Letters to the Editor
1789 14 Rapin N, Bagger FO, Jendholm J, Mora-Jensen H, Krogh A, Kohlmann A et al. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients. Blood 2014; 123: 894–904.
15 Marstrand TT, Borup R, Willer A, Borregaard N, Sandelin A, Porse BT, Theilgaard-Mönch K. A conceptual framework for the identification of candidate drugs and drug targets in acute promyelocytic leukemia. Leukemia 2010; 24: 1265–1275.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
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 current WHO classification recognizes the existence of aggressive B-cell lymphomas that share morphological, immunophenotypic and gene expression profile-based features intermediate between Burkitt lymphomas (BLs) and diffuse large B-cell lymphomas (DLBCLs) and created the provisional category of ‘B-cell lymphoma, unclassifiable, with features intermediate between BL and DLBCL (BCL-U)’.1 From a clinical perspective, however, the introduction of the diagnostic BCL-U category appears problematic, because current therapeutic concepts differ significantly between BL and DLBCL. Moreover, many patients with BCL-U have a dismal prognosis. Translocations of the MYC oncogene are common in BCL-U, usually in the context of a complex genetic background, and 35–50% of BCL-U lymphomas, next to MYC aberrations, harbor additional translocations involving BCL2 and/or BCL6. These cases are referred to as ‘double hit’/’triple hit’ (DH/TH) lymphomas. However, neither MYC translocations nor a ‘double hit’/triple hit’ genetic constellation are unifying genetic features of BCL-U, as ~ 10% of DLBCL carry MYC translocations or show DH/TH features as well.1 Nonetheless, according to current concepts, these B-cell nonHodgkin lymphomas (B-NHLs) are categorized as DLBCL on the basis of their morphological features, although there is evidence that these genetic alterations may predict inferior outcome amongst DLBCL patients.2 The recent rapid progress in next generation sequencing technologies has re-defined the genetic landscape of B-NHL including BL and DLBCL.3–11 To sharpen the diagnostic gray zone between BL and DLBCL, we sequenced relevant genes that are frequently mutated in BL (ID3, TCF3, CCND3 and MYC) and DLBCL (BCL2, EZH2, CREBBP, EP300, MEF2B and SGK1) in 108 aggressive B-cell lymphomas including 31 BL, 24 BCL-U and 53 DLBCL cases, the latter of which were enriched for germinal center-derived (GCB) DLBCL cases and DLBCL with MYC translocations. DLBCL of the activated B-like type and their characteristic mutations (NFkB, B-cell receptor signaling) were not studied, as these cases usually do not fall into the gray zone between BL and DLBCL. Formalinfixed and paraffin-embedded lymphoma specimens were selected from the archives of the Institute of Pathology (University of Würzburg, Würzburg, Germany) and the Department of Clinical Pathology (Robert-Bosch-Krankenhaus, Stuttgart, Germany). The 53 DLBCL cases were subcategorized into three groups, namely 28 DLBCL cases without MYC translocation (DL (GCB)), 16 DLBCL cases with a MYC-only translocation (DL-MYC) and 9 DLBCL with MYC translocation plus additional breaks in the BCL2 and/or BCL6 loci (DL-DH/TH). Representative morphological features for each of
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 brief, each amplicon was processed on the 48.48 Fluidigm Access Array System (Fluidigm Corporation, San Francisco, CA, USA). After attaching barcodes for sample identification and enrichment of the libraries, the samples were pooled and run on the Roche (Mannheim, Germany) GS Junior instrument. Data was analyzed with the GS Amplicon Variant Analyzer Software (Roche) and variant allele frequencies of 420% were validated by Sanger sequencing (detailed methods are provided in the Supplementary Information). The final mutational results of all cases are displayed in Figure 1 and Supplementary Table 1. The mutation frequency of ID3 and/or TCF3 (ID3/TCF3) was comparably high in BL and BCL-U (65% vs 67%). However, the rate of biallelic mutations in ID3 or a mutation in TCF3 was higher in BL (70%) than in BCL-U (37.5%; Supplementary Table 2, Supplementary Figure 3). The frequency of ID3 mutations was lower in the DL-DH/TH (22%) and DL-MYC (37%) subgroups, whereas only one DL (GCB) without a MYC rearrangement (3.5%) carried an ID3 mutation. Initial investigations suggested that ID3 mutations that occur in up to 65% of BL might constitute a discriminatory marker to distinguish between bona fide BLs and non-BLs.3–5 However, the recent report by Gebauer et al.12 already demonstrated the presence of ID3 mutations in 25% of BCL-U cases. In our study, the frequency of ID3 mutations even reached 67% in the BCL-U subgroup thus showing a similar frequency compared with the BL cohort (65%). We even detected ID3 mutations in some GCB DLBCL cases carrying MYC translocations. Of note, all BCL-U cases with MYC (‘single hit’) translocations carried concomitant ID3 mutations, albeit with a lower rate of biallelic mutations. The presence of additional mutations characteristic of DLBCL in many of these cases argues against the idea that these B-NHL might represent ‘true’ BLs. It must therefore be concluded that ID3 mutations do not exclusively occur in BL cases and that the presence or absence of this mutation is not helpful in discriminating BL from non-BL cases. Mutations of CCND3 were observed in all five subgroups at a rate between 11 and 29%. There was no clear enrichment of CCND3 mutations in BL cases (26%), as reported previously,5 as mutations occurred also quite frequently in BCL-U (29%) and DL-MYC (25%), whereas the mutation rate dropped in DL (GCB) without MYC translocations (11%). Thus, CCND3 mutations appear to be enriched in aggressive B-NHL carrying MYC translocations. MYC mutations were highly enriched in aggressive B-NHL cases carrying a MYC translocation. In these lymphomas, the frequency of MYC mutations ranged between 33 and 69%, whereas only few DL (GCB) without MYC translocations showed MYC mutations (7%, Po0.0002, Fisher´s
Accepted article preview online 12 February 2015; advance online publication, 13 March 2015
© 2015 Macmillan Publishers Limited
Leukemia (2015) 1779 – 1797
Letters to the Editor
1790 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) in MYC ‘single hit’ (b) and in MYC ‘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 MYC translocation was highly associated with concomitant MYC mutations (P = 0.0006, Fisher´s exact test). Across all studied subgroups, aggressive B-NHL cases with a MYC-only translocation were more frequently MYC-mutated compared with cases with a ‘double hit’ or ‘triple hit’ constellation (P = 0.0417, Fisher´s exact test). EZH2 mutations were detected in all investigated subgroups except in BL. All mutations in EZH2 altered the tyrosine residue Leukemia (2015) 1779 – 1797
641, which is identical to the most common mutation in previous reports.6,8 The mutation rate of EZH2 in GCB DLBCL (14%) was on the lower side of the reported range (11.7–38.5%).5,6,8,11,14 As EZH2 mutations were found in similar frequencies (around 12%) in DL (GCB), DL-MYC, DL-DH/TH and BCL-U cases, a mutational screening in the diagnostic setting in all of these subgroups would be necessary to identify the candidates for targeted EZH2 inhibition. We even detected two BCL-U cases with concurrent mutations in © 2015 Macmillan Publishers Limited
Letters to the Editor
ID3 and EZH2, which was not observed among pure BL cases. As mutations in ID3 contribute to the activation of PI3K pathway,5 a subset of BCL-U patients might benefit from PI3K and/or EZH2 inhibitors, which are currently tested in preclinical trials.15 BCL2 mutations were most frequent in BCL-U cases (58%) and lowest in BL and DL (GCB) with a rate of 16% and 18%, respectively. Mutations in CREBBP/EP300 were detected in all subgroups, with a clear enrichment in BCL-U (29%) and DL-DH/TH (33%). MEF2B and SGK1 mutations were predominantly detected in DL (GCB) (18% and 21%, respectively) and were rare or absent in other subgroups. We next investigated mutational patterns that occur in each of the subgroups with the aim to determine whether any combination of morphological, immunophenotypic, genetic and mutational features could sharpen the borderline within the spectrum ranging from BL, gray zone lymphoma and GCB DLBCL (especially with MYC rearrangements). On the basis of previously reported data, we assigned mutations of ID3, TCF3, CCND3 and MYC to the Burkitt mutational pattern (‘mutBL’) and mutations of BCL2, EZH2, CREBBP1, EP300, SGK1 and MEF2B to the DLBCL mutational pattern (‘mutDL’). Recognizing the ‘oversimplification’ of this approach, a given lymphoma could then be characterized as having a ‘mutBL’ pattern, a ‘mutDL’ pattern, a combined ‘mutBL/DL’ pattern or none of these patterns. As can be seen in Figure 2a, the scenario was quite homogenous in the BL and DL (GCB) subgroups. As expected, the ‘mutBL’ pattern dominated in the BL subgroup, whereas the ‘mutDL’ pattern dominated amongst DL (GCB) cases thus separating those two groups with regard to their mutational spectrum. In contrast, the other three subgroups (BCL-U, DL-DH/TH and DL-MYC) showed overlapping features with the majority of cases showing a combined ‘mutBL/DL’ pattern. Of note, the vast majority (almost 80%) of DL-MYC cases that are currently diagnosed as DLBCL harbored a ‘mutBL’ or a ‘mutBL/DL’, questioning the current WHO concept that B-NHL with morphological features of DLBCL should be classified as such, regardless of an underlying MYC translocation or a ‘double/triple hit’ constellation. Given that the MYC translocation is considered a dominant oncogenic event in lymphomagenesis, we sought to investigate whether the mutational spectrum of 10 relevant genes correlates with the presence of single or non-single MYC translocations in the B-NHL subgroups studied here. Figure 2b displays the scenario in MYC ‘single hit’ lymphomas including 26 BL, 8 BCL-U and 16 DLMYC cases. As expected, the ‘mutBL’ pattern predominated among BL cases, but approximately one-third of BCL-U and DLMYC cases with MYC translocation (‘single hit’) also showed this feature. Similar results were obtained when MYC ‘double/triple hit’ lymphoma cases were analyzed (Figure 2c), although the ‘mutDL’ pattern occurred more frequently compared with MYC ‘single hit’ lymphomas. In conclusion, our results show that the morphological, immunophenotypic and genetic gray zone between BL and DLBCL is also a gray zone of the mutational spectrum. Thus, it will be difficult to more precisely define molecular subgroups and relevant clinicopathologic entities within this spectrum of aggressive B-cell lymphomas.
CONFLICT OF INTEREST The authors declare no conflict of interest.
1791
ACKNOWLEDGEMENTS We wish to thank Heike Brückner, Eva Bachmann and Tina Grieb for their excellent technical support. This study was supported by the German Ministry for Education and Science (BMBF) in the framework of the ICGC MMML-Seq (01KU1002A-J) Network and by the Robert-Bosch-Stiftung, Stuttgart, Germany.
AUTHOR CONTRIBUTIONS SM, GO and AR designed the research, prepared the figures and wrote the paper. SM, SW, TN, MR, AS and EB performed the experiments. SM, JP and AR analyzed and interpreted the data. AR, GO, MR, EG and SM reviewed and classified the cases.
S Momose1,2, S Weißbach1,2, J Pischimarov1,2, T Nedeva1,2, E Bach3, M Rudelius1,2, E Geissinger1,2, AM Staiger4, G Ott4,5 and A Rosenwald1,2,5 1 Institute of Pathology, University of Würzburg, Würzburg, Germany; 2 Comprehensive Cancer Center Mainfranken (CCC MF), Würzburg, Germany; 3 Department of Human Genetics, University of Würzburg, Würzburg, Germany and 4 Department of Clinical Pathology, Robert-Bosch-Krankenhaus and Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany E-mail: [email protected] 5 Co-senior authors. REFERENCES 1 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC: Lyon, France, 2008. 2 Horn H, Ziepert M, Becher C, Barth TF, Bernd HW, Feller AC et al. MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma. Blood 2013; 121: 2253–2263. 3 Love C, Sun Z, Jima D, Li G, Zhang J, Miles R et al. The genetic landscape of mutations in Burkitt lymphoma. Nat Genet 2012; 44: 1321–1325. 4 Richter J, Schlesner M, Hoffmann S, Kreuz M, Leich E, Burkhardt B et al. Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing. Nat Genet 2012; 44: 1316–1320. 5 Schmitz R, Young RM, Ceribelli M, Jhavar S, Xiao W, Zhang M et al. Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics. Nature 2012; 490: 116–120. 6 Bodor C, O'Riain C, Wrench D, Matthews J, Iyengar S, Tayyib H et al. EZH2 Y641 mutations in follicular lymphoma. Leukemia 2011; 25: 726–729. 7 Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci USA 2012; 109: 3879–3884. 8 Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 2010; 42: 181–185. 9 Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 2011; 476: 298–303. 10 Pasqualucci L, Trifonov V, Fabbri G, Ma J, Rossi D, Chiarenza A et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 2011; 43: 830–837. 11 Zhang J, Grubor V, Love CL, Banerjee A, Richards KL, Mieczkowski PA et al. Genetic heterogeneity of diffuse large B-cell lymphoma. Proc Natl Acad Sci USA 2013; 110: 1398–1403. 12 Gebauer N, Bernard V, Feller AC, Merz H. ID3 mutations are recurrent events in double-hit B-cell lymphomas. Anticancer Res 2013; 33: 4771–4778. 13 Robbiani DF, Nussenzweig MC. Chromosome translocation, B cell lymphoma, and activation-induced cytidine deaminase. Annu Rev Pathol 2013; 8: 79–103. 14 Ryan RJ, Nitta M, Borger D, Zukerberg LR, Ferry JA, Harris NL et al. EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas. PLoS One 2011; 6: e28585. 15 Vaidya R, Witzig TE. Prognostic factors for diffuse large B-cell lymphoma in the R (X)CHOP era. Ann Oncol 2014; 25: 2124–2133.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
© 2015 Macmillan Publishers Limited
Leukemia (2015) 1779 – 1797
1789 14 Rapin N, Bagger FO, Jendholm J, Mora-Jensen H, Krogh A, Kohlmann A et al. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients. Blood 2014; 123: 894–904.
15 Marstrand TT, Borup R, Willer A, Borregaard N, Sandelin A, Porse BT, Theilgaard-Mönch K. A conceptual framework for the identification of candidate drugs and drug targets in acute promyelocytic leukemia. Leukemia 2010; 24: 1265–1275.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
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 current WHO classification recognizes the existence of aggressive B-cell lymphomas that share morphological, immunophenotypic and gene expression profile-based features intermediate between Burkitt lymphomas (BLs) and diffuse large B-cell lymphomas (DLBCLs) and created the provisional category of ‘B-cell lymphoma, unclassifiable, with features intermediate between BL and DLBCL (BCL-U)’.1 From a clinical perspective, however, the introduction of the diagnostic BCL-U category appears problematic, because current therapeutic concepts differ significantly between BL and DLBCL. Moreover, many patients with BCL-U have a dismal prognosis. Translocations of the MYC oncogene are common in BCL-U, usually in the context of a complex genetic background, and 35–50% of BCL-U lymphomas, next to MYC aberrations, harbor additional translocations involving BCL2 and/or BCL6. These cases are referred to as ‘double hit’/’triple hit’ (DH/TH) lymphomas. However, neither MYC translocations nor a ‘double hit’/triple hit’ genetic constellation are unifying genetic features of BCL-U, as ~ 10% of DLBCL carry MYC translocations or show DH/TH features as well.1 Nonetheless, according to current concepts, these B-cell nonHodgkin lymphomas (B-NHLs) are categorized as DLBCL on the basis of their morphological features, although there is evidence that these genetic alterations may predict inferior outcome amongst DLBCL patients.2 The recent rapid progress in next generation sequencing technologies has re-defined the genetic landscape of B-NHL including BL and DLBCL.3–11 To sharpen the diagnostic gray zone between BL and DLBCL, we sequenced relevant genes that are frequently mutated in BL (ID3, TCF3, CCND3 and MYC) and DLBCL (BCL2, EZH2, CREBBP, EP300, MEF2B and SGK1) in 108 aggressive B-cell lymphomas including 31 BL, 24 BCL-U and 53 DLBCL cases, the latter of which were enriched for germinal center-derived (GCB) DLBCL cases and DLBCL with MYC translocations. DLBCL of the activated B-like type and their characteristic mutations (NFkB, B-cell receptor signaling) were not studied, as these cases usually do not fall into the gray zone between BL and DLBCL. Formalinfixed and paraffin-embedded lymphoma specimens were selected from the archives of the Institute of Pathology (University of Würzburg, Würzburg, Germany) and the Department of Clinical Pathology (Robert-Bosch-Krankenhaus, Stuttgart, Germany). The 53 DLBCL cases were subcategorized into three groups, namely 28 DLBCL cases without MYC translocation (DL (GCB)), 16 DLBCL cases with a MYC-only translocation (DL-MYC) and 9 DLBCL with MYC translocation plus additional breaks in the BCL2 and/or BCL6 loci (DL-DH/TH). Representative morphological features for each of
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 brief, each amplicon was processed on the 48.48 Fluidigm Access Array System (Fluidigm Corporation, San Francisco, CA, USA). After attaching barcodes for sample identification and enrichment of the libraries, the samples were pooled and run on the Roche (Mannheim, Germany) GS Junior instrument. Data was analyzed with the GS Amplicon Variant Analyzer Software (Roche) and variant allele frequencies of 420% were validated by Sanger sequencing (detailed methods are provided in the Supplementary Information). The final mutational results of all cases are displayed in Figure 1 and Supplementary Table 1. The mutation frequency of ID3 and/or TCF3 (ID3/TCF3) was comparably high in BL and BCL-U (65% vs 67%). However, the rate of biallelic mutations in ID3 or a mutation in TCF3 was higher in BL (70%) than in BCL-U (37.5%; Supplementary Table 2, Supplementary Figure 3). The frequency of ID3 mutations was lower in the DL-DH/TH (22%) and DL-MYC (37%) subgroups, whereas only one DL (GCB) without a MYC rearrangement (3.5%) carried an ID3 mutation. Initial investigations suggested that ID3 mutations that occur in up to 65% of BL might constitute a discriminatory marker to distinguish between bona fide BLs and non-BLs.3–5 However, the recent report by Gebauer et al.12 already demonstrated the presence of ID3 mutations in 25% of BCL-U cases. In our study, the frequency of ID3 mutations even reached 67% in the BCL-U subgroup thus showing a similar frequency compared with the BL cohort (65%). We even detected ID3 mutations in some GCB DLBCL cases carrying MYC translocations. Of note, all BCL-U cases with MYC (‘single hit’) translocations carried concomitant ID3 mutations, albeit with a lower rate of biallelic mutations. The presence of additional mutations characteristic of DLBCL in many of these cases argues against the idea that these B-NHL might represent ‘true’ BLs. It must therefore be concluded that ID3 mutations do not exclusively occur in BL cases and that the presence or absence of this mutation is not helpful in discriminating BL from non-BL cases. Mutations of CCND3 were observed in all five subgroups at a rate between 11 and 29%. There was no clear enrichment of CCND3 mutations in BL cases (26%), as reported previously,5 as mutations occurred also quite frequently in BCL-U (29%) and DL-MYC (25%), whereas the mutation rate dropped in DL (GCB) without MYC translocations (11%). Thus, CCND3 mutations appear to be enriched in aggressive B-NHL carrying MYC translocations. MYC mutations were highly enriched in aggressive B-NHL cases carrying a MYC translocation. In these lymphomas, the frequency of MYC mutations ranged between 33 and 69%, whereas only few DL (GCB) without MYC translocations showed MYC mutations (7%, Po0.0002, Fisher´s
Accepted article preview online 12 February 2015; advance online publication, 13 March 2015
© 2015 Macmillan Publishers Limited
Leukemia (2015) 1779 – 1797
Letters to the Editor
1790 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) in MYC ‘single hit’ (b) and in MYC ‘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 MYC translocation was highly associated with concomitant MYC mutations (P = 0.0006, Fisher´s exact test). Across all studied subgroups, aggressive B-NHL cases with a MYC-only translocation were more frequently MYC-mutated compared with cases with a ‘double hit’ or ‘triple hit’ constellation (P = 0.0417, Fisher´s exact test). EZH2 mutations were detected in all investigated subgroups except in BL. All mutations in EZH2 altered the tyrosine residue Leukemia (2015) 1779 – 1797
641, which is identical to the most common mutation in previous reports.6,8 The mutation rate of EZH2 in GCB DLBCL (14%) was on the lower side of the reported range (11.7–38.5%).5,6,8,11,14 As EZH2 mutations were found in similar frequencies (around 12%) in DL (GCB), DL-MYC, DL-DH/TH and BCL-U cases, a mutational screening in the diagnostic setting in all of these subgroups would be necessary to identify the candidates for targeted EZH2 inhibition. We even detected two BCL-U cases with concurrent mutations in © 2015 Macmillan Publishers Limited
Letters to the Editor
ID3 and EZH2, which was not observed among pure BL cases. As mutations in ID3 contribute to the activation of PI3K pathway,5 a subset of BCL-U patients might benefit from PI3K and/or EZH2 inhibitors, which are currently tested in preclinical trials.15 BCL2 mutations were most frequent in BCL-U cases (58%) and lowest in BL and DL (GCB) with a rate of 16% and 18%, respectively. Mutations in CREBBP/EP300 were detected in all subgroups, with a clear enrichment in BCL-U (29%) and DL-DH/TH (33%). MEF2B and SGK1 mutations were predominantly detected in DL (GCB) (18% and 21%, respectively) and were rare or absent in other subgroups. We next investigated mutational patterns that occur in each of the subgroups with the aim to determine whether any combination of morphological, immunophenotypic, genetic and mutational features could sharpen the borderline within the spectrum ranging from BL, gray zone lymphoma and GCB DLBCL (especially with MYC rearrangements). On the basis of previously reported data, we assigned mutations of ID3, TCF3, CCND3 and MYC to the Burkitt mutational pattern (‘mutBL’) and mutations of BCL2, EZH2, CREBBP1, EP300, SGK1 and MEF2B to the DLBCL mutational pattern (‘mutDL’). Recognizing the ‘oversimplification’ of this approach, a given lymphoma could then be characterized as having a ‘mutBL’ pattern, a ‘mutDL’ pattern, a combined ‘mutBL/DL’ pattern or none of these patterns. As can be seen in Figure 2a, the scenario was quite homogenous in the BL and DL (GCB) subgroups. As expected, the ‘mutBL’ pattern dominated in the BL subgroup, whereas the ‘mutDL’ pattern dominated amongst DL (GCB) cases thus separating those two groups with regard to their mutational spectrum. In contrast, the other three subgroups (BCL-U, DL-DH/TH and DL-MYC) showed overlapping features with the majority of cases showing a combined ‘mutBL/DL’ pattern. Of note, the vast majority (almost 80%) of DL-MYC cases that are currently diagnosed as DLBCL harbored a ‘mutBL’ or a ‘mutBL/DL’, questioning the current WHO concept that B-NHL with morphological features of DLBCL should be classified as such, regardless of an underlying MYC translocation or a ‘double/triple hit’ constellation. Given that the MYC translocation is considered a dominant oncogenic event in lymphomagenesis, we sought to investigate whether the mutational spectrum of 10 relevant genes correlates with the presence of single or non-single MYC translocations in the B-NHL subgroups studied here. Figure 2b displays the scenario in MYC ‘single hit’ lymphomas including 26 BL, 8 BCL-U and 16 DLMYC cases. As expected, the ‘mutBL’ pattern predominated among BL cases, but approximately one-third of BCL-U and DLMYC cases with MYC translocation (‘single hit’) also showed this feature. Similar results were obtained when MYC ‘double/triple hit’ lymphoma cases were analyzed (Figure 2c), although the ‘mutDL’ pattern occurred more frequently compared with MYC ‘single hit’ lymphomas. In conclusion, our results show that the morphological, immunophenotypic and genetic gray zone between BL and DLBCL is also a gray zone of the mutational spectrum. Thus, it will be difficult to more precisely define molecular subgroups and relevant clinicopathologic entities within this spectrum of aggressive B-cell lymphomas.
CONFLICT OF INTEREST The authors declare no conflict of interest.
1791
ACKNOWLEDGEMENTS We wish to thank Heike Brückner, Eva Bachmann and Tina Grieb for their excellent technical support. This study was supported by the German Ministry for Education and Science (BMBF) in the framework of the ICGC MMML-Seq (01KU1002A-J) Network and by the Robert-Bosch-Stiftung, Stuttgart, Germany.
AUTHOR CONTRIBUTIONS SM, GO and AR designed the research, prepared the figures and wrote the paper. SM, SW, TN, MR, AS and EB performed the experiments. SM, JP and AR analyzed and interpreted the data. AR, GO, MR, EG and SM reviewed and classified the cases.
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Leukemia (2015) 1779 – 1797
