short review

memo (2017) 10:248–254https://doi.org/10.1007/s12254-017-0367-8

Aggressive lymphoma 2016: revision of theWHOclassification

Christine Beham-Schmid

Received: 21 August 2017 / Accepted: 2 November 2017 / Published online: 30 November 2017© The Author(s) 2017. This article is an open access publication.

Summary Aggressive lymphomas are a heteroge-neous group of malignancies reflecting clinical, bio-logical and pathological diversity. Diffuse large B-celllymphoma is the most common histological subtypeand therefore will constitute the key aspect in thisarticle. This lymphoma affects patients of all agegroups with wide range presentations concerning lo-calization, morphology and molecular mechanisms.The median age at presentation is about 60 yearswith a slight male preponderance. Up to 50% of pa-tients present with advanced disease. About 70% ofthese lymphomas occur nodal, about 30% extranodal,the most common sites of the latter being the gas-trointestinal tract, Waldeyer’s ring, skin, cerebrum,mediastinum, testis, salivary gland, thyroid and bone.However, diffuse large B-cell lymphoma can involvevirtually any organ.Since the last WHO classification2008 the adoption of new genomic technologies hasprovided new insights into the biology of these lym-phomas and led to the identification of distinct sep-arate molecular entities and novel pathogenic path-ways. These findings induced an expanding numberof entities in the new WHO classification of 2016, theknowledge of which is essential concerning treatmentoptions and survival of the patients. Therefore, theclinicians request an accurate diagnosis from the in-vestigating pathologist, which can be quite challeng-ing. The diagnosis of lymphomas requires multipleimmunohistochemical studies, and often additionaltests, such as fluorescent in situ hybridization and/orpolymerase chain reaction techniques and occasion-ally, in particular cases, next generation sequencingfor identification of recurrent somatic mutations.

C. Beham-Schmid (�)Institute of Pathology, Medical University Graz, NeueStiftingtalstr. 6, 8010 Graz, Austriachristine.beham@medunigraz.at

This review summarizes relevant aspects of the newWHO classification in aggressive B-cell lymphomas,especially from a haematopathologist’s point of view.

Keywords DLBCL · GCB · ABC · MYC · Double-hit

Introduction

Aggressive B-cell lymphomas consist of precursorlymphoid neoplasms (B-lymphoblastic leukaemia/lymphoma NOS and B-lymphoblastic leukaemia/lymphoma with recurrent genetic abnormalities) andnumerous mature B-cell neoplasms, such as mantlecell lymphoma, Burkitt lymphoma, primary effusionlymphoma, diffuse large B-cell lymphoma (DLBCL)with its numerous subtypes, B-cell lymphoma un-classifiable with features between DLBCL and Burkittlymphoma, and the new variant Burkitt-like lym-phoma with 11q aberrations (see Table 1).

Diffuse large B-cell lymphoma (DLBCL) is an ag-gressive B-cell lymphoma histologically characterizedby dense proliferation of neoplastic B-blasts. DLBCLis the most common histological subtype of non-Hodgkin lymphomas (NHL) accounting worldwidefor about 30% of adult NHL [1]. Within the groupof DLBCL, many disparate entities with enormousdifferences concerning the clinical behaviour, mor-phology, immunophenotype and molecular mecha-nisms are known. Since the response to treatmentregimens is different in the subtypes of DLBCLs, in-tensive research has specialized on the identificationof prognostically or biologically distinct groups aim-ing at tailored treatment regimens. Thus, prognosticmarkers are of great importance [2]. The WHO clas-sification 2008 [3] already recognized the importanceof the cell-of-origin classification into germinal cen-tre B-cell-like (GCB) and activated B-cell-like (ABC)molecular subgroups of DLBCLs not otherwise speci-

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short review

Table 1 WHO classification of mature large B-cell lymphoid neoplasms

Diffuse large B-cell lymphoma (DLBCL), NOS– Germinal centre B-cell typea

– Activated B-cell typea

T-cell/histiocyte-rich large B-cell lymphoma

Primary DLBCL of the central nervous system (CNS)

EBV+ DLBCL, NOSa

EBV+ mucocutaneous ulcera

DLBCL associated with chronic inflammation

Lymphomatoid granulomatosis

Primary mediastinal/thymic large B-cell lymphoma

Intravascular large B-cell lymphoma

ALK+ large B-cell lymphoma

Plasmablastic lymphoma

Primary effusion lymphoma

HHV8+ DLBCL, NOSa

Burkitt lymphoma

Burkitt-like lymphoma with 11q aberrationa

High grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangementsa

High grade B-cell lymphoma, NOSa

B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma

The provisional entities are listed in italicsaChanges from the 2008 classificationNOS not otherwise specified, EBV Epstein Barr virus

Fig. 1 Cytology sample(vitreous preparation) withinfiltration by ocular dif-fuse large B-cell lymphoma(DLBCL).aH&E;bTumourcells stained with an anti-body to CD20

fied (NOS) based on gene expression profiling (GEP).In addition a group of cases that could not be put intoeither category (unclassifiable) was found. The GEP-defined GCB and ABC subtypes are different concern-ing chromosomal alterations, activation of signalingpathways and clinical outcome [4]. Since the esti-mation of GEP-defined subgroups requires fresh orfrozen tissue for extraction of sufficient RNA, whichis difficult to implement in clinical practice, the useof immunohistochemistry with special antibodies,which represent different stages of B-cell differentia-tion, has been proposed. Various immunohistochem-ical algorithms predicting the prognosis of DLBCL andguiding treatment options have been reported [5, 6],revealing the Choi algorithm as the most predictive ofGEP results [7]. Accumulated knowledge of the molec-ular pathogenesis of these 2 subgroups resulted in theinvestigation of more specific therapeutic strategies to

appease the poorer outcome among those with ABC(non-GCB) type DLBCL reported in most studies [8].The revised WHO classification 2016 now requires thecell of origin identification.

In addition, the WHO update recognizes immuno-histochemical coexpression of MYC and BCL2 pro-teins within DLBCL NOS as a new adverse prog-nostic marker, but not a separate category. TheseDLBCLNOS are called “double-expressor lymphomas”and are strongly associated with poor outcome in pa-tients treated with R-CHOP. According to Green et al.[9] a double-hit score (DHS) can be assigned to allpatients with DLBCL.

A worse outcome than double-expressor lym-phomas show high grade B-cell lymphomas (HGBL)with rearrangements of MYC and BCL2 and/or BCL6,the so-called double-hit or triple-hit lymphomas,

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Fig. 2 Schematic representation of immunohistochemical al-gorithm according to Choi et al. [6]

which constitute a single category in the updatedWHO classification.

Primary DLBC in central nervous system, testisand stomach

Approximately 95% of primary central nervous sys-tem lymphomas (PCNSL) are large B-cell lymphomas.They reveal a unique pathobiology with dissemina-tion of the lymphoma within the brain, cranial nerves,leptomeninges, cerebrospinal fluid, intraocular struc-tures (Fig. 1) and spinal cord, without overt systemicdisease. Most of PCNSL cases are of an activated B-cellimmunophenotype [10]. Since the brain is largely as-sumed to be an immunologically quiescent or “privi-leged site”, PCNSL require different treatment options[11].

Primary lymphomas of the testis with morphologyof DLBCL in most cases are of an activated B-cell im-munophenotype. The testis is also considered a “priv-ileged” localization with special treatment regimens[12].

Treatment of primary gastric diffuse large B-celllymphoma is controversial; however, regression ofhigh-grade gastric lymphomas after the cure of Heli-cobacter pylori infection have been described [13].

The clinical differences between nodal and extra-nodal DLBCL thus require the distinction betweennodal and extranodal DLBCL.

Cell of origin classification

Within the large category of DLBCL NOS two molec-ular subtypes have been recognized by GEP, termedgerminal centre B-cell (GCB) and activated B-cell(ABC) with a small proportion of patients (about 15%)remaining unclassifiable. The ABC subtype showsa worse outcome with R-CHOP treatment [8]. Thesesubtypes are provoked by various different oncogenicpathways which might be important for therapeuticbenefits [14].

GCB DLBCL

GCB DLBCL derives from centroblasts and thus ex-press genes which can be detected in germinal centreB-cells, mainly GCET1, CD10 and BCL6. About onethird of GCB DLBCL reveal a c-rel amplification ora t(14;18) translocation and 10–20% show mutationsof the histone methyltransferase EZH2 or a deletionof PTEN. These abnormalities are not found in ABCsubtypes. PTEN, which is an essential tumour sup-pressor gene encoding a phosphatase protein that an-tagonises the PI3K/Akt/mTOR antiapoptotic pathway,might be susceptible to new treatment options [15].

Different oncogenic pathways give rise to an over-expression of BCL2, an antiapoptotic protein, in GCBand also ABC DLBCL. Whereas BCL2 overexpressionin GCB subtype is caused by t(14;18) translocation, inABC subtype gene amplification and transcriptionalupregulation provokes BCL2 overexpression. Appli-ance of BCL2 inhibitors in DLBCL is an auspicioustherapy option [16].

ABC DLBCL

ABC DLBCL originates from plasmablastic cells priorto germinal centre exit and shows a gene expressionsimilar to that of mature plasma cells, like MUM1andFOXP1. Typical for ABC DLBCL is the constitutive ac-tivation of the NF-kappaB signalling pathway whichis essential for cell survival and proliferation and in-hibition of apoptosis. More than 50% of ABC DLBCLshow mutations concerning the regulation of the NF-kappaB cascade: Activating mutations of CARD11arefound in 10% of ABC subtype, 20% show mutationsin CD79A or CD79B, and about 30% reveal recurringmutations in MYD88.

Identification of the cell of origin

Since GEP is not routinely available, a reliable methodof determining the molecular subtype is the use of im-munohistochemically (IHC) based algorithms. Sincethe first published Hans algorithm in 2004 [5], whichuses three markers (CD10, BCL6 and MUM1), severalimproved IHC algorithms have been developed. TheWHO classification does not specify which methodshould be used. The algorithm developed by Choiet al. ([6]; Fig. 2) is consistent in 93% with results ofGEP. IHC algorithms are easily applicable and are a re-liable way for the evaluation of GCB or ABC subtype(Figs. 3 and 4).

Recent techniques using NanoString technology onparaffin embedded tissue are possibly the future wayfor estimation of GCB or ABC subtype [17].

Role of the microenvironment and GEP

Concerning the outcome of DLBCL patients GEP stud-ies revealedmolecular signatures in themicroenviron-

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Fig. 3 DLBCL, GCB type:adense lymph node infil-tration by lymphatic blasts(H&E).bThe blasts arestrongly CD20-positive.cMany tumour cells areGCET1-positive. dNo re-activity with an antibody toMUM1

Fig. 4 DLBCL, ABC type:adense lymph node infil-tration by lymphatic blasts(H&E).bStrong CD20-positivity in the tumour cells.cMany tumour cells areGCET1-positive. dStrongnuclearexpressionofMUM1

ment of DLBCL NOS. High numbers of macrophagesand extracellular matrix deposition (stromal-1 signa-ture) is associated with a better prognosis than stro-mal-2 signature with marked angiogenesis. Both sig-natures could be detected in GCB and ABC subtypes.Furthermore, genetic aberrations concerning antigenpresenting functions and immune recognition have

been identified, probably providing therapeutic pos-sibilities [18].

Importance of BCL2 and MYC

MYC rearrangement, typically detected by fluores-cence in situ hybridization (FISH), is characteristic

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Fig. 5 DLBCL, “double-expressor”: aDense in-filtration of a lymph nodeby lymphatic blasts (H&E).bStaining with an antibodyto CD20 revealing B-cellorigin. cMore than 40% ofthe blasts reveal nuclearMYC-positivity. dThe blastsare strongly BCL2-positive

for Burkitt lymphoma. However, in about 10% ofDLBCL NOS, rearrangement of MYC is found and hasbeen shown to be associated with a poor outcomein patients treated with R-CHOP. Frequently, MYCrearrangement is combined with BCL2, and/or lessfrequent with BCL6 translocation. These “double-hit”or “triple-hit” lymphomas [19] are included in theupdated WHO classification in the category of high-grade B-cell lymphoma (HGBL), with rearrangementsof MYC and BCL2 and/or BCL6. Surprisingly, mostof these cases are of GCB type, which, accordingly tothe cell of origin classification, usually show a betterprognosis than ABC types with R-CHOP treatment.Routinely, the status of MYC, BCL2 and BCL6 is ana-lyzed by FISH techniques. An immunohistochemicaloverexpression of MYC is found more frequently thanMYC translocation. Up to 30% of patients with DLBCLoverexpressMYC protein. A poor prognostic impact ofpatients with MYC protein expression is found whenBCL2 protein is coexpressed (Fig. 5). These so-called“double-expressors” [9], seen in up to 25% of DLBCLpatients, are an independent predictor of poor sur-vival, and might be important to stratify patients forrisk-adapted therapies. Immunohistochemical evalu-ation of MYC, BCL2 and BCL6 has been extensivelydiscussed [20]. In most studies a DLBCL is consid-ered MYC positive when ≥40% of blasts show MYCexpression. The cutoff for BCL2 positivity is ratherdiscordant with values ranging from 30 to 70% asconsidered being positive [21].

Burkitt-like lymphoma with 11q aberration

For a long time it was a matter of debate whethertrue Burkitt lymphoma (BL) without MYC transloca-tion does exist. Many studies reported lymphomaswith clinical, histologic, immunophenotypic, or geneexpression features of BL, but no detectable MYCtranslocation by FISH analysis. One should be ex-tremely cautious to diagnose a true MYC-negative BLbecause the scattering of breakpoints in the MYC andIG loci along with small insertions of one locus intothe other can render MYC breaks undetectable evenif several sets of FISH probes are applied. Salaverriaet al. [22] described a subset of lymphomas with geneexpression and pathological characteristics of Burkittlymphomas but absence of MYC translocation. Theselymphomas carry chr 11q proximal gains and telom-eric losses, suggesting coderegulation of oncogenesand tumour suppressor genes. Although only a smallnumber of these lymphomas have been reported, theclinical course is similar to that of BL.

Conclusion

Risk stratification of the heterogeneous group ofDLBCL NOS is an evolving procedure. Ongoing ef-forts to tailor therapy based on the different onco-genic pathways of GCB and ABC DLBCL demand anoptimal cell of origin classification. Furthermore, thedetection of MYC gene translocation and MYC proteinexpression has shown to be of increasing importancein the prognosis and treatment of DLBCL patients.It is recommended to investigate for both dual or

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trifold translocation (MYC, BCL2 and BCL6) and dualprotein expression status, since treatment optionsmay be different. It is important to mention that im-munohistochemical MYC protein expression does notequate to MYC rearrangement. However, a correlationhas been shown between immunohistochemical MYCprotein expression and MYC gene abnormalities inDLBCL [23]. Routinely, the diagnosis of DLBCL NOSincludes the cell of origin classification and the dou-ble-hit score (“double-expressor”) according to Greenet al. [9]. The next step, if MYC-protein expressionhas been detected, is FISH investigation for MYC, andthe partner genes BCL2 and BCL6. The results arereported subsequently.

To identify relevant disease drivers that are thera-peutically targetable, concerted efforts generated theso-called “lymphopanel”. This targeted gene sequenc-ing panel enables the detection of mutations and sub-type-enriched gene alterations in DLBCL hopefullyyielding to the development of new and effective tar-geted treatment approaches [24].

Take home message Diagnosis of DLBCL must in-clude cell of origin classification (GCB, ABC) and im-munohistochemical double hit score (MYC, BCL2).

Coexpression of MYC and BCL2 is considered a newprognostic marker.

The understanding of the mutational landscapemight become part of the foundation for optimaltreatment options.

Funding Open access funding provided by Medical Univer-sity of Graz.

Conflict of interest C. Beham-Schmid declares that she hasno competing interests.

Open Access This article is distributed under the terms ofthe Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which per-mits unrestricted use, distribution, and reproduction in anymedium, provided you give appropriate credit to the origi-nal author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made.

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7For latest news from interna-tional oncology congresses see: http://www.springermedizin.at/memo-inoncology

254 Aggressive lymphoma 2016: revision of the WHO classification K

Aggressive lymphoma 2016: revision of the WHO classificationSummaryIntroductionPrimary DLBC in central nervous system, testis and stomachCell of origin classificationGCB DLBCLABC DLBCLIdentification of the cell of origin

Role of the microenvironment and GEPImportance of BCL2 and MYCBurkitt-like lymphoma with 11q aberrationConclusionReferences