The Diagnostic Gray Zone Between Burkitt Lymphoma and Diffuse Large B-Cell Lymphoma Is Also a Gray Zone of the Mutational Spectrum

Letters to the Editor 1789 14 Rapin N, Bagger FO, Jendholm J, Mora-Jensen H, Krogh A, Kohlmann A et al. 15 Marstrand TT, Borup R, Willer A, Borregaard N, Sandelin A, Porse BT, Comparing cancer vs normal gene expression profiles identifies new disease Theilgaard-Mönch K. A conceptual framework for the identification of candidate entities and common transcriptional programs in AML patients. Blood 2014; 123: drugs and drug targets in acute promyelocytic leukemia. Leukemia 2010; 24: 894–904. 1265–1275.

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The diagnostic gray zone between Burkitt lymphoma and diffuse large B-cell lymphoma is also a gray zone of the mutational spectrum

Leukemia (2015) 29, 1789–1791; doi:10.1038/leu.2015.34 the five groups are displayed in Supplementary Figure 1. The sequencing approach included a combination of next-generation sequencing and Sanger validation (Supplementary Figure 2). In The current WHO classification recognizes the existence of brief, each amplicon was processed on the 48.48 Fluidigm Access aggressive B-cell lymphomas that share morphological, immu- Array System (Fluidigm Corporation, San Francisco, CA, USA). After nophenotypic and gene expression profile-based features inter- attaching barcodes for sample identification and enrichment of mediate between Burkitt lymphomas (BLs) and diffuse large the libraries, the samples were pooled and run on the Roche B-cell lymphomas (DLBCLs) and created the provisional category (Mannheim, Germany) GS Junior instrument. Data was analyzed of ‘B-cell lymphoma, unclassifiable, with features intermediate with the GS Amplicon Variant Analyzer Software (Roche) and between BL and DLBCL (BCL-U)’.1 From a clinical perspective, variant allele frequencies of 420% were validated by Sanger however, the introduction of the diagnostic BCL-U category sequencing (detailed methods are provided in the Supplementary appears problematic, because current therapeutic concepts Information). The final mutational results of all cases are displayed differ significantly between BL and DLBCL. Moreover, many in Figure 1 and Supplementary Table 1. patients with BCL-U have a dismal prognosis. Translocations of The mutation frequency of ID3 and/or TCF3 (ID3/TCF3) was the MYC oncogene are common in BCL-U, usually in the context comparably high in BL and BCL-U (65% vs 67%). However, the rate of a complex genetic background, and 35–50% of BCL-U of biallelic mutations in ID3 or a mutation in TCF3 was higher in lymphomas, next to MYC aberrations, harbor additional trans- BL (70%) than in BCL-U (37.5%; Supplementary Table 2, locations involving BCL2 and/or BCL6. These cases are referred to Supplementary Figure 3). The frequency of ID3 mutations was as ‘double hit’/’triple hit’ (DH/TH) lymphomas. However, neither lower in the DL-DH/TH (22%) and DL-MYC (37%) subgroups, MYC translocations nor a ‘double hit’/triple hit’ genetic constella- whereas only one DL (GCB) without a MYC rearrangement (3.5%) tion are unifying genetic features of BCL-U, as ~ 10% of DLBCL carried an ID3 mutation. Initial investigations suggested that ID3 carry MYC translocations or show DH/TH features as well.1 mutations that occur in up to 65% of BL might constitute a Nonetheless, according to current concepts, these B-cell non- discriminatory marker to distinguish between bona fide BLs and – Hodgkin lymphomas (B-NHLs) are categorized as DLBCL on the non-BLs.3 5 However, the recent report by Gebauer et al.12 already basis of their morphological features, although there is evidence demonstrated the presence of ID3 mutations in 25% of BCL-U that these genetic alterations may predict inferior outcome cases. In our study, the frequency of ID3 mutations even reached amongst DLBCL patients.2 67% in the BCL-U subgroup thus showing a similar frequency The recent rapid progress in next generation sequencing compared with the BL cohort (65%). We even detected ID3 technologies has re-defined the genetic landscape of B-NHL mutations in some GCB DLBCL cases carrying MYC translocations. including BL and DLBCL.3–11 To sharpen the diagnostic gray zone Of note, all BCL-U cases with MYC (‘single hit’) translocations between BL and DLBCL, we sequenced relevant genes that are carried concomitant ID3 mutations, albeit with a lower rate of frequently mutated in BL (ID3, TCF3, CCND3 and MYC) and DLBCL biallelic mutations. The presence of additional mutations char- (BCL2, EZH2, CREBBP, EP300, MEF2B and SGK1) in 108 aggressive acteristic of DLBCL in many of these cases argues against the idea B-cell lymphomas including 31 BL, 24 BCL-U and 53 DLBCL cases, that these B-NHL might represent ‘true’ BLs. It must therefore be the latter of which were enriched for germinal center-derived concluded that ID3 mutations do not exclusively occur in BL cases (GCB) DLBCL cases and DLBCL with MYC translocations. DLBCL of and that the presence or absence of this mutation is not helpful in the activated B-like type and their characteristic mutations (NFkB, discriminating BL from non-BL cases. B-cell receptor signaling) were not studied, as these cases usually Mutations of CCND3 were observed in all five subgroups at a rate do not fall into the gray zone between BL and DLBCL. Formalin- between 11 and 29%. There was no clear enrichment of CCND3 fixed and paraffin-embedded lymphoma specimens were selected mutations in BL cases (26%), as reported previously,5 as mutations from the archives of the Institute of Pathology (University of occurred also quite frequently in BCL-U (29%) and DL-MYC (25%), Würzburg, Würzburg, Germany) and the Department of Clinical whereas the mutation rate dropped in DL (GCB) without MYC Pathology (Robert-Bosch-Krankenhaus, Stuttgart, Germany). The translocations (11%). Thus, CCND3 mutations appear to be enriched 53 DLBCL cases were subcategorized into three groups, namely 28 in aggressive B-NHL carrying MYC translocations. MYC mutations DLBCL cases without MYC translocation (DL (GCB)), 16 DLBCL were highly enriched in aggressive B-NHL cases carrying a MYC cases with a MYC-only translocation (DL-MYC) and 9 DLBCL with translocation. In these lymphomas, the frequency of MYC mutations MYC translocation plus additional breaks in the BCL2 and/or BCL6 ranged between 33 and 69%, whereas only few DL (GCB) without loci (DL-DH/TH). Representative morphological features for each of MYC translocations showed MYC mutations (7%, Po0.0002, Fisher´s

Accepted article preview online 12 February 2015; advance online publication, 13 March 2015

BL BCL-U DL-DH/TH DL-MYC DL (GCB) MYC Translocation BCL2 BCL6 ID3 TCF3 CCND3 CMYC Non_synonymous BCL2 mutation EZH2 CREBBP EP300 MEF2B SGK1 Figure 1. Translocation status of MYC, BCL2 and BCL6 and mutational status of the 10 selected genes for individual cases (columns) in each subgroup. Black rectangles denote the presence of a translocation. For nonsynonymous mutations, red and pink rectangles denote the mutation of BL-associated genes and yellow rectangles denote mutations of DLBCL-associated genes. For ID3, a red rectangle indicates a biallelic or homozygous mutation, whereas a pink rectangle indicates a monoallelic mutation.

Figure 2. Mutational patterns across B-NHL subgroups studied (a)inMYC ‘single hit’ (b)andinMYC ‘double/triple hit’ lymphomas (c). The ‘mutBL’ pattern includes mutations in BL-associated genes (ID3/TCF3, CCND3 and MYC), the ‘mutDL’ pattern includes mutations in DLBCL-associated genes (BCL2, EZH2, CREBBP, EP300, MEF2B and SGK1). ‘mutBL/DL’ indicates an overlapping pattern of mutations.

exact test: DL (GCB) vs other categories).13 Overall, the presence of a 641, which is identical to the most common mutation in previous MYC translocation was highly associated with concomitant MYC reports.6,8 The mutation rate of EZH2 in GCB DLBCL (14%) was on mutations (P = 0.0006, Fisher´s exact test). Across all studied the lower side of the reported range (11.7–38.5%).5,6,8,11,14 As EZH2 subgroups, aggressive B-NHL cases with a MYC-only translocation mutations were found in similar frequencies (around 12%) in DL were more frequently MYC-mutated compared with cases with a (GCB), DL-MYC, DL-DH/TH and BCL-U cases, a mutational screening ‘double hit’ or ‘triple hit’ constellation (P = 0.0417, Fisher´s exact test). in the diagnostic setting in all of these subgroups would be EZH2 mutations were detected in all investigated subgroups necessary to identify the candidates for targeted EZH2 inhibition. except in BL. All mutations in EZH2 altered the tyrosine residue We even detected two BCL-U cases with concurrent mutations in

Leukemia (2015) 1779 – 1797 © 2015 Macmillan Publishers Limited Letters to the Editor 1791 ID3 and EZH2, which was not observed among pure BL cases. ACKNOWLEDGEMENTS 5 As mutations in ID3 contribute to the activation of PI3K pathway, We wish to thank Heike Brückner, Eva Bachmann and Tina Grieb for their excellent a subset of BCL-U patients might benefit from PI3K and/or EZH2 technical support. This study was supported by the German Ministry for Education inhibitors, which are currently tested in preclinical trials.15 and Science (BMBF) in the framework of the ICGC MMML-Seq (01KU1002A-J) Network BCL2 mutations were most frequent in BCL-U cases (58%) and and by the Robert-Bosch-Stiftung, Stuttgart, Germany. lowest in BL and DL (GCB) with a rate of 16% and 18%, respectively. Mutations in CREBBP/EP300 were detected in all AUTHOR CONTRIBUTIONS subgroups, with a clear enrichment in BCL-U (29%) and DL-DH/TH SM, GO and AR designed the research, prepared the figures and wrote the (33%). MEF2B and SGK1 mutations were predominantly detected paper. SM, SW, TN, MR, AS and EB performed the experiments. SM, JP and AR in DL (GCB) (18% and 21%, respectively) and were rare or absent analyzed and interpreted the data. AR, GO, MR, EG and SM reviewed and classified in other subgroups. the cases. We next investigated mutational patterns that occur in each of the subgroups with the aim to determine whether any S Momose1,2, S Weißbach1,2, J Pischimarov1,2, T Nedeva1,2, combination of morphological, immunophenotypic, genetic and E Bach3, M Rudelius1,2, E Geissinger1,2, AM Staiger4, mutational features could sharpen the borderline within the G Ott4,5 and A Rosenwald1,2,5 1 spectrum ranging from BL, gray zone lymphoma and GCB DLBCL Institute of Pathology, University of Würzburg, Würzburg, Germany; 2 (especially with MYC rearrangements). On the basis of previously Comprehensive Cancer Center Mainfranken (CCC MF), Würzburg, Germany; reported data, we assigned mutations of ID3, TCF3, CCND3 and 3 MYC to the Burkitt mutational pattern (‘mutBL’) and mutations of Department of Human Genetics, University of Würzburg, BCL2, EZH2, CREBBP1, EP300, SGK1 and MEF2B to the DLBCL Würzburg, Germany and 4Department of Clinical Pathology, Robert-Bosch-Krankenhaus and mutational pattern (‘mutDL’). Recognizing the ‘oversimplification’ Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, of this approach, a given lymphoma could then be characterized ‘ ’ ‘ ’ Stuttgart, Germany as having a mutBL pattern, a mutDL pattern, a combined E-mail: [email protected] ‘ ’ mutBL/DL pattern or none of these patterns. As can be seen in 5Co-senior authors. Figure 2a, the scenario was quite homogenous in the BL and DL (GCB) subgroups. 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Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)