were significantly higherwhen compared with patients without pCR (61 and 45%,respectively, p = 0.004). Ten-year survivals were 68 and29% with and without pCR, respectively. After a medianfollow-up of 52.2 months, less recurrences occurred inpatients with pCR (41 versus 62% for patients withoutpCR, p = 0.03). A noteworthy percentage (11%) of pCRswas reported in the group of 37 patients who underwentsecondary R0 resection after FOLFOXIRI.

Such results strongly support the choice of FOLFOXIRIregimen as a very active ‘conversion’ therapy, able to inducerelevant tumoral shrinkage and, therefore, particularly appro-priate for patients with potentially resectable metastases, inorder to provide them not only a chance of resection [34],but also a chance of cure [35,36], absolutely unexpected untila few years ago.

Nevertheless, the setting of patients with potentially ormarginally resectable metastases does not represent the solecontext, in which the choice of a highly active regimen mightrepresent the preferred option.



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In fact, in the Phase III GONO trial, excluding from theanalysis patients who had undergone radical surgery on metas-tases, the FOLFOXIRI arm retained a significant advantage inPFS (median PFS: 9.5 months versus 6.6 months; HR: 0.59,95% CI 0.45 -- 0.76, p < 0.0001) and a trend toward longerOS (median OS: 20.2 months versus 15.9 months, HR:0.80, 95% CI 0.61 -- 1.05, p = 0.12) [37]. With regard tothe wide spectrum of clinically and biologically differentdiseases included in the definition of ‘never resectable’mCRC, a significant tumoral shrinkage should representthe major aim also in those patients with widespread, aggres-sive, symptomatic diseases, sometimes responsible for a deepdeterioration of patients’ quality of life.

In fact, although clinical trials typically enroll a small per-centage of poor-prognosis patients, Sargent et al. [38] haveobserved in a pooled analysis of nine studies in the first-line metastatic setting, that the relative benefit of combinationregimens was the same in PS 2 patients compared with PS0 -- 1 patients, both in terms of survival (HR: 0.89, 95% CI0.82 -- 0.96, p = 0.003 in PS 0 -- 1 patients and HR: 0.79,95% CI 0.62 -- 0.99, p = 0.04 in PS 2 patients, withp = 0.18 for PS--treatment interaction) and likelihood ofresponse (odds ratio (OR): 2.71, 95% CI 2.34 -- 3.14,p = 0.0001 in PS 0 -- 1 and OR: 2.85, 95% CI 1.61 -- 5.02,p = 0.0003 in PS 2, with a p = 0.71 for a PS--treatmentinteraction). Although PS 2 patients presented an increa-sed risk of toxicities and 60-day mortality, it should be notedthat a significant treatment--PS interaction is lacking intoxicity analysis.

Consistently with above reported results, in the Phase IIIGONO trial, among patients defined as ‘high-risk’ accordingto the Kohne scoring system, those treated with FOLFOXIRIachieved significantly longer PFS (median PFS: 8.3 versus4.4 months, HR: 0.44, 95% CI 0.24 -- 0.82) and longer,although not significantly, OS (median OS: 14.1 versus11.7 months, HR: 0.78, 95% CI 0.43 -- 1.41) [37].

Nowadays, the relevance of molecular determinants in driv-ing therapeutic decisions is well-recognized. KRAS mutationsburst on the scene of mCRC, leading not only to a more accu-rate selection of patients to be treated with anti-EGFR mono-clonal antibodies, but also to launching a sort of ‘molecularrevolution’, with a strong influence on physicians’ mental atti-tudes. At the same time, a considerable amount of data havefirstly suggested and then corroborated the awful effect ofBRAF mutation as a poor prognostic factor for mCRCpatients [11,39]. A soaring amount of data about tumors’ biol-ogy suggests that BRAF-mutated tumors represent an almosthomogenous group with peculiar genotypic and also pheno-typic features [40-44], altogether responsible for their aggressivebehavior and poor prognosis.

As a consequence of these attainments of knowledge, besideclinical and biochemical parameters, such as PS, number ofmetastatic sites, white blood cell count, serum alkaline phos-phatase and lacticum dehydrogenase, hemoglobin levels andtime to metastases, the knowledge of BRAF mutational status

has acquired an important meaning as a relevant tool to betterestimate tumor aggressiveness.

Though in the absence of specific data from prospective tri-als, the choice of an intensive and very active first-line regimenmight represent an appropriate option also for patients withBRAF-mutated disease.

Therefore, the presence of major clinical, but also molecu-lar prognostic indicators of marked aggressiveness mightinfluence the choice of the treatment toward an intensivefirst-line regimen, such as FOLFOXIRI, with the aim ofrapidly reducing tumor burden, thus potentially improvingpatients’ symptoms and prolonging survival.

2.3 Increasing complexity: the achievement

of biologicsThree biologic agents have entered the clinic for the treatment ofmCRC during the last five years. All them are monoclonal anti-bodies belonging to two distinct classes: anti-EGFRs (cetuximaband panitumumab) and anti-VEGF (bevacizumab). Summariz-ing the main findings of various clinical studies [4,6,7,45,46] we canargue that VEGF inhibition with bevacizumab is an effectivestrategy in combination with chemotherapy in the first- andsecond-line, while the anti-EGFRs have been demonstrated tobe active and efficacious even in the third-line as monotherapyin chemorefractory patients.

Looking at the clinical effect of these molecules, especiallyin terms of OS, it is evident that it hasn’t been as huge asexpected while their costs are certainly not negligible consider-ing both side-effects and economic expenses. Notwithstand-ing these relatively small clinical achievements, the studyand the use of these drugs has dramatically changed our wayof looking to mCRC from a biomolecular point of view [47].It is nowadays globally accepted [2,48,49] that mCRC shouldbe categorized according to the mutational analysis of theKRAS oncogene since this feature precludes patients fromderiving any benefit with anti-EGFRs. The first assessmentof KRAS mutations as predictors of resistance to cetuximabderives from a small retrospective analysis [50]. This initial sug-gestion has been verified and confirmed in randomizedPhase III studies aimed at demonstrating anti-EGFRs’ efficacyacross different lines of treatment [3,4,8,10]. The Cetuximabcombined with irinotecan in first-line therapy for metastaticcolorectal cancer (CRYSTAL) first-line trial of FOLFIRIplus or minus cetuximab, aimed at verifying an improvementin PFS with the addition of the mAb. The experimental armgained a 15% relative risk reduction for progression (HR:0.85, 95% CI 0.72 -- 0.99, p = 0.048) in the intention-to-treat population. Looking at the results according to KRASmutational status it is clearly evident that only wild-typepatients benefited (HR for PFS: 0.68; 95% CI 0.50 -- 0.94,p = 0.02) while for KRAS-mutant patients there was no advan-tage (HR: 1.07, 95% CI 0.71 -- 1.61, p = 0.75) [4]. Similarresults have been obtained in the Phase II randomized Oxali-platin and Cetuximab in First-Line Treatment of mCRC(OPUS) trial that compared folinic acid leucovorin and



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oxiplatin (FOLFOX)-4 plus cetuximab to chemotherapyalone. Objective response rate (ORR) was the primary end-point. In the overall population the addition of cetuximabmarginally improved the ORR (46 versus 36%, OR: 1.52,95% CI 0.975 -- 2.355, p = 0.064), but among KRAS-wild-type patients the difference was quite wider (61 versus 37%,OR:2.54, 95% CI 1.238 -- 5.227, p = 0.011) while KRASmutant patients even experienced a detrimental effect of theanti-EGFR (33 versus 49%, OR: 0.507, 95% CI 0.223 --1.150, p = 0.106) [3]. Analogous results have been obtainedwith the combination of panitumumab with FOLFOX in arecently published Phase III randomized trial [51].The pooled analysis of the two above mentioned studies

with cetuximab provided a clear estimation of the effect ofadding it to first-line chemotherapy in KRAS-wild-typepatients: EGFR inhibition doubled the odds of achieving aresponse (OR: 2.16, 95% CI 1.64 -- 2.86, p < 0.0001), signif-icantly reduced the risk of disease progression by 34% (HR:0.66, 95% CI 0.55 -- 0.80, p < 0.0001) and improved OS(HR: 0.81, 95%CI 0.69 -- 0.94, p = 0.006) [9].This last finding in terms of OS is strikingly similar to that

obtained in a meta-analysis of five randomized first-line trialslooking at the efficacy of bevacizumab in addition to chemo-therapy in molecularly unselected patients (HR: 0.81, 95%CI 0.73 -- 0.90, p < 0.0001) [52]. It should be noted in factthat bevacizumab exerts its effects independently from KRASmutational status [53]. In the meta-analysis, even in terms ofPFS the results with the anti-VEGF in the overall populationare similar to those obtained with cetuximab in KRAS-wild-type patients (HR: 0.61, 95% CI 0.45 -- 0.83, p = 0.002),but the overall effect of bevacizumab in improving tumoralshrinkage is less evident (OR: 1.41, 95%CI 0.92 -- 2.15,p = 0.11) and doesn’t reach statistical significance. This lastobservation coupled with another retrospective analysis com-paring the waterfall plot of two randomized Phase II stud-ies [54] of capecitabine (Xeloda) plus oxaliplatin (XELOX) orcapecitabine plus irinotecan (XELIRI) with cetuximab [55] orbevacizumab [56] led some authors to suggest anti-EGFR inhi-bition as a more productive strategy when response is the pri-mary objective [57]. In fact, in that analysis the activity ofcetuximab as measured by the average and the median reduc-tion in tumor burden (ARTB and MRTB) seemed to begreater than that of the anti-VEGF (ARTB 38.9 versus29.9%, MRTB 35 versus 30%; p = 0.009). In the absenceof head-to-head comparisons of bevacizumab with the anti-EGFRs all retrospective and indirect conclusions should beinterpreted cautiously and first-line decision-making shouldbe driven also by clinical considerations that take into accounttoxicity profiles, patients’ preferences, and the global stategyto be pursued across subsequent lines [58].In oncology practice the most reliable and unquestionable

end-point to which all efforts are aimed at is and shall bealways overall survival. The increasing complexity of theclinical scenario of mCRC patients cleared the way for newend-points potentially indirectly related to OS, that could be

important for research but still don’t have sure clinical impli-cations. It has been suggested, for example, that bevacizumabmay improve the pathologic response in liver metastases [59,60].On the other hand recent studies questioned the reliability oftraditional response rate as good estimator of bevacizumab’sactivity and the radiological evaluation of tumor responseseems to be more accurate when derived from the observationof lesions’ morphological changes according to new evaluationcriteria [61].

In the attempt to further refine our knowledge aboutmCRC the search for other useful molecular markers progres-sively gained more and more attention, especially after theunforeseen demonstration that the concomitant use of up-front anti-EGFRs with bevacizumab plus chemotherapy isfutile [62,63] and the question of which antibody may be betterfor KRAS-wild-type patients came to the fore. Researcherslooked at effectors of EGFR signaling pathway other thanRAS as possible predictors to maximize the benefit fromEGFR inhibitors. Most interesting results derived fromBRAF mutational status. Such mutations are mutually exclu-sive with those of KRAS. After the first description of the pos-sible role as negative predictor of anti-EGFRs’ activity for theBRAF-mutated gene [64], also other confirmatory experimentswere conducted [65]. The vast majority of data was derivedfrom patients in advanced lines of treatment. The widest anal-ysis has been recently published by De Roock et al., who col-lected samples from 649 chemorefractory patients treatedwith cetuximab plus chemotherapy at 11 European centers.Among 350 KRAS-wild-type patients assessed for response,BRAF mutants had a significantly lower response rate (8.3%(2 out of 24) versus 38.0% (124 out of 326), OR: 0.15,95% CI 0.02 -- 0.51, p = 0.0012), shorter PFS (median 8 ver-sus 26 weeks; HR: 3.74, 95% CI 2.44 -- 5.75, p < 0.0001) andOS (median 26 versus 54 weeks; HR: 3.03, 95% CI1.98 -- 4.63, p < 0.0001) [66]. No data from the randomizedtrials of anti-EGFR monotherapy versus best supportive carewith respect to BRAF mutational status have been publishedyet. As regards first-line of treatment, given the relative rarityof BRAF mutation, data from randomized trials of chemo-therapy plus or minus anti-EGFRs are inconclusive due totheir low statistical power [67]. Many other trials looked atthe pure prognostic effect of BRAFmutational status indepen-dently from treatment with anti-EGFRs. As mentioned else-where in this review all data consistently confirm thatBRAF-mutant tumors have an extremely bad prognosis inthe metastatic phase. Due to the strength of such observationsand their possible implications special attention has beenrecently given to the molecular profile of BRAF-mutantCRC, suggesting that such tumors have a specific gene expres-sion pattern especially when BRAF mutation occurs inmicrosatellite-stable tumors [42].

As suggested in the previous paragraph, in the near futureclinicians will want to know what would be the best approachto face this new category of aggressive mCRC with traditionaltreatments whilst waiting for new targeted drugs.



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2.4 Triplet plus biologics: preliminary findings and

ongoing trialsThe clinical relevance of up-front combined regimens, withparticular reference to the results of the GONO-FOLFOXIRIregimen and to the more and more extensive amount of dataconfirming targeted therapies’ safety and efficacy, have beeninterpreted as promising foundations for developing evenmore intensive combination schedules, including three-drug regimens, combined with a biologic agent. Althoughno conclusive findings are available to date, here we lookinto preliminary evidence and current experiences. A list ofongoing trials is included in Table 2.

A Phase II trial (FOIB study), conducted by the GONOgroup, has assessed the safety and activity of the combinationof GONO-FOLFOXIRI regimen plus bevacizumab in previ-ously untreated, unresectable mCRC patients [68]. Accordingto a Phase II single-stage Fleming design, assuming a nullhypothesis of 10 months-progression free rate (10m-PFR) of50% and an alternative hypothesis of 10m-PFR of 70%,with alpha and beta-errors of 0.05 and 0.10, the experimentaltreatment would have been judged to be promising if at least33 patients, out of 53 evaluable, had been free of progressionat 10 months.

At a median follow-up of 28.8 months, 42 (74%) out of57 treated patients were actually free of progression at10 months, with a median PFS of 13.1 months and a medianOS of 30.9 months (Figure 1). In terms of activity, promisingresults were reported, with a RR of 77% and a disease controlrate of 100%. Such a considerable activity translated into aradical resection rate of 26%, rising to 40% among patientswith liver-only metastases. A pCR was observed in the 20%of patients who underwent radical resection.

The safety profile was absolutely consistent with expectedtoxicities and no unforeseen adverse events were reported.

Such results gain higher prominence when consideringclinical characteristics of the study population, including ahigh percentage of patients with synchronous metastases(86%) and a relatively underrepresented proportion ofpatients with liver-only disease (53%). Consistently withsuch poor prognostic indicators, a higher than expectedfrequency of BRAF mutations (18%) was reported.

The interpretation of study results according to KRAS andBRAF mutational status revealed no differences in terms ofboth PFS and OS between patients with KRAS-wild-typeand KRAS-mutated tumors, or, even more relevantly, betweenpatients with BRAF wild-type and BRAF mutated tumors.Also patients with BRAF-mutated tumors, in fact, achievedmeaningful results in terms of survival (median PFS:12.8 months and median OS: 23.8 months), which did notsignificantly differ from results for the BRAF-wild-type sub-group (HR for PFS: 0.89, 95% CI 0.41 -- 1.91, HR for OS:0.76, 95% CI 0.26 -- 2.21).

Though keeping in mind the small sample size and the ret-rospective nature of considerations about the BRAF-mutatedsubgroup, one could argue that the adoption of an intensive

regimen might allow containment of the aggressive behaviorof these peculiar diseases.

Based on promising results of the Phase II FOIB trial, aPhase III multicenter study, comparing FOLFOXIRI plusbevacizumab versus FOLFIRI plus bevacizumab (Tripletplus Bevacizumab (TRIBE) trial), is currently ongoing [69].Preliminary safety data from the first 150 enrolled patientsconfirmed the safe profile reported in the FOIB trial,without revealing unexpected toxicities or significant diffe-rences between arms, except for oxaliplatin-specific adverseevents [70]. Such forthcoming results will probably clarify theeffect of the up-front triplet plus bevacizumab, potentiallythrowing light also on clinical and molecular features, usefulfor defining the population more likely to benefit from suchan intensive regimen.

A Phase II randomized trial is currently evaluating thesafety and efficacy of first-line FOLFOX plus bevacizumaband FOLFOXIRI plus bevacizumab in a population ofmCRC patients with liver-only disease, in order toexplore the potentiality of such regimen in a ‘neoadjuvant’setting [71]. Similarly, another Phase II trial, that adoptssurgically complete resectability as the primary endpoint, isevaluating the activity of FOLFOXIRI plus bevacizumab ina population of mCRC patients with not optimally resectableliver or lung metastases [72].

A less extensive amount of data has been published untilnow about the safety and efficacy of the combination of athree-drug regimen with an anti-EGFR monoclonal antibody.A Phase I dose-escalating study has explored the safety offirst-line FOLFOXIRI (according to the GONO schedule)plus cetuximab, revealing neutropenia and diarrhea as themost common treatment-related toxicities and identifying125 mg/m2 as the recommended irinotecan dose to be furtherinvestigated in following experiences. Besides treatment’sfeasibility, the study attested treatment’s activity reporting apromising RR of 75% in a cohort of 20 mCRC patients,not selected for EGFR expression or for KRAS mutationalstatus [73].

A group of 42 molecularly unselected, initially unresectablemCRC patients have been treated with the first-line triplet(according to a schedule differing from GONO-FOLFOXIRIonly by a higher planned dose of irinotecan) in a Phase IItrial [74]. According to preliminary results, 22 out of 37 evalu-able patients achieved a response, with a RR of 82% and a dis-ease control rate of 97%. The feasibility of the treatment’sschedule was demonstrated by the acceptable safety profile,that included diarrhea and neutropenia as the most commongrade 3 -- 4 toxicities, with awaited frequencies. Final efficacydata are not available yet.

Another Phase II trial is currently ongoing, evaluating thecombination of HORG-FOLFOXIRI plus cetuximab in apopulation of unresectable mCRC patients, with no molecularselection criteria [75].

The combination of a chrono-modulated schedule ofthree-drug chemotherapy with cetuximab has been tested in



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Table

2.Ongoingclinicaltrials

evaluatingintensivetreatm

ents

ofthree-chemoterapeuticagents

plusabiologic.

Name

Clinical

Trials.govID

Studydesign

Main

inclusion

criteria

Primary

endpoint

Status

Location

APhase

IIIrandomizedtrialofFO

LFOXIRI+

BEVACIZUMABversusFO

LFIRI+BEVACIZUMAB

asfirst-linetreatm

entformetastaticcolorectal

cancerTRIBE[69]

NCT00719797

Phase

III,Open-label,

Two-arm

s,Randomized

Patients

with

unresectable

mCRC

PFS

Recruiting

Italy

AMulticentreRandomizedPhase

IIStudyto

Assess

theSafety

andResectablityin

Patients

WithPrimarily

Unresectable

LiverMetastases

Secondary

toColorectalCancerReceiving

Treatm

entWith5-FU,Leucovorin,Oxaliplatin

andBevacizumabWithorWithoutIrinotecan

asFirstLineTreatm

ent[71]

NCT00778102

Phase

II,Open-label,

Two-arm

s,Randomized

Patients

withliver

onlymCRC

Surgical

resectability

Recruiting

Austria,

France,Spain

Phase

IIStudyonCurative

Resectability

of

NotOptimally

Resectable

Liverand/orLung

MetastasesFrom

ColorectalCarcinoma(CRC)

UnderIntensifiedChemotherapy(FOLFOXIRI/

Bevacizumab)[72]

NCT01126866

Phase

II,Open-label,

Single-arm

Patients

withnot

optimally

resectable

mCRC

(liverand/orl

ungmetastasesonly)

Surgical

resectability

Recruiting

Germ

any

Phase

IItrialofFO

LFOXIRIplusPanitumumab

asfist-linetreatm

entforKRAS-and

BRAF-wild-typemetastaticcolorectalcancerTRIP

NA

Phase

II,Open-label,

Single-arm

Patients

with

unresectable

mCRC

Response

rate

Recruiting

Italy

ATripletCombinationWithIrinotecanPlus

Oxaliplatin,ContinuousInfusion5-Fluorouracil

AndLeucovorinPlusCetuximabAsFirstLine

Treatm

entIn

MetastaticColorectalCancer.

APilotPhase

IITrial[75]

NCT00689624

Phase

II,Open-label,

Single-arm

,Non-Randomized,

ActiveControl

Patients

with

unresectable

mCRC

Response

rate

Recruiting

Greece



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a Phase II trial (preoperative chemotherapy for hepatic resec-tion (POCHER) trial) reporting a response rate of 79% anda resection rate of 63% in a population of 43 unresectablemCRC patients with liver-only metastases [76].

The GONO group is currently conducting a Phase II trial,with the aim of evaluating the safety and activity of the combi-nation of GONO-FOLFOXIRI plus panitumumab in a popu-lation of unresectable mCRC patients with KRAS and BRAFwild-type tumors. The adoption of these molecular inclusioncriteria allows maximization of treatment efficacy, by exploringits potential in a population with favorable predictors ofdisease’s sensitivity to anti-EGFR mAbs. Conversely, the exclu-sion of patients with BRAF-mutated tumors does not permitdetection of a mild activity of such an intensive strategy inthis poor-prognosis subgroup.

3. Conclusion

Ongoing trials will certainly provide further insights into‘four-drug’ regimens. In particular, efficacy results from

TRIBE trial are awaited with great interest, in order to reallyunderstand the effect of the addition of oxaliplatin to first-line FOLFIRI plus bevacizumab. Some urgent issues need tobe handled, to really carve out the most appropriate spacefor these regimens in clinical practice. The availability oftargeted agents might represent a very appealing chance torestrict the use of chemotherapy to a short, initial period, inwhich the achievement of a meaningful tumoral shrinkagerepresents the major goal of the therapeutic strategy. In thisregard, the choice of an intensive up-front chemotherapy,whose activity might even be optimized by the combinationwith a biologic drug, would be extremely appropriate. Resultsof the Phase III randomized Maintenance Bevacizumab afterInduction Therapy in Metastatic Colon cancer (MACRO)trial, that compared XELOX plus bevacizumab until diseaseprogression with XELOX plus bevacizumab administeredfor 6 cycles and followed by maintenance with bevacizumabalone, suggest that the earlier discontinuation of chemother-apy does not substantially impair treatment’s efficacy in termsof both PFS (HR: 1.11, 95% CI 0.89 -- 1.37) and OS(HR:1.04, 95% CI 0.81 -- 1.32) [77]. The adoption of anintensive first-line treatment as a four-drug regimen wouldtherefore allow concentrate of the greatest cytoreductive activ-ity to a limited period, thus potentially allowing patients to bespared of a part of the chemorelated toxicities.

Moreover, to really optimize the adoption of such regimensin clinical practice, efforts in translational research will beindispensable, not only to identify molecular markers, to bepotentially applied as tools to better orient therapeuticchoices, but also to acquire an essential amount of know-ledge, to disclose the ‘backstage’ of diseases with extremelydifferent behaviors.

4. Expert opinion

The choice of the up-front treatment of mCRC patients isnowadays an intriguing challenge for medical oncologists,made complex and crucial not only by the availability of thewide spectrum of therapeutic options, but also by the influ-ence of this first choice on the following steps of the globaltherapeutic strategy.

In our opinion, while waiting for new drugs, to furtherimprove results achieved until now, CRC research shouldfocus on three main topics:

1) The optimization of the global strategy;2) The refinement of the use of available cytotoxic and

targeted agents;3) The identification of molecular tools, able to drive

therapeutic decisions.

First of all, in the definition of the global strategy for treat-ment, modern oncologists can take advantage of an amount oflocoregional and systemic approaches, whose best integrationstill needs to be established. Moreover, in the era of targeted

100

75

50

25

00

100

75

50

25

0

12

Number of patients: 57Number of events: 47Median follow up: 28.8 months

Number of patients: 57Number of events: 26Median follow up: 28.8 months

Median survival: 30.9 months

Median progression-free survival: 13.1 months

24 36

Time (months)

0 12 24 36

Time (months)

Pro

gre

ssio

n f

ree

surv

ival

(%

)O

vera

ll su

rviv

al (

%)

A.

B.

Figure 1. Progression-free A. and overall survival B. curves of

the Phase II FOIB trial (GONO-FOLFOXIRI plus bevacizumab).



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agents, the possibility of alternating induction phases, mainte-nance periods and chemotherapy ‘holidays’ further widensthe range of practicable techniques. For all these reasons,nowadays, the global strategy for treatment deserves itselfa prominent role, thus promoting a shift in medical oncolo-gists’ mental attitudes from the concept of a ‘step by step’designed therapeutic route to a ‘continuum of care, [78], whosefoundations are laid during the up-front decisions.Secondly, the more and more extensive adoption of bio-

logic drugs in current practice, as well as the growing amountof results from clinical trials, underline the need for a deeperawareness of the best integration of available biologic drugsin the therapeutic route. Having ascertained the detrimentaleffect of the double inhibition of VEGF and EGFR, despiteencouraging preclinical and early clinical evidence, it wouldbe reasonable to investigate the use of biologics in a sequentialmanner. In KRAS-wild-type patients, for example, the evalua-tion of the early inhibition of EGFR could be suggestedwith the aim of achieving the best tumor shrinkage, followedby the anti-VEGF for best exploiting the efficacy of the latteras maintenance. In this complex scenario, also the choice ofintensive treatments, like four-drug regimens, should beweighted up by a global point of view focusing on the

objective of concentrating the greatest cytoreduction in an ini-tial short phase of treatment, to be then maintained by lessintensive regimens while considering the possibility of a‘re-induction’ phase with all active drugs. This approach willobviously overcome the fixed scheme of lines of treatmentwith the traditional endpoint of PFS, instead looking at theobjective of extending the time to strategy failure.

Finally, in order to deliver to each patient the best thera-peutic option, not only in terms of first-line regimen, butalso in terms of global strategy across all the lines of treatment,an essential aid is awaited from the attempts of translationalresearch to disclose the phenotypic and genotypic features ofmolecularly and clinically different diseases. The pharmaco-dynamic approach might be useful to provide insights intomechanisms of intrinsic and acquired resistance, thus contrib-uting to building a comprehensive ‘continuum of molecularcharacterization’, able to drive the therapeutic ‘continuumof care’.


The authors declare no conflict of interest and have receivedno payment in preparation of this manuscript.

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AffiliationFotios Loupakis†1,2, Chiara Cremolini2,

Marta Schirripa2, Gianluca Masi1,2 &

Alfredo Falcone1,2

†Author for correspondence1University of Pisa,

Department of Oncology, Transplants and New

Technologies in Medicine, Pisa, Italy

E-mail: [email protected] Ospedaliero-Universitaria Pisana,

Polo Oncologico,

Pisa, Italy



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1. Introduction

2. Discussion

3. Expert opinion

Review

The impact of biologic responsemodifiers on hepatitis B virusinfectionMatthew B Carroll301 Fisher Avenue, Keesler AFB, MS, USA

Introduction:Thebiologic responsemodifiers are adiversegroupofmedications

thathaveemergedover the last decade. They targetpro-inflammatory cytokines

or cell surfacemolecules that drive illnesses such as rheumatoid arthritis. Despite

the greater control afforded they have also ushered in a new spectrum of side

effects. As the same immunologic machinery that helps control infections such

as HBV contributes to the pathogenesis of rheumatologic diseases, persistence

or reactivation of the virus remains an evolving concern.

Areas covered: A systemic literature review was performed using the

PubMed and Medline databases (1996 to January 2010) searching for the

index term ‘Hepatitis B’ combined with the terms ‘tumor necrosis factor’,

‘B cell’, ‘rituximab’, ‘IL-1’, ‘anakinra’, ‘IL-6’, ‘tocilizumab’, ‘CTLA-4’, and

‘abatacept’. All relevant articles in English were reviewed and secondary

references of interest were also retrieved. This paper addresses the role of

the various cytokines and cluster of differentiation molecules in controlling

HBVinfection and the currently known effect that the biologic response

modifiers have on viral control by the host immune response.

Expert opinion: The risk of HBV reactivation is greatest in HBsAg positive

patients. These patients should start antiviral therapy one week before receiv-

ing a biologic response modifier. The risk of HBV reactivation in HBsAg nega-

tive patients appears very low but when HBsAb titers are low use of rituximab

or TNF-a antagonists may increase the risk of reactivation.

Keywords: ankylosis spondylitis, B cells, cytokines, hepatitis B virus, rheumatoid arthritis,

tumor necrosis factor


1. Introduction

Chronic infection with HBV remains a significant public health problem affectingmore than 350 million people worldwide [1]. The incidence of chronic hepatitis Bvirus (HBV) infection is disproportionately higher in areas of the world such asAsia, sub-Saharan Africa, and the Amazon river basin of South America [2]. In theseareas where the prevalence can exceed 8% perinatal infection is the most frequentmode of transmission. While the perinatal mode of HBV transmission does notlead to acute hepatitis it does result in the establishment of chronic infection whichcarries a 15 -- 40% lifetime risk of developing liver failure, cirrhosis or hepatocellularcarcinoma. In the USA and western Europe, the prevalence of HBV infection is lessthan 2% as spread of the virus is facilitated by sexual contact and injected drug use [1,2].

Patients chronically infected with HBV may present in one of four phases, buttransition through all phases may not occur in everyone [3]. Those exposed to the virusperinatally or during their childhood develop immune tolerance. This stage is charac-terized by the presence of the hepatitis B envelope antigen (HBeAg) and high serumlevels of HBV DNA with mild to no inflammatory changes noted on liver biopsy [3].While these patients are at a low risk of progressing to cirrhosis or hepatocellular


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carcinoma, they should be monitored for progression to theimmune clearance stage. This stage marks maturation ofthe host immune response to the virus and is characterized bythe presence of HBeAg, elevated serum levels of HBV DNA,elevation in serum alanine aminotransferase (ALT) levels, andnecroinflammatory activity on liver biopsy [3]. When HBeAgseroconversion occurs a patient enters into the inactive hepatitisB surface antigen (HBsAg) carrier stage. Inactive carriersform the largest group of patients chronically infected withHBV [3]. These patients have little to no HBV DNA detectablein their serum and little to no inflammatory activity on liverbiopsy however 20 -- 30% can have episodic or sustainedHBV reactivation, which can fuel progressive liver damage [3].Progression to this fourth or reactivation phase of chronicHBV infection can occur spontaneously or during immunesuppression [3]. Separate from these four phases is occultHBV infection. Occult HBV infection is marked by theabsence of HBsAg and HBV DNA in the serum; however,low-level HBV replication may persist with HBV DNA foundin the liver [4]. Hepatitis B core antibodies (HBcAb) with orwithout hepatitis B surface antibodies (HBsAb) are detectablein the serum [4]. Immunosuppression may lead to HBVreactivation in these patients [4].The key to containment and eradication of HBV is a robust

immune response. While such a response is complex andrecruits multiple cells and cytokines, the innate arm of hostimmunity is activated first. It eradicates infected hepatocytesand releases cytokines, which inhibit viral replication [5].Though not directly infected within hours of initial HBV infec-tion liver macrophages (Kupffer cells) recognize the virus andactivate pathways releasing pro-inflammatory cytokines suchas IL-6, TNF-a, IL-1b and IL-8 [6]. The rapid release of

IL-6 controls HBV gene expression and replication at a tran-scriptional level [6]. Infected hepatocytes release IFN-a and bwhich activates NK cells [7]. NK cells eliminate HBV-infected hepatocytes and also release IFN-g and TNF-a whichinhibits further viral replication without triggering hepatocytedestruction [7]. Dendritic cells are also activated during initialHBV infection [7]. These cells capture viral particles throughToll-like receptors, secrete cytokines such as IFN-a, TNF-a,IL-12, and IL -10 which help polarize naıve T-cells and processantigens for presentation to T-cells via MHCmolecules [7]. Theadaptive arm of host immunity is then recruited as eliminationof HBV infection and ultimately disease resolution depends ona robust polyclonal T-cell response [5]. Mature dendritic cellsmigrate from the liver to lymph nodes to activate T-cells. Asfacilitated by various pro-inflammatory cytokines and the inter-action between the T-cell receptor and MHC--antigen com-plex, T-cell activation occurs [7]. Cytotoxic CD8+ T-cellscontinue the generation of pro-inflammatory cytokines. NaıveCD4+ T-cells differentiate under the direction of B-cells andcytokines into both a TH1 phenotype, which generates cyto-kines such as IL-2, IFN-g and TNF-a, thus enhancing thehost cytotoxic response and a TH2 phenotype releasing IL-4IL-10, and IL-12 to boost the host humoral response toHBV [7]. When the host immune response is unable to eradi-cate the virus and chronic infection results, HBV-specificT-cell responses gradually wane but the humoral response issustained and vigorous [8].

By their nature chronic inflammatory rheumatologicaldiseases such as rheumatoid arthritis (RA) and the seronega-tive spondyloarthropathies represent aberrant manifestationsof the host immune response [9]. A majority of the samecells and cytokines that organize the host immune responseto infections such as HBV are activated and chronicallyrecruited, thus contributing to the pathogenesis of theserheumatological diseases. Several decades of research haveconvincingly demonstrated that the excessive production ofpro-inflammatory cytokines such as IL-1, IL-6 and TNF-aare critical to the initiation and sustainment of these dis-eases [9,10]. Attempts at refining pharmacological therapies tomore effectively treat rheumatological diseases have lead tothe emergence of a class of medications known as biologicresponse modifiers [11]. This diverse group of medicationshas the unifying goal of targeting a specific cytokine, cell-membrane-bound molecule, or lymphocyte vital to the induc-tion and/or perpetuation of the immune response. Over thelast 10 years multiple biological response modifiers haveemerged to treat chronic inflammatory rheumatological dis-eases. Some antagonize cytokines such as TNF-a (infliximab,etanercept, adalimumab, certolizumab and golimumab),mimic the natural action of the IL-1 receptor antagonist (ana-kinra), or antagonize the IL-6 receptor (tocilizumab). Othersinhibit cell signaling pathways or promote cell cytolysis suchas the antagonist of the cluster of differentiation (CD)80/86 molecules (abatacept) and an anti-CD20 monoclonalantibody (rituxmab) respectively. Having the ability to target

Article highlights.

. Chronic infection with hepatitis B virus progressesthrough several stages, each of which carries differentrisks of reactivation.

. Overlap in cells and cytokines recruited to controlchronic HBV infection also play a role in thepathophysiology of rheumatological diseases such asrheumatoid arthritis.

. The risk of reactivation of chronic or latent viral andmycobacterial infection varies with each biologicresponse modifier.

. Stratification of patients chronically infected withhepatitis B who will be treated with a biologic responsemodifier for a rheumatologic disease should undergoevaluation by a hepatologist or infectious diseasespecialist and serological testing for the presence of thevirus and associated liver injury.

. It is recommended to initiate antiviral therapy 1 weekbefore and continue for 6 months after treatment witha biologic response modifier.


The impact of biologic response modifiers on hepatitis B virus infection


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molecules or cells of the immune response the biologicresponse modifiers have made stopping the clinical, serologi-cal and radiographic manifestations an attainable goal, espe-cially when administered in conjunction with traditionaldisease-modifying medications such as methotrexate [11].

While the biologic response modifiers have ushered in anew era of control over chronic inflammatory rheumatologicdiseases, they have also been associated with a new spectrumof adverse events. A longstanding concern associated withthe use of these agents has been the risk of infection. As aclass the biologic response modifiers appear to be associatedwith higher rates of infection and serious infection, althoughthis risk may only be elevated for a brief period (16 weeks)after starting some of these agents [12-14]. The reactivationof latent or opportunistic bacterial, viral or fungal infectionshave also been problematic. This issue gained attentionshortly after the introduction of the TNF-a antagonistswhen post-marketing surveillance demonstrated higher ratesof reactivation of Mycobacterium tuberculosis even thoughclinical trials did not indicate such a risk existed [11,13,15].Opportunistic infection with atypical Mycobacteria, Histo-plasmosis, Listeria, Aspergillus, Nocardia, and Cytomegalo-virus have also been associated with the use of TNF-aantagonists [11,13]. Amongst the other biologic responsemodifiers treatment with rituximab has been associatedwith reactivation of the JC virus (manifesting as progressivemultifocal leukoencephalopathy) and abatacept has beenassociated with higher rates of herpes simplex infec-tions [12,16,17]. Anakinra does not consistently appear to beassociated with such infections [12,16,17].

The effect that the biologic response modifiers have on thehost immune response to contain and/or eradicate HBV is anevolving area of interest. While a growing body of data sug-gests that select biological response modifiers may be associ-ated with an increased risk of HBV reactivation, this risk isnot as well defined for the other therapies in this group [18].The purpose of this review is to examine the current literatureand summarize the data that exists about the effect that bio-logic response modifiers prescribed by rheumatologists mayhave on patients chronically infected with HBV. Also incor-porated in the review of each biologic response modifier onchronic HBV infection are the currently known effects thatpolymorphisms of the various cytokines and cell signalingpathways have on control and eradication of HBV.

2. Discussion

2.1 TNF-a antagonistsTNF-a is a cytokine composed of three identical 17 kDa unitsand forms part of the initial cytokine response when cells areexposed to a diverse number of infectious, physical, and immu-nological stimuli [19-22]. Initial secretion of TNF-a comes frompreformed stores cleaved from membrane-bound TNF-a resid-ing on inflammatory or antigen-presenting cells (APC) suchas macrophages. Subsequent release reflects new synthesis as

TNF-a is inserted into the cell membrane and then cleaved [9].TNF-a triggers a host of diverse effects on the immune systemand can amplify its own synthesis [9,11]. Soluble TNF-aincreases expression of adhesion molecules, stimulates releaseof other pro-inflammatory cytokines such as IL-1 and IL-6,and can induce apoptosis [9,11]. While the majority of TNF-asynthesis is by monocytes and macrophages under various cir-cumstances other cells may produce TNF-a such as hepato-cytes and T-cells [19,22,23]. In RA, cells containing TNF-a arefound in the synovial lining, juxta-articular blood vessels, andat the cartilage--pannus junction [19]. Levels of TNF-a are oftenmuch greater in the synovial space as compared with the serumin RA patients [20]. Local production of TNF-a directly stimu-lates osteoclast formation and leads to the characteristic boneresorption identified radiographically [19].

The TNF-a (TNFA) allele is found on chromosome 6 inthe class III region of the MHC [24,25]. Genetic polymorphismsthat lead to lower amounts of TNF-a secretion have been asso-ciated with an increased risk of progression to chronic HBVinfection [24,26]. Multivariate analyses of Korean, CaucasianGerman and Chinese populations have demonstrated that the-238GA haplotype is associated with a higher risk of progress-ing to chronic HBV infection [24,27-29]. Other polymorphismssuch as the -308GG haploytpe [30,31], the -857CC haplo-type [27,28,32], and combination -308G/-238 G homozygotes [30]have also been associated with an increased risk of developingchronic HBV infection. Lower levels of TNF-a attributableto these polymorphisms have several adverse effects on thehost immune system response to HBV: the cytokine cascadeinitiated and sustained by TNF-a is less potent [33], hepatocyteclearance via Fas--Fas-ligand mediated apoptosis is decrea-sed [34], and CD8+ T-cell responses are dampened by therelative imbalance between TNF-a and higher levels ofIFN-g [26,35].

An evolving body of literature suggests that patients withchronic HBV infection treated with TNF-a antagonists with-out concomitant antiviral therapy are at increased risk ofexperiencing reactivation of the virus. In patients who areHBsAg-positive a review of 35 case reports suggested thattreatment with infliximab, in the absence of concomitantantiviral therapy, was associated with the highest rates ofdeveloping clinically symptomatic hepatitis, greater than atwofold increase in aspartate aminotransferase (AST)/ALT,greater than 1000-fold increase in HBV DNA levels, andrisk of death [18]. The authors of this review recommendedthat patients who are HBsAg-positive and would benefitfrom treatment with a TNF-a antagonist should be startedon antiviral therapy 1 -- 2 weeks prior to initiating such ther-apy [18,36]. Such patients should remain on antiviral therapyfor 6 months after ceasing TNF-a antagonist therapy asimmune reconstitution could lead to a flare of HBV [37].Close serologic monitoring of trends in AST/ALT and HBVDNA was also recommended and infliximab was suggestedas a second-line agent to treat the inflammatory disease ofpatients who were HBsAg-positive [18]. A consensus statement

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from the American College of Rheumatology (ACR) in2008 stated that use of TNF-a antagonists was contraindi-cated in patients with HBV infection who had Child-Pugh Class B or C liver disease regardless of the concomitantuse of antiviral agents [38]. A consensus statement from theEuropean League Against Rheumatism recommended thatpatients should be screened for HBV infection prior tostarting TNF-a antagonists [39].In patients who are HBsAg-negative but HBcAb-positive

the risk of HBV reactivation appears very low when treatmentwith a TNF-a antagonist is started. While several casereports [40-42] and a retrospective case series of 88 Koreanpatients followed over 6 ½ years [43] suggested that somepatients had serological evidence and occasionally clinical evi-dence of HBV reactivation, two recent prospective cohortstudies comprising 88 European patients reported no casesof HBV reactivation [44,45]. In a case report [41] and one ofthese prospective studies [45] a decrease in HBsAb titers wereobserved in some of the patients. It was recommended inthe prospective study that a decrease in HBsAb titers, espe-cially in patients starting TNF-a antagonists with baselinelow HBsAb titers, should have close monitoring for evidenceof HBV reactivation [45]. Routine antiviral prophylaxis hasnot been recommended in patients who are HBsAg-negative but HBcAb-positive when treated with TNF-aantagonists [44]. Close clinical and serologic follow-up ofsuch patients would nonetheless be prudent.

2.2 IL-1 receptor antagonistsIL-1 is a 17-kDa protein secreted mainly by monocytes andmacrophages and has inflammatory effects, which includethe induction of IL-6 and COX-2 [9,16]. It can also be pro-duced by endothelial cells, B-cells and activated T-cells [9].IL-1 can bind to two types of cell surface receptors. Type IIL-1 receptors (IL-1R) have a cytoplasmic tail, which facili-tates intracellular signaling, whereas Type II IL-1R bindIL-1 without signal transduction [9]. A naturally producedIL-1 receptor antagonist (IL-1RA) binds Type I IL-1R withhigh affinity, competing with IL-1 and its ability to activatetarget cells [9]. Anakinra is almost identical to the non-glycosylated form of the naturally occurring IL-1RA exceptfor an additional N-terminal methionine [16]. In RA theIL-1RA is found at lower levels in inflamed joints than wouldbe expected [16].Genetic studies of select populations suggest that certain

polymorphisms of the IL-1 receptor antagonist gene(IL-1RN) may impart resistance to the development ofchronic infection with HBV. Intron 2 of the IL-1RN geneis an 86 base pair variable number tandem repeat located inthe regulatory region of the IL-1 genes and has potential func-tional importance by modulating IL-1 protein produc-tion [46,47]. Five different alleles of varying repeating lengthshave been identified (alleles 1 through 5) [47]. In a study of190 mainland Chinese patients with chronic HBV infectiona lower number had allele 2 of IL-1RN [46]. The authors

suggested that allele 2 imparted greater resistance to HBVinfection as they were able to generate a more robust responseto clear the virus through increased IL-1b production [46].However, a study of 80 Iranian patients did not demonstratea similar finding, though the authors suggest that the absenceof a correlation in their population could have been due to thesmaller number of patients in the study [47].

To date no reports of HBV reactivation have been pub-lished of patients being treated with anakinra. An observa-tional study of the safety of anakinra in 2006 did not reportany cases of HBV reactivation with use of the medication [48].The most recent package label for the medication availablethrough the FDA does not comment on any known relation-ship to the reactivation of HBV infection [49]. The manufac-turer of anakinra reported no cases of reactivation of HBVto date [50].

2.3 IL-6 receptor antagonistsIL-6 is a 20-kDa protein predominantly synthesized andsecreted by cells of macrophage lineage and T-cells, althoughsynthesis and secretion of this cytokine can occur in other tis-sues throughout the body [51]. It is a pleiotropic cytokine witha diverse array of biological activities and regulates critical cellfunctions such as proliferation, differentiation and gene acti-vation [51,52]. IL-6 controls the response of both the innate [6]

and adaptive arms of host immunity in response to an infec-tion as well as regulating the acute phase response [6,52,53]. Italso induces T-cell proliferation and promotes the differentia-tion of cytotoxic T-cells. IL-6 exerts its numerous effectsthrough interactions with membrane-bound IL-6 receptor(IL-6R) and the recruitment of gp130, a protein that isubiquitous to most cells [51]. Soluble forms of IL-6R andgp130 modulate the systemic effects of IL-6 [51]. In RA serumlevels of IL-6 are elevated and overproduction contributes tothe pathogenesis of this illness [52]. IL-6 promotes and sustainsinflammation in the synovium through leukocyte recruitmentand contributes to joint destruction through endothelial cell,synovial fibroblast and osteoclast activation [52]. The constitu-tional and systemic effects seen in RA reflect the activity ofIL-6 as a pyrogen [52]. Tocilizumab is the sole monoclonalantibody currently available which targets the IL-6 receptorto antagonize the effects of IL-6 in RA.

In chronic HBV infection, serum IL-6 levels have corre-lated with the extent of hepatocyte damage and the develop-ment of cirrhosis [54,55]. IL-6 secretion is increased duringacute exacerbations of chronic HBV infection [55]. Addition-ally, elevated serum IL-6 levels antedated the developmentof hepatocellular carcinoma in patients chronically infectedwith HBV [56]. Despite these observations, several studiesacross multiple ethnic groups have failed to establish a linkbetween IL-6 gene promoter polymorphisms and outcomesof chronic infection although one study of an Europeanpopulation suggested a link between IL-6 -174 G/C and thecourse of chronic HBV infection [5,26,57-59]. While this studyfocused on the role of the polymorphism IL-6 -174, a Korean



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study found no relationship with two other polymorphisms(positions -572 and -597) [58].

Given the role of IL-6 in acute exacerbations of chronicHBV infection it has been suggested that therapeutic neutral-ization of IL-6 could pose a risk in chronic HBV infectedpatients [6]. To date two case reports, both of Japanesepatients chronically infected with HBV who were subse-quently treated with tocilizumab, have been published [60,61].The first case was a 60 year old female who had seropositiveRA resistant to multiple disease-modifying agents [60]. Shewas found to be in the immune tolerant phase (HBeAg-positive, elevated HBV DNA level and normal ALT) whilereceiving tocilizumab. She received tocilizumab for morethan 5 years without an exacerbation of hepatitis, so she wasplaced on entecavir and continued on tocilizumab [60]. Thesecond case was a 40 year old male with adult onset Still’s dis-ease complicated by amyloid A amyloidosis [61]. His inflam-matory arthritis was also refractory to multiple diseasemodifying agents [61]. During the course of treating his arthri-tis with immunosuppressive medications he had a mild persis-tent elevation in ALT and evidence of chronic active hepatitisB infection (HBsAg-positive, HBeAg-positive, and HBVDNA over 107 copies/ml) [61]. The patient was started onentecavir (0.5 mg daily) with his HBV DNA level fallingbelow the limit of detection. He was then started on tocilizu-mab (8 mg/kg every 4 weeks) [61]. No reactivation of hisarthritis or his chronic HBV infection was observed [61]. TheJapan College of Rheumatology in 2009 published guidelinesabout the use of tocilizumab in HBV-infected patients [62]. AsJapan has had access to tocilizumab for almost a decade, rec-ommendations were made to exclude patients with activeHBV infection from receiving this medication [62].

2.4 Anti-CD20 therapyThe dynamic role of B-cells in coordinating the host immuneresponse to a pathogen extends well beyond that of anti-body production [63]. B-cells are also efficient APCs, up to1000 times more potent in processing and presenting antigensthan other APCs such as macrophages or dendritic cells [63].B-cells are very effective in presenting antigens when foundat low concentrations [63]. B-cells also provide key costimula-tory signals for CD4+ cells thus activating cellular immunityand dictating the extent of the initial expansion these cellsundergo. Additionally B-cells can secrete or respond to therelease of cytokines such as IL-6, IL-1, IFN-g and TNF-a.Such cytokines not only establish or enhance a pro-inflammatory state, they also exert a regulatory role on futureT-cell functions [63].

One of the most immunogenic proteins of HBV is the hep-atitis B core antigen (HBcAg) [64]. HBcAg at very low concen-trations can activate numerous naıve B-cells throughcrosslinking of their surface immunoglobulin receptors [64].This T-cell-independent activation helps initiate produc-tion of HBcAb IgM antibodies. HBcAg can also bind tonon-HBcAg-specific B-cells akin to the immune activation

observed to a superantigen [64]. B-cells acting in the capacityof APC phagocytize HBcAg as well as HBsAg. These antigensare then processed and presented to cognate CD4+ cells withappropriate costimulatory molecules to recruit the host cellu-lar immune response [8,64]. As the host immune response con-tains initial HBV infection they produce increasing quantitiesof HBsAb [2]. It has been observed that B-cell depletion leadsto decreased HBsAb serum titers with an increase in HBVDNA and HBV reactivation [65].

Rituximab is a chimeric monoclonal antibody with affinityfor the CD20 molecule. The CD20 molecule is neither shednor internalized and the main mechanism of action of rituxi-mab is through antibody dependent cell-mediated cytotoxic-ity [66]. The CD20 molecule is widely expressed on B-cells,ranging from pre-B-cells to those later in differentiation, butit is absent from plasma cells [66]. It was originally developedand later approved for the treatment of B-cell non-Hodgkin’slymphoma as used in conjunction with cyclophosphamide,hydroxydaunorubicin, vincristine and prednisone (CHOP)chemotherapy [39,65]. It has since been approved for use withmethotrexate in the treatment of moderate to severe RA inpatients with an inadequate response to TNF-a inhibitors [39].

A growing body of literature supports the risk of HBV reac-tivation with rituximab therapy [65]. From the oncology liter-ature rituximab has the ability to induce HBV reactivationwhether administered alone or with CHOP chemotherapy [65].The risk of this reactivation is increased in males and withvery low HBsAb titers [65]. The prevalence of such reactivationvaries between 20 and 55% in HBsAg-positive patients [67],thus the recommendation for HBsAg-positive patientswho need rituximab is to start antiviral prophylaxis [65,67].Resistance to some antiviral therapies, specifically lamivu-dine, may decrease with some chemotherapy regimens butincreases when concomitant steroids or fludaravine areadministered [65].

For patients who are HBsAg-negative a prevalence forHBV reactivation of 3% has been reported [65]. In HBsAg-negative, HBcAb-positive, HBsAb-negative patients, regard-less of HBV DNA levels at the start of rituximab therapy,HBV reactivation appears to be a rare occurrence. Suchreactivation however has been associated with prolongeduse of chemotherapy, lower efficacy of chemotherapyagainst lymphoma, and death from HBV hepatitis [65].A case mortality rate of 30 -- 38% associated with HBV reac-tivation has been observed in this group of patients, thusantiviral prophylaxis has also been recommended [65]. InHBcAb-negative, HBsAb-positive, HBsAb-positive patientsclose clinical and serological monitoring for HBV reactiva-tion has been recommended. It has been proposed thatmonitoring levels of HBsAb titers with HBV DNA levelsmay provide early clues to HBV reactivation [65]. WhenHBsAb titers fall, HBV DNA levels can rise and so willthe risk of HBV reactivation [65]. The authors of one reviewrecommended starting lamivudine when HBsAb titersdeclined to or started below 300 mIU/ml with close

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monitoring of trends in HBsAb titers and HBV DNA levelsfor titers that were otherwise stable, although they did notethat cases existed where HBV reactivation occurred evenwith high (over 1000 mIU/ml) HBsAb titers [65]. Whenlamivudine therapy is started, in the absence of resistance,such therapy should be continued for 6 months after thelast cycle of chemotherapy [37,65].Two case reports have been published that describe HBV

reactivation in patients chronically infected with the viruswho were subsequently treated with rituximab for a rheuma-tologic illness. Both patients were HbsAg-positive [68,69].One patient had severe RA [68] and was treated with severalTNF-a antagonists sequentially as well as cyclosporine, abata-cept and steroids. During her treatment with TNF-a antago-nists she was found to have chronic HBV infection [68]. Shewas started on lamivudine and tolerated other disease-modifying agents with a low but detectable HBV DNAlevel [68]. One month after receiving rituximab the patienthad clinical manifestations of HBV reactivation with anincrease in AST/ALT and dramatic increase in HBV DNAlevels [68]. Tenofovir was added to lamivudine and one monthlater the patient’s elevated AST/ALT normalized [68]. Theother case was a patient with ankylosing spondylitis unableto tolerate multiple disease-modifying agents who later wasfound to be chronically infected with HBV [69]. Lamivudineprophylaxis was started 3 months prior to receiving rituxi-mab [69]. Despite significant improvement in her ankylosingspondylitis and no elevation in AST/ALT after starting treat-ment with rituximab she did have an asymptomatic increasein HBV DNA about 5 months after her dose was adminis-tered [69]. The 2008 practice guidelines from the ACR recom-mended against starting or resuming rituximab in patientschronically infected with HBV who were Child-Pugh classB or C regardless of whether or not they were receiving anti-viral therapy, the same contraindication given for abateceptand the TNF-a antagonists [38].

2.5 Anti-CD80/86 therapyOne of the bridges that links innate immunity with adaptiveimmunity is the stimulation of T-cells by APC. T-cell stim-ulation requires two signals from the APC. The first signaloccurs when the antigen processed by the APC is presentedin a MHC molecule, which interacts with a T-cell receptorthat recognizes the antigen. The second signal is a costimu-latory signal. The best characterized costimulatory signalinvolves the interaction between the CD80 (B7-1) andCD86 (B7-2) molecules on the APC and the CD28 mole-cule on the T-cell [70]. Expression of CD80 and CD86 isenhanced by the presence of various microbes as well as bythe cytokines released in response to microbial invasion.Most CD4+ T-cells and about half of CD8+ T-cells constitu-tively express CD28. Signals working through CD28 canenhance the production of multiple cytokines (specificallyIL-2), promote energy metabolism, and turn on cell cycleprogression [70]. The development and survival of regulatory

T-cells (Treg), cells with the critical role of inhibitingimmune responses, have also been linked to CD80/CD86and CD28 signaling. Cytotoxic T-lymphocyte antigen-4(CTLA-4) is a regulatory protein similar to CD28 in struc-ture which undergoes inducible production after T-cell acti-vation [70]. It exists in both a membrane-bound and solubleform, with the latter enabling the molecule to exert effectsbeyond direct cell-to-cell interaction. CTLA-4 extinguishesT-cell responses by inhibiting production of IL-2 and arrest-ing cell cycle progression. It is constitutively expressed onTreg cells [71]. Abatacept is a soluble fusion protein whichlinks the CTLA-4 extracellular domain to the Fc region ofIgG molecule. Through the inhibition of the costimulatorysignaling of T-cells in RA, abatacept has demonstratedclinical efficacy in multiple trials [12,70].

Recent studies have demonstrated that some CTLA-4polymorphisms influence the ability of the immuneresponse to clear HBV infection [71]. A study of severalhundred Caucasian and African American patients in theUSA linked the presence of the +49G polymorphism ofCTLA-4 to an increased chance of HBV clearance [71]. Ithas been proposed that this outcome is attributable to thealtered polarity of the CTLA-4 molecule (as comparedwith that produced by the +49A polymorphism) [71]. Addi-tionally the +49G polymorphism has been associated withT-cells that express less membrane-bound CTLA-4, prolif-erate under conditions of suboptimal activation, and areless responsive to CTLA-4 directed inhibition [71]. Theseeffects of the +49G polymorphism were noted regardlessof the -1722C polymorphism, a polymorphism associatedwith decreased CTLA-4 production due to altered transcrip-tional activity in the gene promoter region [71]. In this samestudy the presence of the +6230A polymorphism was associ-ated with HBV persistence, an outcome ascribed todecreased T-cell responsiveness related to levels of solubleCTLA-4 [71]. A similar relationship between these CTLA-4polymorphisms and outcome of HBV infection was notedin an Iranian study of 51 patients chronically infectedwith HBV [72]. Beyond the effect on clearance of HBVthe +49G polymorphism of CTLA-4 has been shownin males from a Han Chinese population to have a protec-tive effect against the development of hepatocellularcarcinoma [73].

To date no case reports have been published of reactiva-tion of HBV infection in patients receiving abatacept. Themanufacturer of abatacept has not received any reports ofcases of HBV reactivation in patients receiving the medica-tion [74]. A recent therapy and safety management publica-tion recommended screening for HBV prior to startingabatacept as the safety of using abatacept in such a patienthas not been established [75]. As noted earlier the ACR in2008 stated that it was contraindicated to start or resumeabatacept in patients chronically infected with HBV withChild-Pugh Class B or C regardless of whether concomitantantiviral prophylaxis was given [38].



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3. Expert opinion

The role each of the biologic response modifiers has on therisk of HBV reactivation in chronic infection varies and insome circumstances remains to be determined. A summaryof the aforementioned known risk of HBV reactivationbased on HBsAg status in patients concomitantly treatedwith biologic response modifiers is summarized in Table 1.Integrating expert opinions, societal practice guidelines,data from clinical trials and known pathophysiology itwould be reasonable to refer all patients with known HBsAgpositivity or detectable levels of HBV DNA to a hepatologistor infectious disease specialist [39] for further evaluation withserious consideration of starting antiviral therapy beforetreatment with a biologic response modifier. Initiation ofantiviral therapy at least 1 week before treatment with a bio-logic response modifier is prudent as data from a random-ized study evaluating HBsAg positive lymphoma patientstreated with chemotherapy demonstrated lower HBV reacti-vation rates, lower rates of hepatitis, and longer survival inthe group treated with lamivudine a week prior to chemo-therapy [36,37,76]. Concomitant glucocorticoid use in patientschronically infected with HBV on a biologic response mod-ifier should be done cautiously as oncology experience withrituximab suggest this predisposes to an increased risk ofvirus reactivation and fosters antiviral resistance [65,77]. If apatient who is actively being treated with a biologic responsemodifier develops clinical or serologic evidence of HBVinfection, prompt consultation with a hepatologist or infec-tious disease expert and institution of an antiviral agentappropriate for detected resistance patterns and HBVDNA levels should be instituted. Therapy with the bio-logic response modifier should be held until clinicalimprovement, a decrease in HBV DNA, improvement in

ALT and durable compliance with antiviral therapy can bedemonstrated. It is unclear as to how long this period shouldbe. Prompt institution of antiviral therapy appears needed tominimize the effects generated by an immune system thatreconstitutes towards the end of the last dose of intrave-nously administered biologic response modifiers such asinfliximab and rituximab [18,65,76] Patients that do not haveclinical or serological evidence of HBV reactivation should,in the opinion of the author, have monitoring performedonce every 4 -- 8 weeks. Serologic monitoring should at aminimum include an ALT, HBV DNA levels, and HBsAbtiter. The development of resistance to antiviral therapyshould be considered in a previously stable patient on a bio-logic response modifier and a nucleot(s)ide analogue whopresents with clinical hepatitis or serologic evidence ofHBV reactivation. Upon cessation of a biologic responsemodifier antiviral therapy should be continued for at least6 months as reconstitution of the immune system mayoccur, an issue more likely to arise after treatment withinfliximab or rituximab [18,65,76]. The practice guidelinesfrom the American Association for the Study of Liver Dis-eases recommend continuing antiviral therapy for 6 monthsafter cessation of immunosuppression [77] whereas guidelinesfrom the European Association for the Study of the Liverrecommend a 12 month course [4]. A summary of theserecommendations is provided in Table 2.

For patients that are HbsAg-negative but HbcAb-positivewithout detectable HBV DNA in the serum it appears rea-sonably safe to start a biologic response modifier withoutthe prior initiation of antiviral therapy, with the exceptionof rituximab. Treatment without concomitant antiviraltherapy may not be appropriate when receiving rituximab,as HBV reactivation in lymphoma patients treated withrituximab and chemotherapy has resulted in a refractory

Table 1. Risk of HBV reactivation in patients chronically infected with HBV treated with biologic response

modifiers for a rheumatologic disease*.

Type of biologic

response modifier

Medications HBsAg+

patients

HBsAg- and

HBcAb+ patients

TNF-a antagonists infliximabetanerceptadalimumabcertolizumabgolimumab

Risk of reactivation exists(aggregate data from 35case reports [18]).

Little to no risk of reactivation(case reports, retrospective caseseries, and prospective case series [40-45])Increased risk associatedwith lower HBsAb titers [41,45].

IL-1 receptor antagonist anakinra No information No informationIL-6 receptor antagonist tocilizumab No cases of reactivation (two case

reports [60,61]).No information

Anti-CD20 therapy rituximab 20 -- 55% prevalence reported(data from oncology literature [65,67]

and two case reports in rheumatologicalliterature [68,69]).

Rare to 3% prevalence reported (data fromoncology literature [65]). Increased riskassociated with lower HBsAb titers [65].

Anti-CD80/86 therapy abatacept No information No information

*In the absence of antiviral therapy

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hepatitis with a 38% mortality rate [65]. The risk of HBVreactivation when treated with a biologic response modifiermay also depend on the HBsAb levels. Patients with persis-tently low HBsAb levels treated with TNF-a antagonists orrituximab may have an elevated risk of future HBV reactiva-tion. Consultation with a hepatologist or infectious diseaseexpert before starting a biologic response modifier in aHBsAg-negative but HbcAb-positive patients would beprudent [39]. Again, in the opinion of the author, clinicaland serologic monitoring for HBV reactivation should beperformed every 4 -- 8 weeks after biologic response modi-fier therapy is started. These recommendations are againsummarized in Table 2.

While all of the currently available literature links clinicaland/or serological reactivation as a possible outcome inpatients chronically infected with HBV treated with a bio-logic response modifier without antiviral therapy, it shouldbe considered that some medications in this class may inthe future demonstrate a beneficial effect on controlling theinflammation generated by the host immune response. As anexample, though the long-term effects remain unknown,short (less than 1 year) courses of TNF-a antagonists arereasonably well tolerated and even a potential treatmentadjunct in patients infected with HCV [39]. No significantchanges in HCV RNA or increases in aminotransferasesare typically observed in patients with chronic HCV

Table 2. Recommendations and their rationale for implementing a biologic response modifier (BRM) in patients

chronically infected with HBV

Issue Recommendation Rationale

Prior to start of BRM . Screen all patients at high risk for HBVinfection prior to immunosuppression [77]

. Recommendation made by the AASLD based onmultiple time series and uncontrolled studies [77]

. Check HBsAg and HBsAb and considervaccination if seronegative [77]

. Recommendation made by the AASLD basedon randomized controlled trials [77]

. Seek consultation with a hepatologist orinfectious disease specialist for patients thathave chronic HBV infection

. Recommendation to seek consultation is basedon expert opinion [39]

Preventing reactivationupon initiation of BRM

. HBsAg (+) patient–start antiviral therapyappropriate for detected resistance patternsand HBV DNA level at least 1 week prior toinitiation of BRM. Review risk and benefits of using rituximabin this group of patients

. Recommendation to start antiviral therapy1 week prior to initiation of biologic is basedon a randomized trial [18,36,37,76]

. Case report data from rheumatology literaturesuggests HBV reactivation may still occur inHBsAg (+) patients treated with rituximab onantiviral therapy [68,69]

. HBsAg (-) but HBcAb (+) patients withoutdetectable HBV DNA----initiation of BRM appearsreasonably safe but:. Review risk and benefits of using rituximabin this group of patients

. Check HBsAb titers

. HBV reactivation in HBsAg (-) but HBcAb (+)patients treated with rituximab have experienceda refractory hepatitis with a 38% mortality rate. [65]

. HBsAg (-) but HBcAb (+) patients with low titers ofHBsAb prior to start of TNF-a antagonist or rituximabappear at higher risk of HBV reactivation [41,45,65]

Monitoring forreactivationduring treatmentwith BRM

Check ALT, HBV DNA, and HBsAb titers every4 -- 8 weeks regardless of whether patient istaking antiviral therapy

Recommendation is based on the opinion of theauthor

Development ofreactivationduring treatmentwith BRM

. Stop BRM and seek consultation with ahepatologist or infectious disease expert. Initiate or change antiviral therapy to mitigaterisk of immune reconstitution as BRM is cleared. Minimize patient exposure to glucocorticoidsas risk of reactivation and antiviral resistanceincreases (data from patients treated withrituximab) [65,77]

Development of antiviral resistance should be consideredin a previously stable patient who presents with clinicalhepatitis or serologic evidence of HBV reactivation

Cessation of BRM . Patient on antiviral therapy: Continue therapyfor 6 months after. Patient not on antiviral therapy: Consider closeclinical and serologic monitoring for HBVreactivation based on the dosing interval of theBRM

. Immune reconstitution as BRM is cleared maytrigger HBV reactivation [18]

. The AASLD recommends continuing antiviraltherapy for 6 months after immunosuppresionends; the EASL recommends continuing antiviraltherapy for 12 months after immunosuppressionends [4,77]

AASLD: American Association for the Study of Liver Diseases; HBsAg: Hepatitis B surface antigen; HBsAb: Hepatitis B surface antibody; HBcAb: Hepatitis B core

antibody; ALT: Alanine aminotransferase; EASL: European Association for the Study of the Liver.



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infection exposed to TNF-a antagonists [39]. Clinical experi-ence with rituximab in treating patients with HCV-relatedcryoglobulinemic vasculitis is growing and thus far appearsto be reasonably efficacious without significant hepatic toxi-city [39]. Drawing on known immunologic mechanisms thecostimulatory molecule antagonist CTLA-4 is known todampen the host immune response to an immunologicalchallenge by inducing and maintaining T-cell tolerance [70].In the context of chronic HBV infection CTLA-4 could playa role in attenuating the inflammation generated duringthe host response to viral reactivation. Abatacept, as aCTLA-4 fusion molecule, may thus have a tolerable oreven have a beneficial effect on the host immune res-ponse during HBV reactivation. The demonstration of abenefit when biologic response modifiers modulate thehost immune response in patients chronically infected withHBV awaits further study.

3.1 ConclusionInfection with HBV remains a significant public health issueworldwide. The same host immune system that may success-fully fight and eradicate infection with HBV also participatesin the pathogenesis of chronic inflammatory rheumatologi-cal diseases. Biologic response modifiers are a diverse groupof medications currently at the disposal of rheumatologiststo control rheumatological diseases such as RA and the sero-negative spondyloarthropathies. The effect that these medi-cations have on the host control of HBV infection whentreated for a rheumatological illness is an evolving area ofinterest. Integrating case reports and case series, expert rec-ommendations and societal consensus statements patientswith clinical hepatitis or serological evidence consistent

with HBV reactivation (with elevated AST/ALT, HbsAg-positive, or with detectable HBV DNA) need to defer orstop treatment with a biologic response modifier, seek inputfrom a hepatologist or infectious disease expert and startantiviral therapy appropriate for detected resistance patternsand serum viral load. Antiviral therapy should continueduring biologic response modifier treatment and continuefor 6 months after the treatment has been stopped. Thiswill mitigate the risk of an immune reconstitution response,which can occur off the biologic response modifier. Forpatients who are HBsAg-negative but HBcAb-positive therisk of HBV reactivation while treated with a biologicresponse modifier appears fairly low, although it appearsdependent on the medication used. Consultation with ahepatologist or infectious disease expert prior to the initia-tion of a biologic response modifier and close clinical andserologic monitoring would be prudent.

Acknowledgements

I am indebted to Jennifer Carroll for her critical review of thismanuscript and dedicate this to my daughter Abigail.


The author has no financial support or other benefits fromcommercial sources to disclose. The author has received nopharmaceutical or industry support in writing this manu-script. The author wishes to state that the views expressed inthis article are his and do not reflect the official policy or posi-tion of the United States Air Force, Department of Defense orthe US Government.

Carroll


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AffiliationMatthew B Carroll MD FACP FACR

301 Fisher Avenue,

Keesler AFB,

MS 39534, USA

Tel: +1 228 376 3629; Fax: +1 228 376 0105;




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1. Introduction

2. Overview of the market

3. Chemistry and preparation of

the TNF-kinoid

4. Pharmacodynamics

5. Pharmacokinetics

6. Effects in animal models

7. Safety and tolerability

8. Conclusions

9. Expert opinion

Drug Evaluation

Kinoid of human tumor necrosisfactor-alpha for rheumatoidarthritisLuca Semerano, Eric Assier, Laure Delavallee & Marie-Christophe Boissier††University of Paris-13, Sorbonne Paris-Cite, EA4222, Li2P, 74 rue Marcel Cachin, Bobigny,

France

Introduction: Anti-TNF-a drugs have dramatically changed treatment of rheu-

matoid arthritis (RA) in terms of both clinical control and articular damage

prevention. Despite this, they hold some important drawbacks, such as fre-

quent therapeutic failures and high costs. Anti-TNF-a active immunization,

with a therapeutic vaccine against TNF-a, is a promising alternative anti-

TNF-a targeting strategy, potentially devoid of treatment limitations of

some of current anti-TNF blocking agents.

Areas covered: This review covers the preclinical proof-of-concept of anti-

TNF-a vaccination with the kinoid of human TNF-a (TNFK) and analyzes the

body of evidence forming the rationale for the application of this strategy

in RA and other TNF-a-dependent diseases. We describe the theoretical bases

of anti-TNF-a active immunization and of experimental data supporting the

applicability of TNFK to human disease in terms of both safety and efficacy.

Expert opinion: Based on preclinical efficacy and safety data supporting its

feasibility in a Phase I -- II trial in Crohn’s disease, anti-TNF-a vaccination

with TNFK has entered the phase of clinical development and promises to

be a valuable anti-TNF-a targeting strategy in human disease. The focus is

made in the first clinical trial in RA (Phase II) on the efficacy in active RA

patients having developed antibodies against anti-TNF mAbs.

Keywords: anti-cytokine vaccination, anti-TNF-a, kinoid, rheumatoid arthritis, TNFK


1. Introduction

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic diseasewith a prevalence ranging from 0.3 to 1.5% in different populations [1]. It is char-acterized by an invasive synovial proliferation that leads to joint damage with painand loss of function, with precocious disability [2]. RA patients have associatedco-morbidities leading to a mortality estimated at almost twofold that of generalpopulation [3]. RA is, therefore, a huge public health problem resulting in highdirect and indirect costs for the community [4].

2. Overview of the market

TNF-a-targeting agents brought a revolution in the treatment of RA, providingunheard of results in terms of disease clinical control and prevention of RA struc-tural damage and consequent disability. TNF-a can be targeted with mAbs or theirfragments (infliximab (IFX), adalimumab, golimumab, certolizumab) or withfusion products carrying a TNF-a soluble receptor (etanercept). Anti-TNF-a drugsfirst opened the perspective of a successful cytokine-targeting strategy in RA. Sales ofthe four anti-TNF-a agents on the market in 2008 (adalimumab, IFX, etanerceptand certolizumab pegol) reached $16 billions. By 2014, analysts forecast the entire


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class of anti-TNF drugs to generate a $25 billion market, withgrowth driven by new entrants and continuing demand forthe incumbents (source: EvaluatePharma�) [5]. In 2008,TNF-a inhibitors accounted for 80% of RA drug sales inthe US, France, Germany, Italy, Spain, the UK and Japan(source: Pharmacor�) [6] within a market that, for all bio-logical therapies for RA, was estimated at $7 billion in2007 (source: Datamonitor� Research Store) [7].Current TNF-a targeting strategies have nevertheless

shown several drawbacks as far as safety, efficacy and costsare concerned. Despite the good safety/efficacy profile inselected patients, the overall risk of infection and possiblyneoplasm is increased in RA patients treated with anti-TNF-a mAbs compared to classic DMARDs [8]. Primaryand secondary failures are not infrequent; moreover, < 50%of responder patients in clinical trials attained disease remis-sion [9]. The treatment with anti-TNF blocking agents hashigh costs for the community [10]. While some of these draw-backs such as the increased risk of infection and neoplasm arepresumably related to the blockade of TNF-a itself, others,such as the high production costs, and the risk of anti-drug antibody (ADA) production with possible loss of efficacyand side effects, are proper to current anti-TNF-a agents,especially mAbs [11], and might be possibly overcome byalternative anti-TNF strategies.An alternative way to target TNF-a is active immunization,

where a TNF-a derivative can be used as the immunogen todevelop an anti-TNF-a active immunotherapy consisting ina vaccine [12]. The immunogen must be capable of disruptingB cell, but not T cell, tolerance to TNF-a, thereby elicitingthe production of high titers neutralizing antibodies [13].This strategy allows the production of polyclonal autologousanti-TNF-a antibodies potentially bypassing the risk of ananti xeno- or allogenic antibody response. Refining of ADAdetection techniques allowed in fact detecting ADA in up to40 and 30% of IFX and adalimumab treated patients, respec-tively [11]. The presence of ADA is associated with low troughdrug levels, infusion-related reactions (for IFX) and therapeu-tic failure [14]. Active immunization offers then the possibilityof overcoming this limitation.

The direct costs for anti-TNF blocking agents, togetherwith the costs of drug administration, monitoring and sideeffect management, result in a heavy economical burden forthe community [15], while the active immunization strategymight potentially be a less expensive alternative. Finally, thelonger persistence of detectable anti-TNF-a antibody titersinduced by active anti-TNF-a immunization draws a lesscumbersome administration scenario for the patient, withpossibly higher treatment acceptance.

3. Chemistry and preparation ofthe TNF-kinoid

The preclinical proof-of-concept of active anti-TNF-a immu-nization with a compound called kinoid of human TNF-a(TNFK) has been established in a TNF-a-dependent animalmodel, the human TNF-a (hTNF-a) transgenic mice(TTG mice) [13,16,17] (Box 1). This has led to subsequent test-ing of TNFK in a Phase I clinical trial in Crohn’s disease.A Phase II clinical trial in previously anti-TNF-a treatedRA patients having developed ADA is currently ongoing.

TNFK belongs to a family of cytokine derivatives capableof acting as anti-cytokine vaccines called ‘kinoids’ [18]. Theirname and preparation recalls those of the toxoids, detoxicatedbut still immunogenic products, derived from bacterial toxinsby formalin treatment at 37�C for several days. At thebeginning of the 1980s, a detoxication procedure usingglutaraldehyde instead of formaldehyde was described forthe preparation of fully atoxic polymerized antigens withhigh immunogenicity [19]. This technology with either glutar-aldehyde or formaldehyde was then applied to cytokines inorder to convert them into derivatives devoid of biologicalactivity but capable, when administered in animals, of induc-ing anti-cytokine antibodies. These derivatives were calledkinoids [20]. TNFK is a heterocomplex of inactivated hTNF-aand a carrier, the keyhole limpet hemocyanin (KLH).

KLH is a heterogeneous copper-containing respiratory pro-tein isolated from the mollusk Megathura crenulata belongingto a group of non-heme proteins called hemocyanins. It con-sists of two subunits isoforms with a molecular mass of 390 �103 and 360 � 103 D, originating, respectively, two differentoligomeric aggregates, KLH1 and KLH2. The molecular massof the oligomers ranges from 4,500,000 to 13,000,000. Dueto its large size and its numerous epitopes KLH is capable ofinducing a substantial immune response; its abundance oflysine residues for haptens coupling, with a high hapten:carrier protein ratio, increases the likelihood of generatinghapten-specific antibodies [21].

For preparing the heterocomplex, glutaraldehyde is used tocouple hTNF-a to the KLH carrier protein. KLH, and thenglutaraldehyde, are added to a solution of hTNF-a treatedwith dimethylsulfoxide, in a mixture of 1 molecule of KLHand 40 molecules of hTNF-a. After 45 min incubation at4�C, the preparation is dialyzed against the working bufferand then treated with formaldehyde for 6 days at 37�C.

Box 1. Drug summary.

Drug name Kinoid of human TNF-aPhase Phase II clinical trial, pre-registrationIndication Rheumatoid arthritisPharmacology Active immunization (vaccination)

against the pro-inflammatorycytokine TNF-a

Route ofadministration

Intramuscular

Pivotal trials TNFK001 (http://clinicaltrials.gov/ct2/show/NCT00808262)TNFK003 (http://www.controlled-trials.com/mrct/trial/772671/TNFK003)

Kinoid of human TNF-a


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Concentration and duration of aldehyde treatments have beenadapted for hTNF-a in order to obtain a strong and persis-tent inactivation of its biological activity. The unreactedaldehyde is quenched by addition of glycine (0.1 M), leadingto complex stabilization. The excess aldehyde is eliminatedby dialysis against Dulbecco’s phosphate buffer solution(PBS) [13].

4. Pharmacodynamics

It is assumed that TNFK is a heterocomplex in which KLHprovides T epitopes and bears at its surface a high density ofhTNF-a preserved B-epitopes. The aim of carrier proteins isto promote carrier-specific T-cell help to a B-cell polyclonalresponse [21]. Given that a high number of hTNF-amoleculesare covalently bound to KLH, kinoid immunocomplexes willpresent a high density of hTNF-a antigens in their nativeconformation to the antibody-producing B cells to crosslinkspecific B-cell receptors [18].

TTG mouse, expressing hTNF-a as a self antigen, is theonly relevant model to study TNF-induced anti-hTNF-aantibody production [13]. In all immunized mice in differentstudy protocols, immunization with TNFK induced specificanti-hTNF-a antibodies as detected by ELISA [13,16,17]. In aprotocol where mice received three injections of TNFK atdays 0, 7 and 28, these antibodies, tested at day 122 afterTNFK first injection, appeared to belong mainly toIgG1 (52%) and IgG2a (48%), with negligible amounts ofIgG3, IgM and IgE [13]. Purified IgG from hyperimmunesera exhibited a high affinity for hTNF-a with Kd valuesranging from 5 � 10-8 to 10-10 M and were able to blockits interaction with the high affinity TNFRI (Kd of0.6 nM) [22], resulting in undetectable circulating hTNF-ain immunized mice.

Anti-hTNF-a antibodies have a neutralizing anti-TNF-aeffect as confirmed both in vitro by L929 cytotoxicity assay,showing cytotoxicity inhibition by hyperimmune sera at dilu-tions up to 10-4, and in vivo, where purified IgG from sera ofimmunized mice prevented TNF-a-galactosamine lethalshock in recipient mice [13].

5. Pharmacokinetics

TNFK is mixed at a 1:1 ratio with the PBS and administeredintramuscularly with the adjuvant ISA51� (Seppic, France).The latter is similar to Freund’s incomplete adjuvant and iscomposed of a mix of mineral oil and a surfactant of themono-oleate family; it is currently used in immunotherapyof cancer and infectious diseases [23]. ISA51 is used in a1:1 ratio with the mix TNFK--PBS to obtain a water-in-oilemulsion [18].

Different administration schedules have been tested inmice, involving two (at days 0 and 7), three (at days 0,7 and 28) or four injections with dose regimens varyingfrom 5 to 30 µg of TNFK [13,16,17].

Whatever the exact administration schedule, all immuniza-tion protocols were able to induce anti-hTNF-a antibodies inTTG mice. In a three injection scheme (30 + 30 + 7 µg at days0, 7 and 28), anti hTNF-a antibodies were detectable at firstbleeding as soon as 5 weeks after TNFK first injection [16];they peaked at 6 -- 8 weeks after first injection [13], witha > 50% decline within 16 weeks.

In a protocol with three injections of TNFK 4 µg at days 0,7 and 28, a TNFK boost given 12 weeks after the TNFKfirst injection induced a significant increase in neutralizinganti-hTNF-a antibodies as soon as 3 weeks after the boost [17].

TNFK was first administered in humans in a Phase I -- IIopen-label dose escalation study on 13 patients with moderateto active Crohn’s disease, the TNFK001 study (http://clinicaltrials.gov/ct2/show/NCT00808262). The administra-tion schedule consisted of three injections of TNFK at days0, 7 and 28 at doses of 60, 180 and 360 µg. Four patientsreceived a fourth boost dose at 6 months. In all immunizedpatients, anti-TNF-a antibodies were detected, with a peakin titers between the fourth and the fifth week after firstTNFK injection, and a 50% reduction within 12 weeks.The boost at 6 month resulted in a new peak in antibody titers3 -- 4 weeks later [24].

As far as RA is concerned, a dose-finding Phase II clinicaltrial is currently ongoing in RA patients previously treatedwith anti-TNF agents having developed ADA. The primarygoal of this trial is to demonstrate that active immunizationwith TNFK is able to induce polyclonal anti-TNF-a antibod-ies in RA patients previously treated with anti-TNF-a mAbswho underwent a secondary therapeutic failure (i.e., loss ofclinical response) and have developed ADA. Among the inclu-sion criteria of these patients having an active RA is the posi-tivity of antibodies against a TNF antagonist at screening oron a sample taken since discontinuation of IFX and/or adali-mumab (http://www.controlled-trials.com/mrct/trial/772671/TNFK003).

6. Effects in animal models

TNFK immunization has proven its efficacy in the spontane-ous arthritis of TTG mice thereby posing the rationale for itsuse in RA.

When given before arthritis development, TNFK markedlyreduced the clinical severity of arthritis and resulted in lesshistological joint inflammation and destruction compared tocontrol mice [13,16].

In an experimental three injection protocol (days 0, 7 and28), a highly significant difference in clinical and histologicalscore was already evident when animals were sacrificed 6 weeksafter the first injection, compared to controls. TNFK-immunized animals showed mild histological inflammationand no histological destruction. The co-administration ofmethotrexate did not change the results [16].

When, with the same experimental protocol, theobservation was prolonged up to 17 weeks, arthritis onset

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happened to be delayed by 9 weeks compared to controls andstill low clinical and histological scores were found inimmunized mice.The therapeutic efficacy, its duration and the effect of a

TNFK boost were better evaluated in a subsequent experi-ment more resembling to a human disease scenario, as TTGmice were immunized after spontaneous arthritis onset [17].In 12 weeks follow-up after TNFK immunization, arthritiswas dramatically ameliorated, and clinical scores did notdiffer from those of mice treated with weekly IFX at a doseof 1 mg/kg over the same time period. These findings werecorroborated by histology, showing low inflammation andno sign of cartilage destruction in immunized animals.The observation was prolonged to 30 weeks after TNFK

first injection in order to study the duration of clinical effectand the kinetics of TNFK-induced anti-hTNF-a antibodies.After the initial amelioration, arthritis clinical score inimmunized mice started to increase from week 12 after firstinjection to the end of the experiment. This trend wasreversed by a TNFK boost given at week 12, before clinicaldegradation ensued. The worsening in clinical control ofarthritis coincided with a decrease in anti-hTNF-a antibodytiters, while the TNFK boost triggered a significant increasein antibody titers 3 weeks after its administration. Mildhistological scores of joint inflammation, destruction andcartilage degradation at the end of the experiment confir-med the long-term prevention of structural damage ofTNFK immunization.

7. Safety and tolerability

Some major safety issues are raised by the novel anti-TNF-aapproach of active immunotherapy, namely:

i) The delivered TNF-a must be devoid of toxicitybut still be immunogenic, and this is the case ofthe TNFK heterocomplex, where aldehyde treat-ment results in a hTNF-a derivative satisfying theserequirements. In all experiments conducted withTNFK, no short-term toxicity linked to its adminis-tration and ascribable to hTNF-a activity-relatedtoxicity was detected [13,16,17]. This was the caseeven in the limited experience in humans.

ii) The anti-TNF-a vaccination must result in ruptureof B-cell but not of T-cell tolerance (i.e., vaccinationmust not induce memory, T cells capable of recog-nizing the native cytokine). In fact, the persistenceof a T-cell population sensitized against a self-cytokine would result in a localized cellular responsein its site of production.

iii) This issue was addressed in an animal study where6 -- 8 weeks old TTG mice received three injectionsof TNFK (days 0, 7, 28 ± a boost at day 90) andwere followed up for 120 days after the firstinjection. Our group showed that the splenocytes

from TNFK-immunized TTG mice did nottrigger any cell-mediated immune response to selfhTNF-a, as tested by T-cell proliferation andIL-2 and IFN-g production in culture supernatants,whatever the administration regimen of TNFK [13].The only detectable cellular response was againstKLH. Conversely in Balb/C mice, a TNFK-inducedanti-hTNF-a cellular response was detected whenhTNF-a (a heterologous antigen for this strain)was administered.

iv) In TNFK001 study in Crohn’s disease patients,stimulation of PBMCs of immunized patients withTNF-a failed to induce proliferation.

v) The rupture of B-cell tolerance must be reversible.Our group demonstrated that when TTG micewere immunized with TNFK before spontaneousarthritis appearance, anti-hTNF-a antibodiespeaked 6 -- 8 weeks after TNFK first injection andhad a > 50% antibody titers decline within12 -- 16 weeks. This kinetics is ascribable to shortlife of B-cell memory in the absence of a specificT-cell help [13]. A long-term study where immu-nized TTG mice were monitored up to 30 weeksafter TNFK first injection immunization confirmedthe same results [17].

vi) A similar kinetics, albeit with the limitation of studydesign and sample size, seems to be confirmed inhumans, based on the results of TNFK001 study.In the 13 immunized patients anti-TNF-a antibodytiters were markedly reduced, and sometimes nolonger detectable, within 12 -- 15 weeks afterfirst injection.

vii) A raise in the levels of TNF-a induced by otherstimuli (infections, tumors) must not elicit theproduction of anti-TNF antibodies after TNFKimmunization. This was demonstrated in a studywhere monthly administration of hTNF-a to TTGmice failed to induce any raise of anti-hTNF-aantibodies [17].

viii) Ideally, the ‘physiological’ activity of hTNF-a innormal tissues should be conserved (see points ii,iii and iv).

8. Conclusions

An important preclinical body of evidence (not inferior tothat which first led to test a monoclonal anti-TNF-a antibodyin 10 RA patients in 1992) supports the feasibility of anti-TNF-a active immunization in TNF-a-dependent humandiseases. The efficacy in TTG mice spontaneous arthritis,the relevant model for TNF-a inhibition, strongly suggestsits potential application in RA. The reversibility of anti-TNF-a antibody levels increase and the absence of memoryT-cells induction are both arguments in favor of a good safetyprofile. The first results of an open-label study in Crohn’s



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disease are consistent with animal data regarding the kineticsof antibodies induction and decrease, and a good toleranceis suggested. A dose-finding randomized trial, ongoing atthe present time in RA, will presumably provide more rele-vant safety and efficacy information determining whether ornot TNFK will access the Phase III of clinical development.

9. Expert opinion

We are presently at an early phase of clinical development,as Phase II studies are ongoing in RA and Phase I -- II inCrohn’s is not ended, yet. The expert opinion is consequentlybased on proof-of-concepts experiments in preclinical andpharmacodynamics studies in mice that allow formulatingsome hypotheses.

The active immunotherapy with TNFK aims to reversiblyvaccinate against TNF-a. Unlike the already marketed anti-TNF-a agents, one can suppose that using TNFK couldhave advantages in terms of simplicity and frequency of injec-tions. The effect would probably be quite durable after eachinjection (several weeks or months). Moreover, TNFK treat-ment is not concerned by a possible reduction of effect dueto ADA. These antibodies, found in up to 40% of IFX-treated and in 30% of adalimumab-treated patients, reducethe therapeutic efficacy of the drugs and are responsible oftherapeutic failures and adverse reactions. So, the ADA-positive patient might be a specific clinical situation in whichTNFK administration could be warranted.

Another advantage is a lower economic burden for thecommunity as the costs of production of the kinoid wouldbe presumably lower than those of current anti-TNF-aagents. Cost reductions are currently requested in developedcountries and appear as a necessary condition for treatingTNF-a-dependent diseases with targeted treatments indeveloping countries. The access for the patients to expensive

biological therapies is strongly limited in many countries byhealth authorities or other third party payers, and the choiceof treatment will be more and more influenced by cost-effectiveness analyses. In this scenario, a less expensive alterna-tive providing ‘value’ and ‘value for money’ in RA treatmentwould certainly be welcomed.

If the safety and efficacy data suggested by animal modelsare confirmed by ongoing human clinical studies, it is con-ceivable that TNFK will have a considerable impact on RAtreatment strategies. Not only TNFK promises to be a directcompetitor of passive anti-TNF-a immunotherapies, butalso future scenarios might be conceived, including combina-tion or sequential treatment with both passive and activeTNF-a-targeting strategies.

The reversibility of anti-TNF-a vaccination with TNFKand lack of induction of immunological memory versusthe native cytokine are the key conditions for a favorablebenefit:risk ratio. All preclinical studies show a bell curve ofanti-TNF-a antibodies levels and preliminary results inhumans confirm this point. The administration of TNF-ato TNFK-vaccinated animals fails to induce an anti-TNF-aresponse and, in addition, the persistence of residuallevels of active TNF-a is probably sufficient to protect thehost against infection and tumors. Nevertheless, all thesesafety considerations, based on animal models data, willhave to be confirmed in ongoing and future clinical trialsin humans.


M-C Boissier has been a consultant for Neovacs, Inc. and hislaboratory has received research grants from Neovacs, Pfizer,UCB Pharma and Roche. This manuscript was written with-out any interactions outside co-authors. The other authorsdeclare no conflict of interest.

Semerano, Assier, Delavallee & Boissier


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AffiliationLuca Semerano1,2, Eric Assier2,

Laure Delavallee2 &

Marie-Christophe Boissier†1,2

†Author for correspondence1Assistance Publique-Hopitaux de Paris (AP-HP),

Hopital Avicenne, Rheumatology Department,

125 rue de Stalingrad,

93000 Bobigny, France2University of Paris-13,

Sorbonne Paris-Cite, EA4222, Li2P,

74 rue Marcel Cachin,

93000 Bobigny, France




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expert opinion on biological therapy

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