Integrative Genomic Analysis Focused on Cell Cycle Genes for MYC-Driven Aggressive Mature B-Cell Lymphoma

lin Journal of clinical and experimental hematopathology JC Vol. 60 No.3, 87-96, 2020 EH xp ematopathol

Original Article

Integrative genomic analysis focused on cell cycle genes for MYC-driven aggressive mature B-cell lymphoma

Takahisa Yamashita,1,2) Claudia Vollbrecht,1,3,4) Burkhard Hirsch,1) Karsten Kleo,1) Ioannis Anagnostopoulos,1) Michael Hummel1)

MYC is a transcriptional factor that regulates growth and proliferation through cell cycle pathways. MYC alterations, in particular MYC rearrangements, are important in assessing the prognosis of aggressive B-cell lymphoma. In this study, we focused on the impact of nine major cell cycle genes for MYC-driven aggressive mature B-cell lymphoma and analyzed the mutational status using targeted next generation sequencing. Our 40 cases of aggressive mature B-cell lymphomas included 5 Burkitt lymphomas, 17 high-grade B-cell lymphomas and 18 diffuse large B-cell lymphomas with MYC breaks in 100%, 88% and 11%, respectively. Our data allowed a molecular classification into four categories partially independent from the histopathological diagnosis but correlating with the Ki-67 labelling index: (I) harboring TP53 and CDKN2A mutations, being highly proliferative, (II) with MYC rearrangement associated with MYC and/or ID3 mutations, being highly proliferative, (III) with MYC rearrangement combined with additional molecular changes, being highly proliferative, and (IV) with a diverse pattern of molecular alterations, being less proliferative. Taken together, we found that mutations of TP53, CDKN2A, MYC and ID3 are associated with highly proliferative B-cell lymphomas that could profit from novel therapeutic strategies.

Keywords: Cell cycle, Diffuse large B-cell lymphoma, High-grade B-cell lymphoma, MYC

not otherwise specified (NOS). HBL-DH/TH harbors chro- INTRODUCTION mosomal rearrangements of MYC at the 8q24 locus and MYC is a transcriptional factor and proto-oncoprotein BCL2 at the 18q21 locus, and/or BCL6 at the 3q27 locus. that mediates apoptosis, differentiation and proliferation, HBL with MYC/BCL2 rearrangements accounts for most deregulation of its gene is detected in 50-70% of all human cases of this entity and has a more dismal prognosis than typ- malignancies.1,2 In particular, MYC rearrangement is often ical DLBCL.6,7 On the other hand, HBL NOS does not har- strongly correlated with a poor prognosis in a variety of bor double rearrangements, but approximately 20-35% of malignant hematological tumors and is known not only as a these have isolated MYC rearrangement with or without copy hallmark of Burkitt lymphoma (BL), but also as a prognostic number alteration or amplification of the BCL2 region. factor in other aggressive mature B-cell lymphomas such as Some cases harbor BCL2 rearrangement with MYC copy diffuse large B-cell lymphoma (DLBCL) and high-grade number alteration or amplification.8-10 However, the precise B-cell lymphoma (HBL).3,4 HBL was established as a new definition of HBL NOS is currently controversial and not disease entity in the current 2017 World Health Organization well defined. Additionally, the differentiation from DLBCL (WHO) classification of Haematopoietic and Lymphoid with MYC abnormalities or BL and HBL NOS is impeded Tissue, and partly replaced the provisional entity of B-cell due to limited knowledge of the biological background, lymphoma, unclassifiable with features intermediate between allowing merely insufficient morphological or immunophe- DLBCL and BL (BCL-U) of the previous classification.5 notypical categorization. Therefore, a more reliable, easily The HBL category is further divided into two subtypes: (1) applicable and accurate method to classify these categories is HBL associated with MYC and BCL2 and/or BCL6 rearrange- required. Next generation sequencing (NGS) of BL has elu- ments [HBL-double hit (DH)/triple hit (TH)], and (2) HBL cidated recurrent somatic mutations in CCND3, ID3, TCF3

Received: May 25, 2020. Revised: June 18, 2020. Accepted: June 22, 2020. Onlune Published: September 25, 2020 DOI:10.3960/jslrt.20021 1)Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Berlin, Germany, 2)Department of Pathology, Saitama Medical Center, Saitama Medical University, Saitama, Japan, 3)German Cancer Consortium (DKTK), Berlin, Germany, 4)German Cancer Research Center (DKFZ), Heidelberg, Germany Corresponding author: Takahisa Yamashita, Department of Pathology, Saitama Medical Center, Saitama Medical University, Kamoda, Kawagoe-shi, Saitama, 350-8550, Japan. E-mail: [email protected] Copyright © 2020 The Japanese Society for Lymphoreticular Tissue Research This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

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and TP53, which add to the malignant potential of BL by Commission of the Charité-Universitätsmedizin (approval activating the cell cycle pathway.11-13 Momose et al. reported number: EA4/129/15). that recurrent somatic mutations of CCND3, ID3, TCF3 and Immunohistochemistry (IHC) MYC were detected not only in BL, but also in the other MYC-driven aggressive mature B-cell lymphomas, like HBL The following antibodies were used for IHC: anti-CD3 and DLBCL with MYC rearrangement.14 Furthermore, inac- (clone: LN10, dilution: 1:100, Novocastra, Leica Biosystems, tivating mutations of tumor suppressor genes (e.g. BTG1, Germany), anti-CD10 (clone: 56C6, dilution: 1:25, BTG2, CDKN2A, and TP53) mediating cell cycle arrest were Novocastra, Leica Biosystems), anti-CD20 (clone: L26, dilu- detected in MYC-driven aggressive mature B-cell lympho- tion: 1:100, DAKO, Agilent-Dako, USA), anti-BCL2 (clone: mas.9,15-18 Recently, many alterations of cell cycle- or apop- 124, dilution: 1:25, Agilent-Dako), anti-BCL6 (clone: tosis-genes were detected in approximately 40% of HBL-DH/ PG-B6p, dilution: 1:25, Agilent-Dako), anti-IRF4 (clone: TH cases.19 In this context, it is conceivable that the molec- MUM1p, dilution: 1:25, Agilent-Dako), anti-MYC (clone: ular change in these specific cell cycle genes affects many Y69, dilution: 1:300, Epitomics, USA) and anti-Ki-67 (clone: MYC-driven aggressive mature B-cell lymphomas. Moreover, MIB-1, dilution: 1:200, Agilent-Dako). Immunohistochemical the mutational status may allow a sub-classification into staining was performed using BOND-MAX (Leica, molecular subgroups independent from the histopathological Germany) according to the manufacturer’s instructions. diagnosis. Protein expression was assessed independently by two In this study, we focused on the mutational status of cell pathologists (I.A and T.Y) and was categorized into two cycle genes (BTG1, BTG2, CCND3, CDKN2A, ID3, MAX, groups according to the proportion of expressing cells in sev- MYC, TCF3 and TP53) to gain deeper insights into the eral areas of the specimens by visual examination. The cut- molecular mechanisms of MYC-driven aggressive mature off value for positive expression of each antibody was set as B-cell lymphoma. follows: ≥90% of lymphoma cells stained for CD20, ≥30% of lymphoma cells stained for CD10, BCL2, BCL6 and IRF4, and ≥40% of lymphoma cells stained for MYC. The defini- MATERIALS AND METHODS tion of double expressor (DE) lymphoma followed the WHO criteria for MYC expression (≥40%) and BCL2 expression Case selection (≥50%). The percentage of lymphoma cells with Ki-67 We selected 168 cases of aggressive mature B-cell lym- nuclear immunostaining was defined as Ki-67 LI. We sepa- phoma diagnosed as BL (n=12), HBL-DH/TH (n=17), HBL rated our cases into 3 groups according to the expression rate NOS (n=8) and DLBCL NOS (n=131) according to the crite- of MYC as follows: M1: 40-59%, M2: 60-79% and M3: ria of the current WHO classification between 2016 and 2018 80-100%. A further subdivision was made according to diagnosed at the Institute of Pathology at the Charité - Ki-67 LI in the following manner: less proliferative K1: Universitätsmedizin Berlin. These cases were reviewed by 80-94% and highly proliferative K2: 95-100% because a two pathologists (I.A and T.Y). Next, we selected 68 of 168 Ki-67 LI over 95% is one of the criteria for highly aggressive cases that fulfilled our criteria for MYC-driven aggressive lymphoma such as BL.24 mature B-cell lymphoma, which were analyzed for not only Fluorescence in situ hybridization (FISH) analysis MYC expression (≥40%), but also the Ki-67 labeling index (LI) (≥80%). As we used the Ki-67 LI for case selection, The interphase FISH analyses were carried out to detect Ki-67 was both a useful prognostic marker and cell cycle- translocations of MYC (Vysis LSI MYC-dual color break- related cellular marker for cell proliferation in the cases of apart rearrangement probe, Abbott, Germany), BCL2 (dual aggressive lymphoma. Recent studies reported that overex- color break-apart probe, DAKO) and BCL6 (Vysis LSI pression of Ki-67 was a predictor of a dismal prognosis in BCL6-dual color break-apart rearrangement probe, Abbott). patients treated using rituximab.20-22 Moreover, MYC The cut-off levels for positivity were 18%, 7% and 11% for expression was independent of Ki-67 and mature B-cell lym- MYC, BCL2 and BCL6 in the routine diagnostic setting, phomas harboring high MYC expression did not always have respectively. The total counted number of target cells was high proliferation rates.23 Our criteria of MYC-driven 50 to 100 cells for the detection of breaks, and all cases were aggressive mature B-cell lymphoma referenced the previous independently evaluated by two or more investigators. report, which compared the expression of MYC with that of Initially, we selected 168 cases consisting of 46 cases (27%) Ki-67.16,23 Finally, we excluded 28 cases, in particular CD5- of MYC rearrangement, 39 cases (23%) of BCL2 rearrange- positive DLBCL, DLBCL with coexistent or transformed fol- ment, 43 cases (26%) of BCL6 rearrangement and 68 cases licular lymphoma (FL), human immune deficiency virus- (40%) without rearrangements (Supplementary figure 1). related lymphoma, primary central nervous system Next generation sequencing (NGS) lymphoma, and cases mostly exhibiting necrotic tissue or DNA preparation insufficient material for our analysis (Supplementary figure 1). DNA was extracted from formalin-fixed and paraffin- This study was conducted in accordance with the embedded (FFPE) tissue sections using the Maxwell 16 Declaration of Helsinki and was approved by the Ethics FFPE plus LEV DNA purification kit (Promega, Germany)

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according to the manufacturer’s protocol, followed by fluo- RESULTS rescence-based quantification using Qubit dsDNA HS assay kit (ThermoFisher Scientific, USA). Immunohistochemistry and FISH Design of B-cell lymphoma gene panel The majority of cases (28 out of 40; 70%) displayed a The B-cell lymphoma specific gene panel spans a germinal center B-cell lymphoma immunophenotype (GCB) genomic region of 6.72 kb covering proportions of the coding according to the Hans algorithm,29 whereby 12 cases (30%) regions of nine genes relevant for cell cycle regulation were assigned to non-germinal center B-cells (non-GCB). (BTG1, BTG2, CCND3, CDKN2A, ID3, MAX, MYC, TCF3 All HBL cases with the exception of one (16 out of 17; 94%) and TP53) and was custom-designed using the Ion Ampliseq were also of the GCB type. Thirty-one (89%) of 35 cases of Designer (Version v7.0.9, Thermo Fisher Scientific) DLBCL or blastoid morphology (not including BL) demon- (Supplementary table 1). strated co-expression of MYC and BCL2 (DE lymphoma), and were divided into subgroups according to their percent- Library preparation, sequencing and data analysis age of MYC or Ki-67-expressing tumor cells. All cases of Amplification of the selected gene regions by multiplex the BL subgroup belonged to M3/K2, whereas a decreasing polymerase chain reaction (PCR) was performed with 40 ng percentage of HBL cases (71% M3, 88% K2) and DLBCL of DNA input for each primer pool. (Supplementary table cases (61% M3, 56% K2) was assigned to these two sub- 2). Subsequently, adaptors were ligated following the groups (Table 1 and Supplementary figure 2). The detection instructions of NEXTflex DNA Sequencing Kit for Ion of chromosomal breaks was performed by FISH for MYC, Torrent, Manual V15.12 (Bioo Scientific, USA). Finally, BCL2 and BCL6 genes. MYC breaks were detectable in all 5 library quality was analyzed by microfluidic electrophoresis cases of BL, 15 out of 17 cases (88%) of HBL and 2 out of using Fragment Analyzer (Agilent Technologies, USA) and 18 cases (11%) of DLBCL. Correlation with MYC expres- quantified using Ion Library TaqMan Quantitation Kit sion demonstrated the presence of MYC breaks in 50% (1/2) (Thermo Fisher Scientific). Libraries were sequenced on the of subgroup M1, 30% (3/10) of subgroup M2 and 64% Ion S5XL system (Thermo Fisher Scientific). Fastq files (18/28) of subgroup M3. BCL6 breaks were detectable in 7 were analyzed using the CLC Genomics Workbench, version cases of DLBCL and in 3 cases of HBL; BCL2 breaks were 5.0.1 (Qiagen, Hilden, Germany) after alignment, mapping to found in 8 cases of HBL and 3 cases of DLBCL. Seventeen the hg19 human reference genome and corresponding target HBL cases were classified as HBL-DH, 9 cases as HBL-TH regions as determined in the custom panel bed file. The and 8 cases as HBL NOS using a combined evaluation of quality of base scoring and minimum depth of coverage met FISH, and morphological and immunohistochemical findings the criteria of Q20, which is equal to an error rate of 1% and (Figure 1). a minimum of > 250 reads.25 The cut-off value of allele fre- Targeted next generation sequencing (NGS) quency for mutation detection was set to 5% for tissue sam- ple analysis according to our results of the limit of detection In order to determine the sensitivity of mutation detection (LoD).25 and uniformity of the amplicon distribution, we employed serial dilutions of DNA of BL cell line Raji extracted from Determining the limit of detection (LoD) non-diseased human tonsils. The resulting allelic frequen- In order to determine the LoD of our NGS assay, DNA cies of mutations, as exemplified by the MYC and TP53 from BL cell line Raji with known mutations (MYC, genes, demonstrated high correlation with the calculated dilu- NP_002458: p.V20A, p.S21T, p.E54D, p.A59V; TP53, tion of cell line DNA ranging from 100% to 5% (Figure 2 NP_000537: p.R213Q, p.Y234H) was serially diluted to 5%, and Supplementary table 3). Non-synonymous mutations 20% or undiluted (100%) with un-mutated tonsil reference determined by NGS of clinical samples consisted of 57 mis- DNA, and analyzed in duplicate by NGS as described sense mutations, 7 frameshifts and 6 nonsense mutations above.13 (Figure 3, Supplementary table 4). All analyzed cell cycle regulating genes were mutated at varying frequencies: MYC Prediction analysis of single nucleotide variants (number of variants/number of samples: 19/10), TP53 To investigate the impact of the DNA variants detected in (11/10), ID3 (8/7), BTG2 (11/5), BTG1 (5/4), CDKN2A (4/4), the histological samples on protein structure/function, we TCF3 (3/3), CCND3 (2/2) and MAX (1/1), respectively. Five used the web-based tools MutationAssessor (cBio@MSKCC, variants were excluded because of non-cancerous change and release 3),26 Functional Analysis through Hidden Markov low impact defined on all predictive analyses. According to Model (FATHMM, version 2.3)27 and Clinical Interpretation our results, almost all cases were able to be classified molec- of Genetic Variants by the 2015 ACMG-AMP Guidelines ularly into four categories (Figure 4). Category I (12/40 (InterVar, version 0.1.7).28 cases) is characterized by the dominance of TP53 and CDKN2A mutations, which were exclusively found in this subgroup. The majority of cases belonged to DLBCL (7 cases), and the remaining cases belonged to HBL (3 cases) and BL (2 cases). For example, case 46 (DLBCL) had three

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Table 1. FISH and IHC results

BL HBL-DH/TH HBS NOS DLBCL Total Case number 5 9 8 18 40 Hans algorithm GCB/non-GCB 5/0 9/0 7/1 7/11 28/12 FISH Rearrangement MYC 5 (100%) 9 (100%) 6 (75%) 2 (11%) 21 (53%) BCL2 0 (0%) 8 (89%) 0 (0%) 3 (17%) 11 (28%) BCL6 0 (0%) 2 (22%) 1 (13%) 7 (39%) 10 (25%) IHC MYC M1 (40-59%) 0 (0%) 0 (0%) 0 (0%) 2 (10%) 2 (5%) M2 (60-79%) 0 (0%) 3 (33%) 2 (25%) 5 (28%) 10 (25%) M3 (80-100%) 5 (100%) 6 (67%) 6 (75%) 11 (61%) 28 (70%) BCL2 0 (0%) 9 (100%) 5 (65%) 17 (94%) 31 (78%) BCL6 5 (100%) 8 (89%) 7 (88%) 17 (94%) 37 (93%) CD10 5 (100%) 8 (89%) 6 (75%) 7 (39%) 26 (65%) IRF4 5 (100%) 2 (22%) 4 (50%) 16 (89%) 27 (68%) Ki-67 K1 (80-94%) 0 (0%) 2 (22%) 0 (100%) 8 (44%) 10 (25%) K2 (95-100%) 5 (100%) 7 (78%) 8 (100%) 10 (56%) 30 (75%) DE lymphoma Positive 0 (0%) 9 (100%) 5 (63%) 17 (94%) 31 (78%)

FISH: Fluorescence in situ hybridization, IHC: Immunohistochemistry, BL: Burkitt lymphoma, HBL-DH/TH: high-grade B-cell lymphoma double hit/triple hit, HBL NOS: high-grade B-cell lymphoma not otherwise specified, DLBCL: diffuse large B-cell lymphoma, GCB: germinal center B-cell lymphoma immunophenotype, DE lymphoma: double expressor lymphoma

Fig. 1. Representative FISH results (a) Case 35 (BL) MYC (+)/BCL2 (-)/BCL6 (-), (b) Case 42 (HBL NOS) MYC (+)/BCL2 (-)/BCL6 (-), (c) Case14 (HBL-TH) MYC (+)/BCL2 (+)/BCL6 (+). FISH: Fluorescence in situ hybridization, BL: Burkitt lymphoma, HBL NOS: high-grade B-cell lymphoma, not otherwise specified, HBL TH: high-grade B-cell lymphoma triple hit.

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Fig. 2. Determining the limit of detection Sequencing results of serial dilutions of mutated Burkitt lymphoma cell line Raji with unmutated tonsil DNA. A high correlation of expected and effective variant allele frequencies was noted, even at tumor cell dilutions down to 5%.

Fig. 3. Summary of non-synonymous variants detected by the B-cell lymphoma specific NGS panel (a) Splitting the number of non-synonymous variants by disease revealed the highest amount for HBL and DLCBL; (b) Number of non-synonymous detected variants by gene; Most alteration types were missense mutations (81%), with MYC as the most frequently altered gene (23 variants). NGS: next generation sequencing, HBL: high-grade B-cell lymphoma, DLBCL: diffuse large B-cell lymphoma. mutations in CDKN2A (p.A148T), BTG1 (p.L37M) and was dominated by mutations in ID3 and MYC, and samples BTG2 (p.L10F), and no chromosomal rearrangements. harbored MYC translocations in the majority of cases (7 BTG1 mutation (p.L37M, MSKCC impact: medium, cases). With the exception of one case, all others belonged FATHMM impact: oncogenic/non-cancer associated) located to HBL (5 cases) or BL (3 cases). Almost all cases of cate- in the potential phosphorylation site commonly found in gory III (9 cases) carried MYC breaks, and 6 cases displayed aggressive B-cell lymphoma has a significant function to additional BCL2 (5 cases) or BCL6 (1 case) breaks. Only accelerate the cell cycle.30 The in silico analysis predicted a three of the investigated genes (BTG2, TCF3 and MAX) were functional impact of the CDKN2A mutation p.A148T associated with mutations. Category IV (10 cases) is com- (MSKCC impact: medium, FATHMM impact: neutral/can- posed of mainly DLBCL cases; only two cases belonged to cer-associated) (Table 2). Category II (9/40 cases) the HBL group. Based on the molecular status, this category

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Fig. 4. Molecular classification of MYC-driven aggressive mature B-cell lymphoma based on IHC, FISH and NGS The block color represents the type of rearrangement and mutational status lined by expression of MYC and Ki-67. Categories I and II are included in the aggressive type (K2 group) and Category III (K1 group) is a less aggressive type than the others. IHC: immunohistochemistry, FISH: fluorescence in situ hybridization, NGS: next generation sequencing, DLBCL: diffuse large B-cell lymphoma.

Table 2. TP53 and CDKN2A mutations

Changed MSKCC FATHMM N Diag. Gene Mut. DBD Func. DNA amino acid impact Impact 5 DL TP53 N c.586C>T p.R196 + LOF NA Onco./ Cancer 11 HBS TP53 M c.738G>A p.M246I + LOF Med. Onco./ Cancer NOS 16 DL TP53 M c.818G>A p.R273H + LOF Med. Onco./ Cancer 28 HBL TP53 M c.417G>T p.K139N + LOF Med. Onco./ Cancer DH c.700T>G p.Y234D LOF 32 BL TP53 M c.843C>A p.D281E + LOF Med. Onco./ Cancer 44 DL TP53 M c.413C>T p.A138V + LOF Med. Onco./ Cancer 52 DL TP53 M c.817C>G p.R273G + LOF Med. Onco./ Cancer 58 DL TP53 M c.701A>G p.Y234C + LOF Med. Onco./ Cancer 62 HBL TP53 M c.847C>T p.R283C + LOF Low Onco./ Cancer NOS 66 DL TP53 M c.542G>C p.R181P + LOF Med. Onco./ Cancer 5 DL CDKN2A M c.442G>A p.A148T - LOF? Med. Neut./ Cancer 35 BL CDKN2A M c.442G>A p.A148T - LOF? Med. Neut./ Cancer 46 DL CDKN2A M c.442G>A p.A148T - LOF? Med. Neut./ Cancer 66 DL CDKN2A M c.442G>A p.A148T - LOF? Med. Neut./ Cancer

N: case number, Diag: diagnosis, DL: diffuse large B-cell lymphoma, HBL NOS: high-grade B-cell lymphoma not otherwise specified, HBL DH: high-grade B-cell lymphoma double hit, BL: Burkitt lymphoma, Mut: mutational type, N: nonsense mutation, M: missense mutation, DBD: DNA-binding region, Func: function, LOF: loss of function, NA: not avail- able, Med: medium impact, Onco: oncogenic impact, Neut: neutral impact, Cancer: cancer-associated variant

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is heterogeneous with few scattered mutations and a predom- reported that the variant p.A148T may affect the function of inance of BCL6 breaks. Categories I, II and III are charac- p16INK4a through the CDKN2A promoter.45,46 Our in silico terized by a very high proliferation rate (Ki-67 LI: ≥95%) analysis demonstrated that this alteration induced cancer- and categories I and II are dominated by a high load of muta- associated ability (MSKCC impact; medium, FATHMM tions in genes involved in proliferation control. impact; cancer-associated). Taken together, these mutations Interestingly, category III had only few mutations in prolifer- of TP53 and CDKN2A may affect the progression, develop- ation-associated genes despite a high proportion of HBL-DH/ ment and survival of MYC-driven aggressive mature B-cell TH cases. Taken together, our approach allows a molecular lymphoma. categorization of MYC-driven aggressive mature B-cell lym- The highly proliferative cases of Category II are charac- phoma into four groups, which is partially independent from terized by MYC rearrangement with MYC and/or ID3 muta- pathological diagnosis. tion, and consisted of 5 HBL cases (63%) and 3 BL cases (37%). Most of the Category II samples had two or more mutations in all nine cell-cycle associated genes. Both HBL DISCUSSION with MYC mono-rearrangement and BL cases of this cate- In this study, we examined the mutational status of nine gory are similar in terms of the mutational status (MYC, ID3 cell cycle genes of MYC-driven aggressive mature B-cell and CCND3). These BL-specific mutations may give the lymphoma, and identified four molecular categories accord- aggressive malignant ability not only for BL, but also for ing to the combined results of targeted NGS and FISH that HBL and DLBCL as previously assumed.14,47 For example, correlated with proliferative activity but only partially with case 30 (HBL NOS) displayed not only BL-like morphologi- the histopathological diagnosis. Of note, the highly prolifer- cal features, but also a BL-like mutational status of MYC ative types (Categories I and II) demonstrated a high muta- (p.E54D, p.S107R, p.G152A), ID3 (p.V67I, p.E68fs) and tional frequency in the analyzed cell cycle-regulating genes. CCND3 (p.L292fs), with MYC rearrangement and, in addi- Category I is mainly based on the mutations of TP53 and tion, a strong expression of BCL2 (Figure 5). In particular, CDKN2A (Table 2). The missense and nonsense mutations both the transactivation domain (aa position: 12-155) in exon of these tumor suppressor genes are known to be driver muta- 2 of MYC and loop-helix-loop (HLH) domain (aa position: tions and prognostic factors in numerous tumors, and directly 43-80) in exon 1 of ID3 commonly mutated in BL (known as affect cell cycle progression.31-34 In particular, the DNA- a hot spot region) and may be important for the pathogenesis binding site of TP53, which ranges from exon 4 to 9, is very of these tumors.12,13,30 Almost all cases of Category III had important for p53 activity and mutations in this region lead to MYC rearrangements – comparable to Category II - but addi- not only loss-of-function due to their ability to act as domi- tional chromosomal rearrangements are found frequently. nant-negative function of wild type p53, but also to a gain-of- The high proliferative activity of this category is likely sup- function independent of the effects on wild type p53.35 In ported by the chromosomal rearrangements and modulated our cases, the missense mutations of this region matched by other molecular changes. Evrard et al. recently reported those described in previous reports.36,37 Moreover, the cases that CREBBP mutations - most frequently mutated in with point mutations in the DNA binding site of TP53 had a HBL-DH/TH cases - lead not only to accelerated BCL6 activ- significantly poorer prognosis than those with mutations in ity, but were also able to suppress p53 function.19 For this the non-DNA binding domain.38 This is in concordance with reason, Category III may be influenced by the other altera- our results, as Category I (highly proliferative) consists of tions of cell cycle pathway. Taken together, Categories I, II cases with mutations in the TP53 DNA-binding domain. and III had increased proliferative activity, which is associ- The other frequent molecular alterations in Category I are ated with characteristic molecular alterations. Lastly, 15 alterations of CDKN2A that encode two cycle-dependent (83%) of 18 DLBCL cases were classified into either kinase inhibitors, p16INK4a and p14AR, which directly neg- Category I (7 cases) or IV (8 cases). Seven cases of 8 atively regulate the cell cycle like p53.39 Deletion of this DLBCL cases in Category IV had at least one rearrangement gene is a prognostic factor in many tumors40 and missense and half had additional alterations (MYC, BTG1, BTG2 and mutations of this gene were detected previously in some TCF3) of cell cycle genes. This suggested that TP53 and DLBCL cases.41 The common CDKN2A polymorphism CDKN2A alterations separate Category I (highly proliferative p.A148T found in 4 cases was previously described in sev- type) from Category IV (less proliferative type) in MYC- eral cancer types such as colon-, lung-, breast-, bladder-can- driven DLBCL cases. cer and melanoma.40,42 This variant and amino acid change In conclusion, our targeted NGS approach using a gene located in exon 2 of CDKN2A and in the open reading frame panel for cell cycle related genes in conjunction with chro- of p16INK4a are not directly involved in binding to cyclin mosomal rearrangements was able to identify four molecular kinases, CDK4 or CDK6. Thus, this variant (p.A148T) may categories of aggressive B-cell lymphomas. Category I not have direct effects on the inhibitor activity, but the func- based on TP53 and CDKN2A mutations, Category II based tional impact is unclear. However, previous functional stud- on MYC rearrangement with MYC and/or ID3 mutation, ies suggested that this alteration (p.A148T) can reduce its Category III based on MYC rearrangement with one addi- ability to inhibit the cell cycle in both melanoma and acute tional molecular alteration, and Category IV based on one lymphoblastic leukemia cell lines.43,44 Debniak et al. rearrangement of BCL2 or BCL6 without TP53, CDKN2A,

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Fig. 5. Histopathological findings in HBL NOS Case 30 (HBL, NOS: Category II) exhibited (a) morphology (H&E stain) and expression of (b) CD20, (c) CD10, (d) MYC, and (e) Ki-67 similar to BL, but (f) with the additional expression of BCL2. HBL NOS: high-grade B-cell lymphoma not otherwise specified.

ID3 or MYC mutations. Thus, our study demonstrated, CONFLICT OF INTEREST despite the limited number of cases and lack of clinical information, that alterations in cell cycles genes provide added The authors declare that they have no conflict of interest. value for a more precise sub-classification of highly aggressive B-cell lymphoma. Consequently, Categories I, II and REFERENCES III having characteristic molecular features and the highest Ki-67 LI should be further evaluated for novel treatment 1 Ellrott K, Bailey MH, Saksena G, et al. Scalable open science strategies. approach for mutation calling of tumor exomes using multiple genomic pipelines. Cell Syst. 2018; 6 : 271-281. e7. 2 Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and AUTHOR CONTRIBUTIONS human neoplastic disease. Oncogene. 1999; 18 : 3004-3016. T.Y., C.V. and M.H. analyzed all categories. T.Y. and 3 Savage KJ, Johnson NA, Ben-Neriah S, et al. MYC gene rear- I.A. analyzed the pathological part. B.H. and K.K. contrib- rangements are associated with a poor prognosis in diffuse large uted to the molecular analysis. B-cell lymphoma patients treated with R-CHOP chemotherapy. Blood. 2009; 114 : 3533-3537. 4 Li S, Weiss VL, Wang XJ, et al. High-grade B-cell lymphoma ACKNOWLEDGMENTS with MYC rearrangement and without BCL2 and BCL6 rear- We thank the molecular pathology team at the Charité, rangements is associated with high P53 expression and a poor especially Anke Sommerfeld, Erika Berg, Edda von der Wall, prognosis. Am J Surg Pathol. 2016; 40 : 253-261. Hedwig Lammert and Arndt Boshof, for their technical 5 Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of assistance. the World Health Organization classification of lymphoid neo- plasms. Blood. 2016; 127 : 2375-2390. 6 Sarkozy C, Traverse-Glehen A, Coiffier B. Double-hit and dou- FUNDING ble-protein-expression lymphomas: aggressive and refractory This study was not supported by any commercial lymphomas. Lancet Oncol. 2015; 16 : e555-e567. foundation. 7 Zeng D, Desai A, Yan F, et al. Challenges and opportunities for high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement (double-hit lymphoma). Am J Clin Oncol.

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96 Analysis of cell cycle gene for lymphoma

Supplementary Materials

Supplementary Fig. S1. Flow diagram for case selection The information of case selection in our study by IHC selection and exception of particular cases. FISH: fluorescence in situ hybridization, Ki-67 LI: Ki-67 labeling index, BL: Burkitt lymphoma, HBL-DH/TH: high- grade B-cell lymphoma double hit/ triple hit, HBL NOS: high-grade B-cell lymphoma, not otherwise classified, DLBCL: diffuse large B-cell lymphoma, HIV: human immune deficiency virus, IHC: immunohistochemistry.

Supplementary Fig. S2. IHC and rearrangement results of all cases According to the Hans algorithm, 28 cases (70%) were assigned to the GCB type and 12 cases (30%) were assigned to the non-GCB type. After exclusion of Burkitt lymphoma, 31 (89%) of 35 cases were double expressors. MYC, BCL2 and BCL6 rearrangements were found in 22 (55%), 11 (28%) and 10 (25%) of 40 cases. BL: Burkitt lymphoma, HBL-DH/TH: high-grade B-cell lymphoma double hit/ triple hit, HBL NOS: high-grade B-cell lymphoma, not otherwise classified, DLBCL: diffuse large B-cell lymphoma, IHC: immunohistochemistry, GCB: germinal center B-cell lymphoma. BCL-U: B-cell lymphoma, unclassifiable with features intermediate between DLBCL and Burkitt lymphoma, DLBCL: diffuse large B-cell lymphoma. Yamashita T, et al.

Supplementary Table S1. Target regions of our panel

Exonregion Targetregion Gene (Accession No.) Chr. Exon Coveroftargetregion(%) Start End Start End MYC (NM_002467) 8 1 128748315 to 128748869 128748829 to 128748879 100 2 128750494 to 128751265 128750483 to 128751275 94.19 3 128752642 to 128753680 128752631 to 128753214 100 MAX (NM_002382) 14 1 65569022 to 65569057 65569011 to 65569067 66.07 2 65568264 to 65568290 65568253 to 65568300 100 3 65560426 to 65560533 65560415 to 65560543 100 4 65544631 to 65544754 65544620 to 65544764 100 5 65541842 to 65543381 65543183 to 65543391 83.65 MAX (NM_001271068) 3 65550457 to 65551017 65550887 to 65551027 100 TP53 (NM_000546) 17 5 7578371 to 7579554 7578371 to 7579554 95.63 6 7578177 to 7578289 7578177 to 7578289 100 7 7577499 to 7577608 7577499 to 7577608 100 8 7577019 to 7577155 7577019 to 7577155 100 CCND3 (NM_001760) 6 5 41902671 to 41903845 41903671 to 41903951 100 CDKN2A (NM_00077) 9 2 21970901 to 21971207 21970901 to 21970991 100 BTG1 (NM_001731) 12 1 92539164 to 92539673 92539164 to 92539312 100 2 92534054 to 92538223 92537856 to 92538223 100 BTG2 (NM_006763) 1 1 203274664 to 203274876 203274735 to 203274876 95.04 2 203276232 to 203278729 203276227 to 203276553 97.06 ID3 (NM_002167) 1 1 23885618 to 23886285 23885607 to 23885927 95 2 23885426 to 23885510 23885440 to 23885520 100 TCF3 (NM_001136139) 19 17 1612206 to 1612432 1612290 to 1612380 100

The information of exon regions, target regions of each gene and the coverage of target regions by each amplicon. Chr: chromosome.

Supplementary Table S2. PCR composition and condition Supplementary Table S3. Limit of detection (Raji)

(A) PCR Component Amount VAF 100% 20% 5% Qiagen Multiplex PCR master mix (Qiagen) 12 μl 9.5 2.7 MYC p.V20A 58.7 Ion ampliseq custom panel primer pool (Thermo Fisher) 8 μl 8.0 2.7 Diluted FFPE DNA 5 μl (40 ng) 8.3 2.1 p.S21T 56.7 Total 25 μl 7.1 1.7 8.5 2.3 p.E54D 45.2 9.2 2.6 (B) PCR condition 11.7 3.5 p.A59V 50.4 First denaturation (95 ºC) 15 min 11.6 3.6 23 cycles Denaturation (94 ºC) 30 sec 5.1 1.2 TP53 p.R213Q 37.7 Annealing (60 ºC) 90 sec 6.5 1.5 Extension (68 ºC) 15 sec 10.7 2.7 p.Y234H 59.7 Final extension (72 ºC) 10 min 10.1 3.1

(A) The information of the amounts of DNA polymerase, DNA extracted The precise percentage (%) of effected VAF of MYC from FFPE and composition of the PCR primers. (B) PCR conditions, (p.V20A, p.S21T, p.E54D, p.A59V) and TP53 including the cycle number, and the degree and time of denaturation, (p.R213Q, p.Y234H). VAF: variant allele fraction. annealing and extension. PCR: polymerase chain reaction, FFPE: formalin- fixed and paraffin-embedded Supplementary Table S4. Pathological and genetic status/prediction-function FATHMM impact FATHMM impact InterVar Cancer-associated MSKCC impact Disease- Pathogenesis/ variant (Cancer)/ High/Medium/ Oncogenic Likely pathogen- non-cancer-associ- Low (Oncogenic)/ esis/Uncertain sig- ated variant Neutral nificance/Benign (Non-Cancer)

Case Rearrangement Hans IHC ID3 (NP_ BTG2 (NP_ CCND3 (NP_ MYC (NP_ CDKN2A (NP_ BTG1 (NP_ MAX (NP_ TP53 (NP_ TCF3 (NP_ num- Diagnosis algo- MYC Ki-67 MYC BCL2 BCL6 BCL2 CD10 BCL6 IRF4 002158) 006754) 001751) 002458) 000068) 001722) 002373) 0000537) 001129611) ber rithm (%) (%)

D37G (Medium/ HBL Oncogenic/Non- 4 + - - GCB 90 - + + - 99 NOS cancer/Uncertain significance) A148T (Medium/ R196* (NA/NA/ 5 DLBCL - - + GCB 99 - + + + 99 Neutral/Cancer/ NA/Pathogenic) Benign) S53I (Medium/ Oncogenic/Non- cancer/Likely pathogenic) P72T (Medium/ Oncogenic/Non- cancer/Likely pathogenic) 6 HBL-DH + + - GCB 70 + + - - 95 L26fs (NA) T73I (Medium/ Oncogenic/Non- cancer/Likely pathogenic) K158N (Medium/ NA/Non-cancer/ Uncertain significance) 9 HBL-DH + + - GCB 80 + + + + 99 H74D (Medium/ non- Oncogenic/Non- 10 DLBCL - - + 99 + - + + 90 GCB cancer/Uncertain significance) M246l (Medium/ HBL Oncogenic/ 11 + - - GCB 90 + + + + 99 NOS Cancer/Likely

pathogenic) Analysis ofcellcyclegenefor lymphoma N551K (Medium/ Oncogenic/Non- 12 HBL-DH + + - GCB 60 + + + - 99 cancer/Likely pathogenic) 13 HBL-DH + + - GCB 80 + + + + 99 P17A (Medium/ Oncogenic/Non- cancer/Uncertain significance) Y27F (Medium/ Oncogenic/Non- 14 HBL-TH + + + GCB 90 + + + - 80 cancer/Uncertain significance) Y47C (Medium/ Oncogenic/Non- cancer/Uncertain significance) Yamashita T,

non- 15 DLBCL - + - 40 + - + + 85

GCB et al. R273H (Medium/ non- Oncogenic/ 16 DLBCL - - - 90 + - - + 95 GCB Cancer/ Pathogenic) HBL 17 + - - GCB 80 - + + - 99 NOS non- 19 DLBCL - - + 60 + - + + 90 GCB L70P (High/ Oncogenic/ 20 BL + - - GCB 90 - + + + 99 Cancer/Uncertain significance) 21 HBL-DH + + - GCB 90 + + + - 95 L20F (Low/ Oncogenic/Non- cancer/Uncertain D63N (Medium/ non- significance) Oncogenic/Non- 27 DLBCL - - + 80 + - + + 95 GCB G117D (Medium/ cancer/Uncertain Oncogenic/Non- significance) cancer/Uncertain significance) Y234D (Medium/ Oncogenic/ Cancer/Likely pathologic) 28 HBL-DH + + - GCB 80 + + + - 95 K139N (Medium/ Oncogenic/ Cancer/Uncertain significance) E54D (Low/ Oncogenic/Non- E68fs(NA) cancer/Likely V67L(Low/ HBL pathogenic) 30 + - - GCB 90 + + + + 99 Oncogenic/Non- L292fs (NA) NOS G152A (Medium/ cancer/Uncertain NA/Non-cancer/ significance) Uncertain significance) D281E (Medium/ Oncogenic/ 32 BL + - - GCB 90 - + - + 99 Q63* (NA) Cancer/Uncertain significance) T73S (Medium/ Oncogenic/Non- 34 BL + - - GCB 90 - + + + 99 L78fs (NA) Q276* (NA) cancer/Likely pathogenic) Q50H (Low/ Oncogenic/Non- cancer/Uncertain significance) E54D (Low/ Oncogenic/Non- cancer/Likely A148T (Medium/ pathogenic) 35 BL + - - GCB 90 - + + + 99 Neutral/Cancer/ F153C (Medium/ Benign) NA/Non-cancer/ Uncertain significance) S264R (Medium/ NA/Non-cancer/ Uncertain significance) Case Rearrangement Hans IHC ID3 (NP_ BTG2 (NP_ CCND3 (NP_ MYC (NP_ CDKN2A (NP_ BTG1 (NP_ MAX (NP_ TP53 (NP_ TCF3 (NP_ num- Diagnosis algo- MYC Ki-67 MYC BCL2 BCL6 BCL2 CD10 BCL6 IRF4 002158) 006754) 001751) 002458) 000068) 001722) 002373) 0000537) 001129611) ber rithm (%) (%)

L64F (Medium/ S21N (Low/ HBL NA/Non-cancer/ Oncogenic/Non- 36 + - - GCB 80 - + + - 99 NOS Uncertain cancer/Uncertain significance) significance) non- 37 DLBCL - - - 80 + - + + 95 GCB P72T (Medium/ Oncogenic/Non- 38 BL + - - GCB 80 - + + + 95 Y76* (NA) cancer/Likely pathogenic) non- 41 DLBCL + - - 99 + - + + 90 E555fs (NA) GCB E54D (Low/ HBL Oncogenic/Non- 42 + - - GCB 80 + - + - 95 NOS cancer/Likely pathogenic) A126- L128delinsV (NA) A138V (Medium/ A129fs (NA) non- Oncogenic/ 44 DLBCL - - + 80 + - + + 95 S149F (Medium/ GCB Cancer/Uncertain Oncogenic/Non- significance) cancer/Uncertain significance) K162I (Medium/ Oncogenic/Non- L10F (Low/ cancer/Uncertain A148T (Medium/ Oncogenic/Non- significance) 46 DLBCL - - - GCB 70 + + + + 95 Neutral/Cancer/ cancer/Uncertain L37M (Medium/ Benign) significance) Oncogenic/Non- cancer/Uncertain significance) L10F (Low/ Oncogenic/Non- cancer/Uncertain significance) G62D (Medium/ L64V (Low/ G132S (Low/ Oncogenic/Non- HBL non- Oncogenic/Non- Oncogenic/Non- 48 - - - 70 + - - + 95 cancer/Uncertain NOS GCB cancer/Uncertain cancer/Uncertain significance) significance) significance) R69C (Medium/ Oncogenic/Non- Analysis ofcellcyclegenefor lymphoma cancer/Uncertain significance) Q140* (NA) non- 49 DLBCL - - + 60 + - + + 80 GCB P75T (Medium/ R273G (Medium/ Oncogenic/Non- Oncogenic/ 52 DLBCL - + - GCB 80 + + + - 98 cancer/Uncertain Cancer/Likely significance) pathogenic) 53 DLBCL + - - GCB 50 + + + + 98 55 HBL-DH + + - GCB 80 + + + - 80 57 DLBCL - + - GCB 90 + + + - 80 D561fs (NA) Y234C (Medium/ non- Oncogenic/ 58 DLBCL - - - 70 + - + + 95 GCB Cancer/Likely pathogenic) Yamashita T,

R283C (Low/

HBL Oncogenic/ et al. 62 - - + GCB 70 + + + + 95 NOS Cancer/Likely pathogenic) A82T (Medium/ non- Oncogenic/Non- 64 DLBCL - - - 70 + - + - 90 GCB cancer/Uncertain significance) 65 DLBCL - - + GCB 80 + + + + 90 A181P (Medium/ A148T (Medium/ Oncogenic/ 66 DLBCL - - - GCB 80 + + + + 95 Neutral/Cancer/ Cancer/Likely Benign) pathogenic) 68 HBL-DH + - + GCB 80 + - + - 99

The case number, clinical diagnosis, type of rearrangement, Hans algorithm, results of IHC and FISH and amino acid changes with functional impact (MutationAssessor and FATHMM) are included in this table. IHC: immunohistochemistry, FISH: fluorescence in situ hybridization.