Pediatr Blood Cancer 2004;43:110–114

Cerebrospinal Fluid Insulin-like Growth Factor (IGF-1) andInsulin-like Growth Factor Binding Protein (IGFBP-2) in

Children With Acute Lymphoblastic Leukemia

Raili Riikonen, MD, PhD,1* Kim Vettenranta, MD, PhD,2 Pekka Riikonen, MD, PhD,1 U. Turpeinen, PhD,3

and Ulla M. Saarinen-Pihkala, MD, PhD2

INTRODUCTION

Antileukemia chemotherapy is known to have adverseeffects on both the peripheral and central nervous systems,most of them being transient The background leading tothe damage is not fully understood.

Insulin-like growth factors, IGF-1 and IGF-2, aremembers of the insulin gene family that stimulate cellularproliferation and differentiation during fetal and postnataldevelopment. IGF-1 is a peptide that binds to the bindingproteins, IGFBP 1–6, in the plasma and CSF. The IGFBPscan modulate the biological actions of IGF-1 and IGF-2.The most important binding protein in the CSF is IGFBP-2.

There is overwhelming evidence today that the IGFshave a definite biological role within the CNS and in theperipheral nervous system. Within the IGF family, IGF-1is the most potent neurotrophic factor. IGF-1 has a specificeffect on axonal growth and myelination [1]; it promotesthe survival of neurons, oligodendrocytes, and pro-oligodendrocytes [2] by inhibiting apoptosis [3], andIGF-1 increases the levels of the myelin basic proteinmRNA and proteolipid in vitro [4–6]. The IGF receptorsand IGF binding proteins are widely distributed in the CNSwithin the neurons and glial cells, predominantly in thefrontal lobe, hippocampus, brain stem, and cerebellum[6–8]. Furthermore, it has been shown that IGF-1 geneknock-out mice have defective myelination in the wholebrain [1]. Consequently, lack of IGF-1 and relatedmolecules might be involved in neurodegenerative

processes also in humans. Low CSF IGF-1 levels havebeen found in some severe neurologic disease [9–11].

The purpose of this study was to explore the effect ofALL chemotherapy on the CSF levels of IGF-1 andIGFBP-2, and to find out whether changes in these levelswould translate in any neurological deficits observed inthese children.

SUBJECTS AND METHODS

Patients

Fourteen children with newly diagnosed ALL, aged3 months–14 years (mean 4 years), were admitted during2002 to the pediatric oncology units of the Children’sHospital, University of Kuopio, or the Hospital forChildren and Adolescents, University of Helsinki,

Background. Insulin-like growth factor-1(IGF-1) has specific effects on axonal growthand myelination, low CSF IGF-1 levels beingfound in some severe neurologic diseases. Westudied the levels of CSF IGF-1 and IGF bindingprotein-2 (IGFBP-2) in children with ALL to findout whether these levels correlated with any ofthe neurological deficits observed. Methods.IGF-1 and IGFBP-2 levels were prospectivelymeasured by radioimmunoassay in the CSF of14 children with ALL throughout the ALLchemotherapy. These were compared with thelevels of 16 control subjects and of patient

groups with severe neurological diseases.Results. During induction, the children withALL had subnormal CSF IGF-1 levels whichimproved after 2 months. In seven individuals,two with severe vincristine polyneuropathy, thesubnormal levels persisted throughout the che-motherapy. Conclusions. Our findings suggestimpairment of the IGF-1 trophic system duringinduction by a mechanism so far unknown. Cor-relation with disturbed neuronal function couldnot be statistically proven. Pediatr Blood Cancer2004;43:110–114. � 2004 Wiley-Liss, Inc.

Key words: ALL; CSF; insulin-like growth factors

——————1Children’s Hospital, University of Kuopio, Finland

2Hospital for Children and Adolescents, University of Helsinki,

Finland

3Laboratory, Helsinki University Central Hospital, Finland

Grant sponsor: This study was financially supported by the University

Hospitals of Kuopio and Helsinki.

*Correspondence to: Dr. Raili Riikonen, Professor of Child Neurology,

University of Kuopio, Department of Child Neurology, P.O. Box 1627,

FIN-70211 Kuopio, Finland. E-mail: Raili.Riikonen@kuh.fi

Received 4 September 2003; Accepted 25 March 2004

� 2004 Wiley-Liss, Inc.DOI 10.1002/pbc.20072

Finland. They were followed up longitudinally with intotal 45 samples from diagnosis to maintenance. Inaddition to this follow-up cohort, sporadic samples froman additional 14 patients were used as a cross-sectionalpilot group.

The antileukemia therapy was given according to theNordic NOPHO-ALL protocol and stratified by riskfactors in standard therapy, intermediate therapy, andintensive therapy [12]. There was a four-drug inductiontherapy including prednisolone, vincristine, doxorubicine,and L-asparaginase plus four intrathecal doses of metho-trexate. The second part of the induction consisted ofcyclophosphamide, low-dose cytarabine, and 6-mercap-topurine [12]. The main stay of consolidation therapy wasrepeated (eight or nine) courses of high-dose methotrex-ate, or in intensive therapy, high-dose methotrexate, andhigh-dose cytarabine alternating, plus a series of intrathe-cal methotrexate [12].

Cerebrospinal fluid sampling for this study wasperformed at times of the intrathecal medications, sche-duled for seven occasions during the course of ALLtherapy, before starting therapy, at 1–2 weeks, 30 days,50–60 days, and later during maintenance.

Control Subjects

‘‘Normal’’ control subjects without severe CNSdisease. Every patient had an age-matched control(n¼ 16) for IGF-1. For IGFBP-2, we used ten of thecontrols. The control subjects had other milder neurolo-gical diseases not involving the white matter or thecerebellum. The diagnoses of the 16 control subjects wereas follows: mental retardation of unknown cause (n¼ 5),epilepsy (n¼ 3), visual symptoms (n¼ 2), hydrocephalus(n¼ 1), psychogenic symptoms (n¼ 1), migraine (n¼ 1),acute fatigue (n¼ 1), facial palsy (n¼ 1), and hemiplegia(n¼ 1). None of the control subjects had malnutrition,endocrine disturbances, acute infection, gastroenteritis,liver disease, renal disease, postoperative state, oroncologic disease.

Control subjects with severe CNS disease. CSFsamples from patients with severe CNS diseases; most ofthem admitted to the Central Hospital, University ofHelsinki and studied by the same methods, were used forcomparison. This group included 8 patients with infantileceroid lipofuscinosis (INCL) aged 15–27 (mean 19)months [9], 11 patients with cerebellar atrophy aged 2–11(mean 5.1) years [10], 8 patients with progressiveencephalopathy, hypsarrhythmia, and optic atrophy(PEHO) syndrome aged 9 months–8.2 (mean 4.7) years[10], 7 patients with PEHO-like syndrome (similar clinicalfeatures, no cerebellar atrophy) aged 7 months–16 (mean5.2) years [10]), and 8 patients with Rett syndrome aged1.8–17.4 (mean 5.8) years [11]. The patients with PEHO-like syndrome and Rett syndrome, unlike patients with

INCL, cerebellar atrophy, and PEHO syndrome, do nothave any cerebellar atrophy or white matter disorder.

The study protocol was approved by the InstitutionalReview Boards of both the participating hospitals. Theappropriate consents were obtained.

Laboratory Methods

The CSF samples of the patients and control subjectswere taken through lumbar puncture as part of the clinicalevaluation or in association with intrathecal therapy. Thepatients with ALL were only included if they were not in asevere catabolic state, for example, sepsis. CSF samplescontaminated with macroscopic or microscopic bloodwere not accepted for analysis. When there was aquantitative limitation of sample volume, priority wasgiven to clinical purposes. Therefore, a sample leftoverwas not always available for this study as scheduled, andonly a few samples were available at the initial diagnosis.

Samples were kept frozen at �708C until analysis. TheIGF-1 and IGFBP-2 levels were determined by radio-immunoassay using commercially available kits (Med-iagnost, Tubingen, Germany) according to the instructionsof the manufacturer. The samples were analyzed induplicate. The inter-assay coefficient of variations (CV)of both assays were less than 10%. The sensitivity of theassay was 0.02 mg/L for IGF-1 and 1 mg/L for IGFBP-2.

Statistical Methods

In comparing CSF concentrations of IGF-1 and IGFBP-2 between the patients with ALL and the control subjects,Student’s two-tailed t-test was used. In testing ourhypothesis of lower IGF-1 levels during induction, withlater return to normal, Bonferroni correction was used toavoid the multiple comparison problem. Spearmancorrelations between CSF IGF-1 and IGFBP-2 concentra-tions were computed.

RESULTS

The concentration of CSF IGF-1 was 0.41� 0.27 mg/L(mean� SD) at diagnosis (n¼ 14), 0.32� 0.16 mg/Lduring the induction therapy (days 1–60) (n¼ 18), and0.47� 0.21 mg/L later during therapy from 2 months to2 years (n¼ 27). In comparing the levels before treatment,during induction and later, there was a significantdifference (P¼ 0.036; Bonferroni correction used) indi-cating a lower level during induction (Fig. 1). The levelsduring induction were significantly lower than the levels ofthe controls without severe CNS disease (P¼ 0.003).

In the 16 control patients without severe CNS disease,the CSF IGF-1 level was 0.49� 0.13 mg/L (mean� SD),which did not differ from the levels of the ALL patientstaken at diagnosis or later, post-induction (Fig. 1). Thelevels of patient groups with severe CNS disease are

CSF Insulin-Like Growth Factors in ALL 111

shown for comparison in Figure 1. The CSF concentration(mean� SD) of the INCL patients (n¼ 8) was0.24� 0.07 mg/L, of patients with cerebellar degeneration(n¼ 11) 0.19� 0.004 mg/L, of PEHO syndrome (n¼ 8)0.26� 0.12 mg/L, PEHO-like syndrome (n¼ 7) 0.51�0.15 mg/L, and of the patients with Rett syndrome0.46� 0.20 mg/L.

The mean CSF IGFBP-2 level of the control patientswas 180 mg/L, which did not differ from the levels of theALL patients at any stage of the therapy (at diagnosis,197 mg/L; during induction 192 mg/L; later post-induction219 mg/L. The CSF IGF-1 levels and IGFBP-2 levels didnot correlate with each other.

Among our follow-up cohort, there were seven childrenwho had continuously low CSF IGF-1 levels throughoutthe therapy (Fig. 2). No factor common to these ALLpatients could be found out as an explanation. Their ages atdiagnosis ranged from 3 months to 9 years (median3 years). High-risk cases were over-presented: four wereof high-risk, two of intermediate risk, and only one ofstandard risk ALL. None had or developed CNS-ALL, andnone had convulsions. The weights and heights of thesechildren remained at the appropriate percentiles. Twochildren, both 2 years of age, developed clinically signi-ficant vincristine polyneuropathy. None of the patients hadseptic infection when the CSF samples were taken.

DISCUSSION

Our data indicate that children with ALL have normalCSF IGF-1 levels at diagnosis and subnormal levels duringthe induction therapy, but the levels return to normal afterinduction and remain so during the rest of the ALLchemotherapy course (Fig. 1). The CSF IGFBP-2 levelsremained normal throughout the study. There were sevenchildren with ALL who had very low IGF-1 levelsthroughout the study course (Fig. 2). No factor could bediscovered to explain these continuously low levels. Twoout of the seven children had clinically significantvincristine polyneuropathy. None had or developed laterCNS-ALL, however the follow-up is not very long, somechildren still being on maintenance chemotherapy.

Very low CNS IGF-1 levels have been demonstrated inpatients with progressive encephalopathy, such as INCLand cerebellar atrophy (Fig. 1) [10–12]. These levels wereclearly lower than those during ALL induction therapy(Fig. 1). In the central nervous system, certain distinct celltypes appear to be selectively susceptible to lack of IGF-1,including cerebellar granule cells, oligodendrocytes, andmotoneurons, while the other cells are independent on thepresence of IGF-1 [4,13,14,15].

The continuous use of pharmacological doses ofcorticosteroids for several weeks might offer one explana-tion for the decrease during ALL induction chemotherapy[16]. Pathological conditions associated with insulinresistance may be associated with low IGF-1 levels.Synthesis of IGF-1 is stimulated by insulin; corticostero-ids have an acute effect counteracting the influence ofhormone excess on insulin. In the neonatal rat, dexa-methasone causes brain atrophy and reduces the expres-sion of IGF-1 [18]. During the ALL induction protocol,some children may have been more sensitive than others indecreasing their IGF-1 levels in the CNS.

Use of vincristine and the development of clinical orsubclinical polyneuropathy might be another factor.

Fig. 1. CSF IGF-1 levels (mg/L) in children with ALL and controls

with and without severe CNS diseases. Cerebrospinal fluid IGF-1 levels

(mg/L) in children with ALL at the initial diagnosis (before therapy),

during induction therapy, and later during ongoing therapy (>2 months

after diagnosis), as well as in groups with severe neurological diseases,

infantile neuronal ceroid lipofuscinosis (INCL), cerebellar atrophy,

progressive encephalopathy, hypsarrhythmia, and optic atrophy (PEHO

syndrome), PEHO-like syndrome, and Rett syndrome. The 16 age-

matched controls without severe CNS disease had other neurological

diseases not involving the white matter or cerebellum (similar to

the patients with PEHO-like syndrome and Rett syndrome). The

means� SD (vertical line) are shown. The difference between ALL

during induction and later is significant (P¼ 0.036 with Bonferroni

correction). The levels of patients during induction were significantly

lower than the levels of the controls (P¼ 0.003).

Fig. 2. Individuals with low CSF IGF-1 levels. During the follow-up,

seven children had continuously low CSF IGF-1 levels during ongoing

therapy.

112 Riikonen et al.

Peripheral neuropathy is characterized by loss of tissueinnervation, decreased neuronal survival, and reducedability to regenerate. Vincristine has been shown to crossthe blood–brain-barrier [17]. Neuropathologically theeffect of vincristine seems to be mainly axonal, butsecondarily demyelination has also been shown to occur[18]. The role of IGF-1 in myelination in the peripheralnervous system is not exactly known. IGF-1 is thought toplay a role in Schwann cell myelin production and survival[6,19]. Furthermore, clinical trials have revealed thebeneficial effects of neurotrophic factors in the treatmentof various neuropathies and neurodegenerative diseases[20–22]. IGF-1 also reduces diabetic neuropathy, sug-gested to be a consequence of loss of IGF-1 activity[23,24]. In rats, co-treatment with IGF-1 has prevented allthe effects of vincristine on the gait and on the sensorymodalities [24].

Plasma concentrations of IGF-1 are regulated bygrowth hormones and by the nutritional status [25]). Inour children with low CSF IGF-1 levels, the weight-to-height ratios were all within the normal range. However,protein-energy malnutrition has been shown to developduring ALL induction therapy, when the muscle mass iswasting and, in contrast, the fat increases [26,27], whichcondition is not necessarily revealed by the weight-to-height but would require ultrasound or CT examination ofthe muscles. Children during ALL induction chemother-apy were likely to have protein-energy malnutrition,which might have contributed to the low CSF IGF-1 levels.We did not find any correlation between CSF IGF-1 andCSF IGFBP-2 concentration which is not surprizingbecause the regulation of IGF-1 synthesis in the brainseems to be a complex one [28,29].

In conclusion, children with ALL had low CSF IGF-1levels during induction which levels later returned tonormal. We suspect the role of chemotherapy, particularlycorticosteroids and vincristine, in the subnormal IGF-1levels. A correlation to disturbed neuronal function couldnot be proven in this selectivity small study. Future studiesshould consider IGF-1 in examining toxic neurologicaladverse effects.

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