medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

1 Title: Mutational landscape of high-grade B-cell lymphoma with -, BCL2 and/or

2 BCL6 rearrangements characterized by whole-exome sequencing

3

4 Authors: Axel Künstner1,2,3*, Hanno M. Witte4,5*, Jörg Riedl4,6*, Veronica Bernard6,

5 Stephanie Stölting6, Hartmut Merz6, Vito Olschewski4, Wolfgang Peter7,8, Julius Ketzer9,

6 Yannik Busch7, Peter Trojok7, Nikolas von Bubnoff3,4, Hauke Busch1,2,3**, Alfred C. Feller6**,

7 Niklas Gebauer3,4**

8

9 Affiliations:

10 1 Medical Systems Biology Group, University of Lübeck, Ratzeburger Allee 160, 23538

11 Lübeck, Germany

12 2 Institute for Cardiogenetics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck,

13 Germany

14 3 University Cancer Center Schleswig-Holstein, University Hospital of Schleswig- Holstein,

15 Campus Lübeck, 23538 Lübeck, Germany

16 4 Department of Hematology and Oncology, University Hospital of Schleswig-Holstein,

17 Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

18 5 Department of Hematology and Oncology, Federal Armed Forces Hospital Ulm, Oberer

19 Eselsberg 40, 89081 Ulm

20 6 Hämatopathologie Lübeck, Reference Centre for Lymph Node Pathology and

21 Hematopathology, Lübeck, Germany

22 7 HLA Typing Laboratory of the Stefan-Morsch-Foundation, 557565 Birkenfeld, Germany

23 8 Institut für Tranfusionsmedizin, Universitätsklinikum Köln. Kerpenerstr. 62, 50937 Köln

24 9 Department of Paediatrics, University Hospital of Schleswig-Holstein, Campus Luebeck,

25 23538 Luebeck, Germany

26

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.1 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

27 Scientific category: Lymphoid Neoplasia

28 Running Title: HGBL-DH/TH mutational landscape

29 Key Words: High-grade B-cell lymphoma, mutational landscape, whole-exome sequencing

30 Word Count (Text): 3.972

31 Word Count (Abstract): 250

32 References: 43

33 Tables: 1

34 Figures: 5

35 Supplementary Tables: 7

36 Supplementary Figures: 7

37 Competing Interests: The authors declare no conflicts of interest.

38

39 *These authors contributed equally to this manuscript

40 **Shared senior authorship

41 Date of submission: July 14th2021

42 Corresponding Author:

43 PD Dr. med. Niklas Gebauer

44 Department of Hematology and Oncology

45 UKSH Campus Luebeck

46 Ratzenburger Allee 160

47 23538 Luebeck

48 Email: [email protected]

49

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50 Abstract

51 High-grade B-cell lymphoma accompanied with MYC and BCL2 and/or BCL6 rearrangements

52 (HGBL-DH/TH) poses a cytogenetically-defined provisional entity among aggressive B-cell

53 lymphomas that is traditionally associated with unfavorable prognosis.

54 To better understand the mutational and molecular landscape of HGBL-DH/TH we here

55 performed whole-exome sequencing and deep panel next-generation-sequencing (NGS) of 47

56 clinically annotated cases. Oncogenic drivers, mutational signatures and perturbed pathways

57 were compared with data from follicular lymphoma (FL), diffuse large-B-cell lymphoma

58 (DLBCL) and (BL).

59 We find an accumulation of oncogenic mutations in NOTCH, IL6/JAK/STAT and NFκB

60 signaling pathways and delineate the mutational relationship within the continuum between

61 FL/DLBCL, HGBL-DH/TH and BL. Further, we provide evidence of a molecular divergence

62 between BCL2 and BCL6 rearranged HGBL-DH. Beyond a significant congruency with the

63 C3/EZB DLBCL cluster in BCL2 rearranged cases on an exome-wide level, we observe an

64 enrichment of the SBS6 mutation signature in BCL6 rearranged cases. Differential set

65 enrichment and subsequent network propagation analysis according to cytogenetically defined

66 subgroups revealed an impairment of TP53 and MYC pathway signaling in BCL2 rearranged

67 cases, whereas BCL6 rearranged cases lacked this enrichment, but instead exhibited showed

68 impairment of E2F targets. Intriguingly, HGBL-TH displayed intermediate mutational

69 features in all three aspects.

70 This study elucidates a recurrent pattern of mutational events driving FL into MYC-driven

71 BCL2 rearranged HGBL, unveiling the mutational pathogenesis of this provisional entity.

72 Through this refinement of the molecular taxonomy for aggressive, germinal-center derived

73 B-cell lymphomas, this calls into question the current WHO classification system, especially

74 regarding the status of MYC/BCL6 rearranged HGBL.

75

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76 Introduction

77 High-grade B-cell lymphoma with MYC-, BCL2 and/or BCL6 (HGBL) rearrangements poses

78 a novel, yet provisional, cytogenetically-defined entity within the current WHO classification

79 of lymphoid tumors. It is presumably allocated in the pathobiological continuum between

80 diffuse large B-cell (DLBCL) and Burkitt lymphoma (BL) (1). The t(8;14)(q24;q32)

81 IgH/MYC rearrangement constitutes the molecular hallmark of BL. This or further derivative

82 chromosomal rearrangements that juxtapose MYC to a genomic enhancer, occur in

83 approximately 10% of DLBCL and have been shown to correlate with inferior clinical

84 outcome (2). The rearrangement is a driver of oncogenesis that is in approx. 50% of cases

85 accompanied by additional rearrangements involving BCL2 and/or BCL6 and referred to as

86 double-hit (DH) or triple-hit (TH) lymphomas (3-11).

87 While the clinical outcome in HGBL-DH/TH patients is generally poor, recent studies have

88 emphasized the significant impact of MYC translocation partners and defined MYC/Ig-

89 rearrangements to be the most reliable predictors of adverse outcome (2, 7).

90 In a prior study, we discovered an elevated frequency of TP53 impairment in MYC-driven

91 DH/TH, whose presence was subsequently demonstrated for a subset of patients with a

92 single-hit MYC translocation as well, indicating inferior outcome (12, 13).

93 By conventional cytogenetics HGBL-DH/TH were shown to recurrently harbor a complex

94 karyotype (4). Data on the genetic basis of this entity, however, remains elusive. Several

95 preliminary studies, predominantly focusing on HGBL-DH/TH with DLBCL morphology,

96 have employed a panel-based next generation sequencing (NGS) approach (14-16). The

97 insights from these studies were all restricted by gene-panel design and the associated

98 clinicopathological data, yet their central assertions included a significant enrichment in

99 mutations affecting CREBBP, BCL2 and KMT2D alongside an overall reflection of the

100 phenotypical gray-zone between DLBCL and BL. Most recently, Cucco et al. elucidated

101 significant aspects of the molecular signature of HGBL in a panel-based sequencing and gene

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102 expression study, employing a 70-gene HaloPlex panel and an array-based gene expression

103 approach. The authors restricted their study to samples with DLBCL morphology that

104 stemmed from a clinical trial and the UK's population-based Haematological Malignancy

105 Research Network (17).

106 A comprehensive, exome-wide assessment of oncogenic driver mutations in HGBL-DH/TH,

107 including cases with BL-like morphology is, however, still warranted and of vital importance

108 to the refinement of the pathogenetic understanding of this clinically challenging entity

109 ultimately enabling targeted therapeutic approaches.

110 We therefore conducted a whole-exome sequencing (WES) study on a large cohort of HGBL-

111 DH/TH, validated by panel-based NGS and supplemented these data with a comprehensive

112 clinicopathological assessment of the study group. Here we report on oncogenic drivers,

113 somatic copy number alterations (SCNAs) and putative pathway perturbations, thus refining

114 the molecular taxonomy of MYC-driven germinal-center-derived aggressive lymphomas.

115

116 Materials and methods

117 Case selection and clinicopathological characteristics

118 In a retrospective approach, we reviewed our institutional database to identify HGBL patients

119 whose primary diagnostic biopsy specimen had been referred to the Reference center for

120 Hematopathology University Hospital Schleswig Holstein Campus Lübeck and

121 Hämatopathologie Lübeck for centralized histopathological panel evaluation between January

122 2007 and December 2019. For additional Information on clinicopathological work-up, please

123 see Supplementary materials and methods and Supplementary Table 1.

124 This retrospective study was approved by the ethics committee of the University of Lübeck

125 (reference-no 18-356) and conducted in accordance with the declaration of Helsinki. Patients

126 had given written informed consent regarding routine diagnostic and academic assessment of

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127 their biopsy specimen including molecular studies at the Reference center for

128 Hematopathology and transfer of their clinical data.

129

130 Whole exome and targeted amplicon-based sequencing

131 Whole exome sequencing of n = 47 HGBL-DH/TH samples was performed by a hybrid

132 capture approach with the Agilent SureSelect Human All Exon V6 library preparation kit

133 (Agilent Technologies) followed by Illumina short read sequencing on a NovaSeq platform

134 (Illumina)

135 to an average depth of 303x (standard deviation ±152x; median 237x) by Novogene (UK)

136 Co., Ltd. Seeking to validate the initial delineation of the exome sequencing-derived

137 mutational landscape in HGBL we employed our in-house custom AmpliSeq panel (Thermo

138 Fisher Scientific, Waltham, Massachusetts, USA) for targeted amplicon sequencing (tNGS),

139 encompassing all coding exons of 43 (see Supplementary Table 2) in 21 cases. Raw

140 paired-end data (fastq format) was trimmed and quality filtered using FASTP (v0.20.0;

141 minimum length 50bp, max. unqualified bases 30%, trim tail set to 1) (18) and trimmed reads

142 were mapped to GRCh37/hg19 using BWA MEM (v0.7.15) (19). Resulting alignment files in

143 SAM format were cleaned, sorted, and converted into BAM format using PICARD TOOLS

144 (v2.18.4). Single nucleotide variants (SNVs), as well as short insertions and deletions

145 (InDels) were identified following the best practices for somatic mutations calling provided

146 by GATK (20). Somatic copy number aberrations (SCNAs) were identified by CONTROL-

147 FREEC (v11.4) (21). For further details on nucleic acid extraction, panel sequencing, single

148 nucleotide and copy number variant calling please see Supplementary methods.

149

150 Mutational deleteriousness and significance, network propagation, gene set enrichment

151 and mutational cluster analysis

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152 The MUTSIGCV algorithm was employed on WES data to delineate significantly mutated

153 genes within the study cohort while deleteriousness was assessed via the CADD v1.3. The

154 acquired genomic data were then processed through the LymphGen algorithm and underwent

155 manual screening for an enrichment in overlapping aberrations with the molecular clusters

156 proposed by Chapuy et al. followed by validation through a logistic regression framework

157 (22, 23). Cytogenetically defined subgroups (HGBL with MYC and BCL2 aberrations, HGBL

158 with MYC and BCL6 aberrations, and HGBL-TH) underwent differential downstream analysis

159 by a network propagation approach simulating a -protein interaction network.

160 Subsequently a gene set variation analysis was performed against HALLMARK gene sets.

161 For details including statistical approaches correlating molecular and clinicopathological

162 findings please see Supplementary methods.

163

164 Data availability

165 Sequencing data in bam format from WES and panel sequencing have been deposited in the

166 European genome-phenome archive (EGA) under the accession number EGAS00001005420.

167

168 Results

169 Clinicopathological characteristics of the study group

170 We collected 47 cases of HGBL-DH/TH at diagnosis with sufficient FFPE tissue samples for

171 molecular studies (median age 71; range 35 – 89 years) all of which were included in the final

172 analysis, following successful library preparation for WES. There was insufficient clinical

173 follow-up in 9/47 (19%) cases. An underlying HIV infection was clinically excluded in all

174 cases. The majority of patients in our study were male (25/47; 53%) and presented with

175 advanced stage disease (24/38 stage III/IV; 63%) and an adverse prognostic constellation

176 (24/38 (63%) R-IPI > 2). Most patients received an intensive CHOP-like therapeutic frontline

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177 approach (25/38; 66%). The overall response rate after first line (immuno-) chemotherapy

178 was 76% resembling a general therapeutic response in 29/38 cases.

179 Table 1 summarizes the baseline characteristics of all HGBL-DH/TH cases included in the

180 current study. Histologically, the predominant morphology was that of DLBCL (NOS)

181 (32/47), however, Burkitt-like morphology and immunophenotype was present in 15/47

182 patients. Cytogenetically 21/47 cases presented with MYC/BCL2, 17/47 with MYC/BCL6

183 double-hit constellation and 9 triple-hit lymphomas were included. MYC translocation partner

184 revealed MYC-Ig rearrangement in 8/17 cases. The treatment outcome in our cohort was

185 unfavorable yet in keeping with previous data reported by Rosenwald and colleagues (2). For

186 confirmatory purposes, we included four cases, which were assessed for TP53 mutation status

187 in a previous study and were able to validate both cytogenetic as well as molecular

188 observations (12).

189

190 The mutational landscape of HGBL-DH/TH identified by WES

191 To characterize the mutational landscape in an extensive cohort of HGBL-DH/TH cases, we

192 successfully performed WES in 47 patient-derived tumor biopsies and matched constitutional

193 DNA in seven cases. We further applied the analytical framework outlined above to analyze

194 WES data in the absence of paired germline DNA in the majority of cases. Following the

195 primary identification of SNVs and indels in individual samples and subsequent filtering to

196 correct for FFPE-derived artefacts and spurious mutations, we applied the MutSig2CV

197 algorithm and thereby identified 22 significant candidate driver genes (p < 0.001; 13 genes

198 with q < 0.1; Supplementary Table 3) (24).

199 All HGBL-DH/TH cases carried mutations in genes of oncogenic relevance according to our

200 bioinformatic annotations. In total, we described 10,092 presumably harmful somatic

201 mutations (cut-off see materials and methods) involving 5,521 genes after variant filtering. Of

202 these, SNVs and InDels represented 74.1% of the mutations (7,479 SNVs). Among them,

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203 missense mutations were the most frequent alterations (85.2%), followed by nonsense (5.7%)

204 and InDels (5.6%), while splice-site mutations posed 3.3% of somatic mutations (Figure 1A).

205 Displaying an overall intermediate tumor mutational burden (median 3.974; range 1.065 –

206 18.234 mutations/Mbase; Figure 1B), HGBL-DH/TH revealed no evidence of MSI-related

207 hypermutations, which is in keeping with observations in DLBCL (0.3%), yet differs from

208 other aggressive lymphomas (e.g., primary mediastinal B-cell lymphoma) (25).

209 Upon comparative analysis of WES and targeted resequencing data, we were able to

210 demonstrate a concordance rate of 92.0% (46/50 in 18 matched samples) of mutational calls,

211 prompting high confidence in mutational calls derived from WES, even in non-germline-

212 paired cases. A comprehensive description of all variants described by WES as well as panel

213 based NGS is provided in Supplementary Tables 4 and 5. Nevertheless, we observed a

214 significant enrichment of non-germline matched samples in non-synonymous SNVs, which

215 prompted us to include significantly mutated genes according to the MUTSIGCV analysis,

216 only. As an exception to this rule, we also included MYC mutations below the statistical

217 significance level due to their previously established clinical and functional relevance.

218

219 Recurrent copy number alterations in HGBL-DH/TH

220 We investigated our HGBL-DH/TH cohort for SCNVs employing the CONTROL-FREEC (21)

221 algorithm in tumor-normal and tumor-only mode, respectively, followed by GISTIC2.0 (26)

222 analysis. The analysis excluded X and Y as well as common benign copy

223 number variants defined by the Broad Institute’s panel of normal. Upon cross-referencing our

224 findings with genomic loci of known oncogenes, tumor-suppressors and elements of

225 significant signaling pathways we identified recurrent copy number gains in oncogenes such

226 as MEF2B and CSF1R, which have previously been implicated in the pathogenesis of

227 malignant lymphomas (27, 28). Further, copy number losses in tumor suppressors like NPM1

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228 were recurrently identified (Figure 2A, B). No significant differences were detected for genes

229 affected by copy number alterations between the three cytogenetically defined subgroups

230 (Fisher exact test p > 0.05 after Bonferroni correction for multiple testing). Common CNVs,

231 as defined by the above referenced panel of normal were encountered at the expected

232 frequencies (29, 30).

233

234 Significantly mutated candidate driver genes and mutational signatures

235 Putative candidate driver genes comprised several genes previously implicated in HGBL-

236 DH/TH pathogenesis, such as KMT2D, CREBBP and TP53 alongside several further mutated

237 genes such as CDKN2A, LNP1 or SI (14). Established mutational drivers known from other

238 B-cell lymphoproliferative disorders (e.g., FL, DLBCL, BL) were recurrently encountered

239 (e.g., CCND3, ARID1A) (Figures 2C; 3A) (31, 32). In our limited cohort, distinctions

240 regarding subtype-specific mutational signatures were found to be marginal among

241 BCL2/BCL6 status or Burkitt-like vs non-Burkitt like morphology. However, we found

242 CCND3 mutations, previously reported as driver mutations in BL pathogenesis, to be

243 significantly enriched in HGBL-DH/TH patients with Burkitt-like morphology (9/15 vs 3/28).

244 This observation hints at partially similar molecular paths of pathogenesis between BL and

245 HGBL with Burkitt-like morphology. Further, an enrichment of mutations affecting CREBBP

246 in HGBL-DH/TH patients with BCL2 rearrangement was observed, which is well in keeping

247 with its proposed fundamental role in FL pathogenesis. Distribution of mutations within

248 selected, significantly mutated genes is depicted in Supplementary Figure 1. Additional

249 profiling of mutational signatures driving HGBL-DH/TH revealed a predominance of the

250 SBS5 signature alongside the emphasized occurrence of the SBS6 signature (implicated in

251 defective DNA mismatch repair) in patients with BCL6 rearrangements (Supplementary

252 Figure 2, Supplementary Table 6).

253

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254 Comparative analysis of mutational landscape in HGBL-DH/TH, related entities and

255 molecular clusters in DLBCL

256 Next, we sought to refine the genomic taxonomy of aggressive germinal-center-derived B-cell

257 lymphomas and to investigate the mutational commonalities and differences between HGBL-

258 DT/TH and other related pathological entities. These were subsequently selected for their

259 similar features of B-cell differentiation (ABC-type DLBCL, FL, GCB-type DLBCL and BL)

260 and a comparative analysis of candidate mutational drivers in HGBL-DH/TH (as described

261 earlier) and cBioPortal cohorts of ABC-type DLCBL (n = 67), GCB-type DLBCL (n = 45

262 (22)), BL (n = 108(33)) and FL (n = 199(33)) was conducted. Interestingly, we identified one

263 overlapping candidate driver common to all entities (KMT2D). Additionally, CREBBP was

264 found in all entities except ABC-type DLCBL. Mutations affecting EZH2, IRF8 and

265 TNFRSF14 were, however, specifically occured in HGBL-DT/TH and FL/GCB-type

266 DLBCL, while CCND3 mutations appeared to be a pathogenetic feature shared between BL

267 and HGBL-DT/TH. Additionally, TP53 mutations posed a predominant feature of aggressive

268 lymphomas present in all HGBL-DH/TH, GCB-type DLBCL and BL types and therefore

269 most likely acquired during high-grade transformation (Figure 3G, H). In basic accordance

270 with previous studies, our data suggest a common origin especially for BCL2 rearranged

271 HGBL-DH/TH and FL/GCB-type DLBCL (14, 17).

272 Upon comparative investigation of our current data and mutational clusters, previously

273 described in DLBCL, we observed a striking predominance of C3/EZB cluster cases in the

274 BCL2 rearranged subgroup according to the integrative molecular classification proposed by

275 Chapuy et al. and Wright et al., respectively. This is in keeping with a significant enrichment

276 of these cases with DLBCL morphology in terms of MYC rearrangement status (Figure 4,

277 Supplementary Figure 3) (22, 23). Complementary to our analysis, employing the

278 LymphGen algorithm (cf. Supplementary Table 7), a logistic regression indicated a

279 significantly different number of mutated genes in C3 between the HGBL subtypes. HGBL

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280 harboring only BCL2 were shown to exhibit the highest number of mutated C3 genes, while

281 HGBL with BCL6 alterations had the lowest number of mutated C3 genes (BCL2/6 cases: (p

282 = 6.059*10-5, adj R2 = 0.3108). In contrast to triple hit cases, HGBL with MYC and an

283 isolated additional BCL6 rearrangement showed a significant decrease in the number of

284 mutated C3 genes (p = 2.00*10-5, estimate: -1.7589) (Supplementary Figure 4,

285 Supplementary Table 8). Within the subgroup of BCL6 rearranged cases, the BN2 cluster

286 was more prominent than the EZB cluster. In keeping with their strong affinity towards the

287 C3/EZB cluster, BCL2 rearranged cases exhibited an enrichment for mutations in CREBBP

288 and KMT2D, while BCL6 rearranged cases were, in contrast, enriched for mutations in

289 ARID1A. The vast majority of triple-hit cases was also classified within the EZB cluster.

290

291 Mutational impairment of NOTCH, RTK-RAS and TP53 signaling in HGBL-DH/TH

292 Cumulatively, we detected genetic lesions, putatively impairing NOTCH signaling in 74% of

293 HGBL-DH/TH patients. Expanding on previously reported CREBBP, EP300 and DTX1

294 mutations in HGBL we further identified recurrent mutations affecting NCOR1 and others

295 (Supplementary Figure 5) (14, 17). NOTCH signaling was thereby the predominant target

296 of somatic mutation in HGBL-DH/TH, albeit with a quite heterogeneous mutational pattern

297 affecting 35/47 patients with lesions in 28/71 genes (Supplemental Figure 6A). Most of

298 these genomic aberrations had been previously reported to be gain-of-function mutations

299 putatively resulting in constitutive NOTCH pathway activation in various types of

300 predominantly germinal-center derived B-cell lymphoma. Several of these mutational hits

301 including NCOR1 and DTX1 have been shown to herald adverse clinical outcome (34, 35).

302 This remained the case when undertaking a differential downstream analysis within the

303 cytogenetically defined subgroups (HGBL with MYC and BCL2 aberrations, HGBL with

304 MYC and BCL6 aberrations and HGBL-TH), which was prompted by their significantly

305 divergent distribution onto molecular clusters. Through this analysis a mutational signature

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306 became apparent that is additionally dominated by impairment of TP53 and MYC signaling in

307 BCL2 rearranged cases. BCL6 rearranged cases lacked this enrichment, while HGBL-TH

308 cases revealed intermediate mutational features. As another remarkable feature throughout all

309 subgroups we observed alterations, putatively resulting in gain-of-function in IL6/JAK/STAT

310 signaling in 74% of patients (Supplementary Figure 5). Activating mutations affecting

311 PIM1 and SOCS1 were most frequently encountered in our case series and have been

312 previously implicated in HGBL-DH/TH pathogenesis (17). These candidate driver genes are

313 supplemented by mutations in LTB and STAT3 (both previously identified in HIV-associated

314 plasmablastic lymphoma) among others (36). Beyond this, we observed a relatively dispersed

315 mutational pattern with putative driver events affecting 28 genes within the NOTCH-pathway

316 (Supplemental Figure 6A).

317 In accordance with previous studies, we found mutations directly impacting NF-kB signaling

318 in 62% of cases (Supplementary Figure 5) (37). While this was among the predominant

319 pathways identified through our network propagation approach, mutations affecting the

320 pathway were narrowly detectable with only BCL2 harboring mutations in more than three

321 patients (34%) followed by recurrent SNVs and indels in PARP1 (6%) and BIRC3 (4%) and

322 CARD11 (4%).

323 Following SNV and InDel evaluation with MUTSIGCV, a network propagation approach

324 (Figures 3B, C) was employed on significantly mutated genes to delineate the functional

325 implications of significant genetic events on neighboring genes. This further underscored the

326 mutational impairment of the aforementioned pathways alongside WNT and PI3K signaling.

327 These observations are in accordance with preliminary impressions derived from targeted

328 sequencing studies, employing panel-based approaches (14, 17). In addition to the divergent

329 results from our mutational pathway analysis, we identified an enrichment of E2F targets

330 impacted by significantly mutated genes in both double- and triple-hit cases affected by BCL6

331 rearrangements (Figures 3D, E, F).

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332

333 Survival analysis

334 Upon integrated analysis of molecular and clinical data we investigated genomic alterations

335 present in > 15% of patients for their impact on overall survival (OS) and progression-free

336 survival (PFS). Hereby we identified ARID1A mutations to be predictive of worse clinical

337 outcome in our cohort (OS: p = 0.0049; PFS: 0.025). Subsequent Bonferroni correction for

338 multiple testing was performed. Thus, we identified a significant impact of mutations

339 affecting ARID1A which was maintained regarding OS when correction for multiple testing

340 was applied while its primarily significant effect on PFS was reduced to a trend of borderline

341 statistical significance (Figure 5). A Cox proportional hazard model revealed this effect to be

342 independent of the established clinical IPI prognosticators (age, LDH, extra nodal

343 manifestations, stage, and performance status; OS: p < 0.001; HR: 13.989; 95%CI: 3.362 –

344 58.205; PFS: p = 0.001; HR: 6.648; 95%CI: 2.098 – 21.061). Within the cytogenetically

345 defined subgroups, we identified no alterations with independent impact on clinical outcome.

346 However, a trend of borderline statistical significance towards inferior outcome in

347 MYC/BCL2 rearranged cases harboring FOXO1 mutations was observed (Supplementary

348 Figure 7).

349

350 Discussion

351 Here we report on whole-exome sequencing data from an extensive cohort of HGBL-DH/TH

352 tumors, which is to the best of our knowledge the hitherto largest cohort and most extensive

353 molecular data set for this entity. Previous reports on HGBL-DH/TH were limited by low

354 sample numbers and/or targeted sequencing approaches. Contrary to this, WES here allowed

355 to systematically define recurrent mutations, predominant mutational signatures and SCNVs

356 in their respective clinicopathological context from which we report three central

357 observations.

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358 Firstly, being the first exome-wide mutational investigation for this rare subtype of

359 lymphoma, we identify a significant overlap of mutational drivers between HGBL-DH/TH

360 and FL as well as GCB-type DLBCL (e.g., TNFRSF14, EZH2 and IRF4) as its high-grade

361 counterpart. Aggressive transformation was associated with the acquisition of mutations in

362 TP53. Moreover, shared features, including CCND3 and CDKN2A mutations underscore a

363 close molecular relation between HGBL-DH/TH and BL (38, 39). This is additionally

364 reflected in the enrichment of HGBL-DH/TH patients with Burkitt-like morphology for

365 CCND3 mutations. Further, we identify a number of significant mutational drivers not

366 captured by previous, panel-based sequencing studies. Most frequently among these, we find

367 SI mutations that have been previously implicated in CLL progression aa well as mutations in

368 POU2AF1, which has been recently found to be an augmented target of mutations during

369 aggressive transformation of FL to DLBCL (40, 41). Although MYC did not meet the

370 predefined MUTSIGCV significance level in our study, we still observed mutations in 19% of

371 cohort samples (Supplementary Figure 8), which is in agreement with previous panel-based

372 studies (17).

373 Secondly, upon screening the mutational landscape in HGBL-DH/TH in comparison to the

374 molecular clusters of DLBCL, proposed by Chapuy et al. and the LymphGen algorithm

375 proposed by Wright et al., we unveil a striking overlap of BCL2 rearranged cases with the

376 C3/EZB cluster, which was previously shown to be enriched for MYC rearrangements and

377 oncogenic drivers implicated in FL pathogenesis (22, 23). We argue that a predominant subset

378 of HGBL-DH/TH most likely corresponds to these transformed FL. This offers a potential

379 explanation for the inferior clinical outcome of C3 DLBCL patients, despite their GCB-

380 phenotype, through an enrichment for MYC rearranged HGBL-DH/TH cases. Of note, we find

381 the predominant impairment of TP53 and to a lesser extent MYC signaling in BCL2

382 rearranged cases to be in keeping with a previous study on an independent set of HGBL-

383 DH/TH, in which we found TP53 mutations to be a recurrent feature of HGBL-DH with

15 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

384 BCL2, but not BCL6 rearrangements (12). Intriguingly, we further observed two molecular

385 subtypes in MYC/BCL6 only rearranged cases. While selected cases were categorized within

386 the EZB cluster, several cases revealed an association with the BN2 cluster, potentially

387 hinting at a MYC-driven high-grade transformation of a precursor lesion with an origin within

388 the marginal zone, as previously described (23). In addition to these observations, we found

389 triple-hit cases to reflect EZB lymphomas in the vast majority of cases, potentially hinting at

390 BCL6 rearrangements as late and non-defining events in HGBL-TH lymphomagenesis.

391 Supporting this assumption, Pedrosa et al. have shown DLBCL with BCL2 and BCL6, but

392 without MYC rearrangements to be exclusively associated with the EZB cluster (42).

393 Considering the significantly mutated genes, our observations underscore previous

394 assumptions regarding a molecular divergence between BCL2 and BCL6 rearranged HGBL-

395 DH (14, 17, 43). The predominant mutational distinction between these groups was the

396 presumably FL-derived enrichment for CREBBP mutations in the BCL2 rearranged subgroup.

397 On an exome-wide level we observed an enrichment of the SBS6 signature (implicated in

398 defective DNA mismatch repair) and a significantly diminished congruency with the C3/EZB

399 DLBCL cluster in the BCL6 rearranged subgroup. Of note, these findings fundamentally

400 dispute the combined characterization in the current WHO classification, despite several

401 shared clinical aspects common to all subtypes of HGBL-DH/TH (1, 2). Beyond de novo

402 DLBCL with BCL6 rearrangement, potential alternative explanations for this phenomenon

403 include both clonal evolution and subsequent aggressive transformation from rare cases of

404 BCL6 rearranged marginal zone lymphomas alongside BCL2 non-rearranged/BCL6

405 rearranged FL, which were previously shown to be characterized by a heterogenous

406 mutational landscape (44, 45). From our data, we further deduce an intermediate role for

407 HGBL-TH, which may indicate two divergent paths of clonal evolution originating from a

408 BCL2 or a BCL6 driven disease with subsequent acquisition of the alternative rearrangement.

409 Lastly, we describe a pronounced mutational impairment of NOTCH, IL6/JAK/STAT and

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410 NFκB signaling pathways and recurrent oncogenetically relevant genes affected by SCNVs

411 (including MEF2B, which was previously shown to be enriched in mutations/aberrations

412 within the C3 DLBCL cluster) thereby systematically characterize the oncogenetic footprint

413 of this subgroup of lymphoma. This is further combined with the identification of novel

414 putative mutational drivers (e.g., NCOR1, DTX1, LTB and STAT3) alongside several

415 previously established mutational hotspots in HGBL-DH/TH. Moreover, among these

416 significantly mutated genes we describe ARID1A which emerges as a potential prognosticator

417 of treatment response and outcome from our correlative assessment of clinical and molecular

418 features of our present cohort, which was found to be independent from previously

419 established clinical prognostic factors.

420 We acknowledge the shortcomings inherent to the retrospective design of the study alongside

421 the limited availability of germline DNA for matched pair analysis. The latter aspect is

422 reflected in a significantly elevated number of mutations in non-matched samples. This

423 prompted us to limit our subsequent analysis to significantly mutated genes (except for MYC

424 and BCL2 mutations, which were additionally included based on their proven relevance in

425 prior studies (14, 17, 22, 46) and thereby equalizing the abovementioned effect. Pairing of our

426 WES-results with RNA-seq data, preferably in an extended, clinically annotated cohort,

427 which was beyond the scope of the present study, would further deepen our molecular

428 understanding of HGBL-DH/TH, especially regarding cases with prominent Burkitt or

429 Burkitt-like morphology.

430 In summary, our identification of distinct mutational landscapes among HGBL-DH/TH,

431 derived from an exome-wide sequencing approach shows both overlapping and distinctive

432 features compared with germinal center derived lymphomas such as GCB-type DLBCL and

433 low-grade FL as well as BL. Our work further underscores the developing notion of a

434 recurrent pattern of mutational events driving a potentially unidentified preexisting FL into

435 MYC-driven HGBL-DH/TH, offering insight into the molecular pathogenesis of this

17 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

436 provisional entity. By refining the molecular taxonomy for aggressive, germinal-center

437 derived B-cell lymphomas, these results call into question the current WHO classification

438 system, especially regarding the status of MYC/BCL6 rearranged HGBL.

439

440 Acknowledgments

441 A.K. and H.B. acknowledge computational support from the OMICS compute cluster at the

442 University of Lübeck. The research was supported by a grant to N.G. by the Stefan-Morsch-

443 Foundation alongside infrastructural support. H.B. acknowledges funding by the Deutsche

444 Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence

445 Strategy— EXC 22167-390884018).

446

447 References

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759 Table 1. Clinical characteristics of the study group.

Characteristics HGBL DHL-BCL2 DHL-BCL6 THL (n = 47) (n = 21) (n = 17) (n = 9) Insufficient FU 9 (19.1%) 4 (19.0%) 2 (11.8%) 3 (33.3%) Age 71 (35 – 89) 73 (35 – 88) 72 (35 – 89) 66 (42 – 76) (yrs.; median + range) Sex Female 22 (46.8%) 8 (38.1%) 11 (64.7%) 3 (33.3%)

27 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Male 25 (53.2%) 13 (61.9%) 6 (35.3%) 6 (66.7%) R-IPI 0 1 (2.6%) 1 (5.9%) - - 1-2 13 (34.2%) 5 (29.4%) 6 (40.0%) 2 (33.3%) >2 24 (63.2%) 11 (64.7%) 9 (60.0%) 4 (66.7%) Stage (Ann Arbor) I 5 (13.2%) 2 (11.8%) 3 (20.0%) - II 9 (23.7%) 4 (23.5%) 3 (20.0%) 2 (33.3%) III 5 (13.2%) 1 (5.9%) 2 (13.3%) 2 (33.3%) IV 19 (50.0%) 10 (58.8%) 7 (46.7%) 2 (33.3%) B-Symptoms Yes 20 (52.6%) 10 (58.8%) 7 (46.7%) 3 (50.0%) No 18 (47.4%) 7 (41.2%) 8 (53.3%) 3 (50.0%) Extranodal sites 0 9 (23.7%) 3 (17.6%) 4 (26.7%) 2 (33.3%) 1-2 28 (73.7%) 14 (82.4%) 10 (66.7%) 4 (66.7%) >2 1 (2.6%) - 1 (6.7%) - ECOG PS 0-1 20 (52.6%) 8 (47.1%) 8 (53.3%) 4 (66.7%) ≥2 18 (47.4%) 9 (52.9%) 7 (46.7%) 2 (33.3%) LDH Normal 7 (18.4%) 3 (17.6%) 3 (20.0%) 1 (16.7%) Elevated 31 (81.6%) 14 (82.4%) 12 (80.0%) 5 (83.3%) CNS involvement at diagnosis Yes 2 (5.3%) - 2 (13.3%) - No 36 (94.7%) 17 (100.0%) 13 (86.7%) 6 (100.0%) Morphology DLBCL-like 32 (68.1%) 17 (80.9%) 12 (70.6%) 3 (33.3%) Burkitt-like 15 (31.9%) 4 (19.0%) 5 (29.4%) 6 (66.7%) Frontline therapy regimen CHOP-like 25 (65.8%) 11 (64.7%) 10 (66.7%) 4 (66.7%) R-based 31 (81.6%) 14 (82.4%) 12 (80.0%) 5 (83.3%) Intensified* 13 (34.2%) 7 (41.1%) 3 (20.0%) 3 (50.0%) Less 9 (23.7%) 4 (23.5%) 4 (26.7%) 1 (16.7%) intensive** Refusal 1 (2.6%) - 1 (6.7%) - CHOP, cyclophosphamide/hydroxadaunorubicin/vincristine/predinislone; CNS, central nervous system; DHL, double-hit lymphoma; DLBCL, diffuse large B-Cell Lymphoma; ECOG; Eastern cooperative oncology group; FU, follow-up; HGBL, high grade B-cell lymphoma; LDH, Lactate dehydrogenase; PS, performance status; R, rituximab; R-IPI, revised International Prognostic Index; THL, triple-hit lymphoma; Yrs., years. *Intensified regimens: B-ALL, GMALL, CHOEP (additional etoposide), EPOCH **Less intensive regimens: Bendamustin, mini-CHOP (50% dose reduction), rituximab mono 760 Figure Legends:

761 Figure 1. Panel (A) shows the number of variants stratified by variant classification while

762 panel (B) delineates the number of mutations per sample with a median of 153 mutations per

763 sample.

28 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

764

765 Figure 2. Co-oncoplot for genes identified as significant driver genes by MUTSIGCV (p <

766 0.001; n = 22) in our cohort stratified by cytogenetical subtypes is shown in Panel (A);

767 different types of mutations are colour coded. Additionally, covariates are shown below the

768 plot for each sample. (B) Location of SCNVs along the genome (red bars denote gains; blue

769 bars denote losses; gene names refer to affected oncogenes and genes related to oncogenetic

770 pathways). Panel (C) displays selected genes (oncogenes and genes implicated in oncogenetic

771 pathways) affected by SCNVs.

772

773 Figure 3. (A) Significance levels for all HGBL-DH/TH MUTSIGCV genes (p < 0.001; gene

774 names in orange indicate q < 0.1) and (B) HALLMARK gene sets and NFκB pathway

775 enrichment for network propagation analysis of significant MUTSIGCV genes (MUTSIGCV p

776 < 0.001). (C) Significant levels for MYC/BCL2 subgroup (MUTSIGCV p < 0.001) and (D)

777 results for enrichment for network propagation analysis against HALLMARK gene sets

778 including NFκB pathway. Panels (E) and (F) show enrichment results for cytogenetical

779 subgroups MYC/BCL6 and triple hit, respectively (MYC/BCL6 genes included: CDKN2A,

780 CD78B, LNP1, KRTAP13-1, UBE2A, CCND3; triple hit genes included: CDKN2A, LNP1,

781 CCND3).

782 UpSet plot (G) showing the overlap of MUTSIGCV genes using our HGBL-DH/TH (D/THL)

783 cohort, the cytogenetical subgroups (BCL2, BCL6, THL), as well as cohorts of ABC-type

784 DLBCL (n = 67(33)), GCB-type DLBCL (n = 45 (22)), BL (n = 108(38)) and FL (n =

785 199(38)) (all retrieved via cBioPortal); (H) shows the overlap between the five lymphoma

786 subtypes and the three cytogenetical subtypes of HGBL-DH/TH.

787

788 Figure 4. Allocation of HGBL-DH/TH samples unto the molecular subgroups/clusters of

789 DLBCL, according to LymphGen based on their mutational signature. Additionally,

29 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

790 covariates are shown above the plot for each sample. Row names refer to chromosomal

791 alterations, genes and fusions and are background coloured by their specific subtype (blue =

792 BN2, orange = EZB).

793

794 Figure 5. Overall (A) and progression-free survival (B) according to ARID1A mutational

795 status. Numbers at risk alongside hazard ratios and p-values according to log-rank testing are

796 provided. Subsequent Bonferroni correction for multiple testing (all genes with a mutational

797 frequency > 15% were investigated) identified a significant impact of ARID1A mutations

798 regarding OS while its primarily significant effect on PFS was reduced to a trend of

799 borderline statistical significance.

30 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Figure 1A Variant Classification

Missense Mutation

Nonsense Mutation

Frame Shift Ins

Splice Site

In Frame Ins

Nonstop Mutation

0 1000 2000 3000 4000 5000 6000 Figure 1B Variants per sample 702 Median: 153

468

234

0 Figure 2A 1.5 12q13.3

1.0 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint 11q13.5 15q24.1 19p13.11 9q34.11 0.5 (which was not certified3p14.3 by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 3p21.31 7p13 12q12 15q24.2 18q21.1 3q22.1 All rights5q32 reserved. No reuse allowed without permission. 18q11.2 2q36.3 4p16.3 6q136q25.3 10q24.1 12q13.11 17q12 22q13.2 2q35 6p21.2 7q22.1 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y 4q24 7q11.23 11q24.2 13q34 15q15.2 17q25.1 19p13.2 22q13.1 1p35.1 1q32.1 2q13 9q33.3 20p12.1 2q33.1 4q12 4q33 6q27 9q33.2 11q23.3 14q22.1 17q24.3 22q11.23 1p36.32 3q25.33 4q31.21 5q35.1 12q21.2 14q32.33 16q12.2 20q13.13 1p22.3 8q24.13 9q32 15q15.3 18q22.1 -0.5 4q13.2 11q22.3 12q24.31 17q24.2 21q22.13 G-Score 4q13.3 5q13.2 9q21.32 11p13 13q14.3 19q13.2 22q12.1 2q31.1

-1.0 1p13.2 12p13.33 14q13.2 18q12.1 2q11.1 2q37.3 7p14.2 13q32.1 14q32.12 20q13.33 -1.5 SI B2M IRF8 TP53 LNP1 BTG1 EZH2 UFY2 TIMP4 AP3S1 GULP1

-2.0 CD79B R KMT2D CCND3 ARID1A CDKN2A CREBBP POU2AF1 TNFRSF14 KRTAP13-1

Figure 2B

MEF2B 17% SCNA 2 ARID3B 17% 1 CD276 17% 0 CSF1R 15% -1 HDAC7 4% -2 FEV 4% INHA 4% PDGFRB 6% DNAH8 4% NPM1 11% TLX3 11% TP53BP1 9% CHEK2 30%

Figure 2C MYC/BCL2 (N = 21) MYC/BCL6 (N = 16) Triple hit (N = 10) CREBBP 52% 6% 10% KMT2D 48% 12% 50% EZH2 33% 6% 20% SI 29% 6% 30% TP53 29% 6% 20% TNFRSF14 24% 12% 0% GULP1 19% 0% 0% AP3S1 14% 6% 0% BTG1 14% 6% 0% CCND3 14% 25% 50% LNP1 14% 12% 20% RUFY2 14% 6% 10% ARID1A 10% 19% 30% B2M 10% 0% 0% CD79B 10% 12% 0% POU2AF1 10% 6% 10% CDKN2A 5% 12% 20% IRF8 5% 6% 40% OR13H1 5% 6% 0% TIMP4 5% 6% 10% KRTAP13−1 0% 12% 10% UBE2A 0% 19% 0% BCL2 FisH BCL6 FisH Prior Fol. Lymphoma Morphology Gender Matched Missense Mutation Frame Shift Ins BCL2 FisH BCL6 FisH Prior Fol Lymphoma Morphology Gender Matched normal tissue Nonsense Mutation Splice Site no no no DLBCL female no Nonstop Mutation Multi Hit yes yes yes Burkitt-like male yes In Frame Ins na medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Figure 3A Figure 3B Figure 3C Figure 3D OXIDATIVE PHOSPHORYL. MYC TARGETS V2 OR13H1 BILE ACID METABOLISM GLYCOLYSIS FATTY ACID METABOLISM ESTROGEN RESPONSE EARLY B2M −log10(adj p) POU2AF1 −log10(adj p) XENOBIOTIC METABOLISM ESTROGEN RESPONSE LATE 50 MYOGENESIS ADIPOGENESIS BTG1 40 40 SPERMATOGENESIS P53 PATHWAY 30 30 ARID1A MTORC1 SIGNALING SPERMATOGENESIS 20 HVCN1 20 UV RESPONSE UP UV RESPONSE UP CREBBP 10 10 APICAL JUNCTION DNA REPAIR UBE2A KRAS SIGNALING UP MTORC1 SIGNALING P53 PATHWAY B2M OXIDATIVE PHOSPHORYL. KRTAP13−1 UNFOLDED PROTEIN RESP. MYOGENESIS UV RESPONSE DN KRAS SIGNALING UP EZH2 E2F TARGETS IL2 STAT5 SIGNALING HYPOXIA TIMP4 HYPOXIA TP53 APICAL JUNCTION EPITHELIAL MESENCH. TRANS. GULP1 UV RESPONSE DN MYC TARGETS V1 IL2 STAT5 SIGNALING DNA REPAIR IRF8 G2M CHECKPOINT COAGULATION AP3S1 APICAL SURFACE RUFY2 COMPLEMENT APOPTOSIS PROTEIN SECRETION CD79B EPITHELIAL MESENCH. TRANS. APOPTOSIS COAGULATION INFLAMMATORY RESPONSE LNP1 SI TNFA SIGNALING VIA NFKB MITOTIC SPINDLE HEDGEHOG SIGNALING POU2AF1 HEDGEHOG SIGNALING MITOTIC SPINDLE TNFA SIGNALING VIA NFKB AP3S1 CREBBP COMPLEMENT ANGIOGENESIS TGF BETA SIGNALING KMT2D TGF BETA SIGNALING INFLAMMATORY RESPONSE CDKN2A NOTCH SIGNALING ANGIOGENESIS INTERFERON GAMMA RESP. GULP1 NOTCH SIGNALING TP53 INTERFERON ALPHA RESP. INTERFERON GAMMA RESP. E2F TARGETS INTERFERON ALPHA RESP. TNFRSF14 ALLOGRAFT REJECTION PROTEIN SECRETION CCND3 PI3K AKT MTOR SIGNALING ALLOGRAFT REJECTION IL6 JAK STAT3 SIGNALING TNFRSF14 WNT BETA CATENIN SIGNAL. LNP1 G2M CHECKPOINT IL6 JAK STAT3 SIGNALING WNT BETA CATENIN SIGNAL. PI3K AKT MTOR SIGNALING 0 3 6 9 Nf kappa B 0 2 4 6 Nf kappa B −log (p) −0.5 0.0 0.5 1.0 −log (p) −0.5 0.0 0.5 1.0 10 Enrichment 10 Enrichment Figure 3E Figure 3F Figure 3G Figure 3H

XENOBIOTIC METAB. XENOBIOTIC METABOLISM SPERMATOGENESIS MYOGENESIS 30 APICAL JUNCTION −log10(adj p) −log10(adj p) 28 CCND3 BILE ACID METABOLISM KRAS SIGNALING UP 60 CDKN2A 60 MTORC1 SIGNALING KRAS SIGNALING DN BTG1 40 UV RESPONSE UP 40 CD79B EPITHELIAL MESENCH. TRANS. IL2 STAT5 SIGNALING 20 20 LNP1 COAGULATION MTORC1 SIGNALING POU2AF1 UV RESPONSE DN TNFA SIGNALING VIA NFKB GULP1 P53 PATHWAY 20 AP3S1 MITOTIC SPINDLE B2M PROTEIN SECRETION P53 PATHWAY KRTAP13-1 OXIDATIVE PHOSPH. UBE2A ANGIOGENESIS APOPTOSIS EZH2 COMPLEMENT ALLOGRAFT REJECTION IRF8

Intersection Size 13 INFLAMMATORY RESP. HIST1H1E INTERFERON GAMMA RESP. APOPTOSIS LRP1B HEDGEHOG SIGNALING INTERFERON ALPHA RESP. CREBBP 10 9 UNFOL. PROTEIN RESP. IL6 JAK STAT3 SIGNALING TNFRSF14 ARID1A MYC TARGETS V2 7 UNFOLDED PROTEIN RESP. KMT2D TNFA SIGNAL. VIA NFKB 6 TP53 MITOTIC SPINDLE PI3K AKT MTOR SIGNALING 4 4 BCL2 IFN GAMMA RESP. OXIDATIVE PHOSPHORYL. 3 MYC IFN ALPHA RESP. 2 2 2 Nf kappa B CSMD1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 DNA REPAIR GNA13 TGF BETA SIGNALING TGF BETA SIGNALING 0 PIM1

NOTCH SIGNALING MYC TARGETS V2 THL FAT1 IL6 JAK STAT3 SIGNALING XIRP2 NOTCH SIGNALING BCL6 ALLOGRAFT REJECTION USH2A DNA REPAIR BCL2 MYC TARGETS V1 TTN PCLO PI3K AKT MTOR SIG. WNT BETA CATENIN SIGNAL. Burkitt MUC4 WNT BETA CATENIN SIG. ABC MYC TARGETS V1 FAT4 G2M CHECKPOINT D/THL G2M CHECKPOINT CSMD3 Nf kappa B FL ACTB E2F TARGETS E2F TARGETS CARD11

GCB ABC GCB Bu FL BCL6 THL D/THL BCL2 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 r

50 40 30 20 10 0 kitt Enrichment Enrichment Set Size MYC/BCL2/BCL6 LymphGen Subtype Prior Fol. Lymphoma Morphology medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint Gender (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Matched normal tissue Number Mutations Number Clones BCL2 Fusion MYC/BCL2/BCL6 BCL6 Fusion MYC/BCL2 BCL2 MYC/BCL6 EZH2 Triple hit TNFRSF14 LymphGen Subtype KMT2D BN2 CREBBP EZB Other REL FAS Prior Fol. Lymphoma IRF8 na EP300 no yes Chrom. 12p MEF2B Morphology CIITA Burkitt-like DLBCL ARID1A GNA13 Gender STAT6 female PTEN male Chrom. 21 Matched normal tissue EBF1 no GNAI2 yes C10orf12 Number Mutations BCL7A 800 HLA−DMB 600 S1PR2 400 MAP2K1 200 FBXO11 0 MIR17HG Number Clones BCL6 6 NOTCH2 4 TNFAIP3 2 DTX1 0 CD70 BCL10 Alterations Mutation UBE2A Truncation TMEM30A Amplification KLF2 Translocation SPEN CCND3 NOL9 TP63 ETS1 HIST1H1D PRKCB HIST1H2BK TRIP12 KLHL21 TRRAP PABPC1 medRxiv preprint doi: https://doi.org/10.1101/2021.07.13.21260465; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Figure 5A Figure 5B

Strata + ARID1A=Mutant + ARID1A=WT Strata + ARID1A=Mutant + ARID1A=WT

1.00 1.00 p = 0.0049 p = 0.025 HR: 3.56 HR: 2.76 al v

0.75 vi 0.75 r al v vi r + + ++ 0.50 0.50 all su r + e

v + + + + ++ ++ O ++ 0.25 0.25 + + + + ++ Progression free su

0.00 0.00 0 30 60 90 0 30 60 90 Time Time

ARID1A=Mutant 7 2 0 0 ARID1A=Mutant 7 0 0 0 ata ata r r

St ARID1A=WT 31 17 11 6 St ARID1A=WT 31 11 7 4

0 30 60 90 0 30 60 90 Time Time