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J.E. Ramis-Zaldivar et al.
as clonal or subclonal if their cancer cell fractions were ≥85% or <85%, respectively.19 Previously published mutational data from 28 BL, 203 PCM and 295 ABC-DLBCL were used for comparisons.24–26
Gene expression analysis
A total of 12 PBL were analyzed using the Nanostring PanCancer Pathways Panel (NanoString, Seattle, WA, USA) to investigate gene expression differences between different subsets of PBL (Online Supplementary Methods, Online Supplementary Table S3).
Statistical analyses
R software version 3.6.2 was used for statistical analyses. Differences in the distribution of individual parameters among subsets of patients were analyzed by the Fisher exact test for cate- gorical variables, and the Student t-test for continuous variables. Nonparametric Wilcoxon tests were applied when necessary. P- values for multiple comparisons were adjusted using the Benjamini-Hochberg correction (false discovery rate). A P value <0.05 was considered statistically significant unless otherwise indi- cated.
Results
Pathological features and fluorescence in situ hybridization
Twenty-four patients were male and ten female with a median age of 52 years (range, 11-87 years). HIV infection was confirmed in 14 out of 16 cases with available data (88%) and one patient (PBL6) had a post-transplant associ- ated immunodeficiency. Seven patients for whom HIV information was not available were older than 60 years (Table 1). Most cases had an extranodal presentation (84%), mainly affecting the head and neck region (44%) and gas- trointestinal tract (30%). As expected, virtually all cases were negative for B-cell markers, and only two cases showed weak expression of PAX5. Conversely, plasma cell markers such as CD138 and MUM1/IRF4 were positive in 68% and 100% of the cases, respectively. CD79a was expressed in 60% (18/30) of the tumors, usually with a focal pattern, and CD56 was positive in 21% (5/24) (Table 1). EBV (as determined by EBV-encoded small RNA; EBER) was positive in 18 out of 30 (60%) cases whereas human herpes virus-8 was negative in all analyzed cases (Table 1; Figure 1A-H). MYC rearrangements were detected in 26 of the 30 (87%) analyzed cases, most of them with IGH as transloca- tion partner (8/9 cases evaluated). Neither BCL2 nor BCL6 translocations were observed (Table 1, Figure 1I, J).
Genomic profiling of plasmablastic lymphomas
Copy number analysis showed a total of 401 alterations in 33 cases with a median of 12.2 imbalances per case, (range, 3-49) (Online Supplementary Table S4). Specifically, 178 gains, 175 losses, 42 amplifications and six homozy- gous deletions were detected. The most recurrently gained regions (present in ≥20%) were 1q21.1-q44, trisomy 7, 8q23.2-q24.21, 11p13-p11.2, 11q14.2-q25, 12p and 19p13.3-p13.13, whereas recurrent deletions (≥20%) were identified at 1p33, 1p31.1-p22.3, 13q and 17p13.3-p11.2 (Figure 2A). Furthermore, 46 regions of CNN-LOH were detected, with 16q11.2-q24.3 being the most frequently affected region (24%) (Figure 2B).
Recurrent amplifications occurred in chromosome 1q, 7
(6 cases each), 11p13-p11.2 and 15q23 regions (2 cases each). Interestingly, the minimal amplified regions of some recurrent amplifications included key genes related to lym- phoma biology such as 1q21.3-q23.2 (IL6R, ADAR, MEF2D and CKS1B), 1q32.1-q41 (TRAF5), 11p13-p11.2 (CD44 and TRAF6) and 15q23 (MAP2K5) (Online Supplementary Figure S2).27,28 Besides, 25 of the 42 amplifications identified were observed in the context of a chromothripsis-like pattern (Online Supplementary Methods). No recurrent homozygous deletions were found but one case (PBL15) had a focal homozygous deletion including the CDKN2A/B tumor sup- pressor gene.
Mutational landscape of plasmablastic lymphomas
Twenty-seven PBL cases were analyzed by next-genera- tion sequencing with a mean coverage of 374x (range, 66- 1688x). One hundred and ninety-four mutations were iden- tified (Online Supplementary Table S5). A total of 133 variants (69%) were predicted as potential driver mutations with a mean of 4.9 driver mutations per case. The most recurrently mutated genes were STAT3 (37%), NRAS and TP53 (33% each), MYC and EP300 (19% each) and CARD11, SOCS1 and TET2 (11% each) (Figure 2C; Online Supplementary Figure S3).
All STAT3 mutations except one were nonsynonymous variants located in the SH2 domain (positions 585 to 688). Two different hotspots were found within the SH2 domain including D661Y/V and Y640F (3 cases each), which have been previously described to upregulate STAT3 down- stream target genes.29 To assess the STAT3 activation status p-STAT3 was studied by immunhistochemistry in six cases (2 mutated and 4 wild-type). RNA expression of genes relat- ed to the JAK-STAT pathway (JAK-STAT pathway score), was also analyzed in 12 cases using the NanoString plat- form (Online Supplementary Methods). p-STAT3 was positive in four cases (67%), including the two mutated cases but also two wild-type cases. The four plasmablastic PCM studied were negative for p-STAT3 (Online Supplementary Figure S4, Online Supplementary Table S6). STAT3 mRNA expression was similar in mutated and unmutated cases but it showed a tendency to be higher in EBV-positive than - negative tumors (Wilcoxon test, P=0.07). (Online Supplementary Results, Online Supplementary Figures S4 and S5). Additionally, there were no significant differences in the expression of JAK-STAT pathway-related genes between STAT3-mutated and wild-type cases (Wilcoxon test; P=0.15), although a tendency to higher expression was observed in EBV-positive cases (Wilcoxon test; P=0.1) (Online Supplementary Figure S6).
All 12 TP53 mutations were located in the DNA binding domain and three of them were stop-gain mutations (Figure 2D). Bi-allelic inactivation of TP53 was observed in six cases, all negative for EBV expression, in which the 17p13/TP53 locus was also affected by losses or CNN- LOH. NRAS mutations mainly affected the known hotspots Q61 (4 cases) and G13 (3 cases). Finally, all but one exonic MYC mutations were located on the transactivation domain, which has been previously demonstrated to induce MYC stability and inhibition of apoptosis (Figure 2D).30 Interestingly, multiple mutations (considering >10 variants and including intronic and synonymous ones) were found in the five cases with MYC driver mutations and in one additional case with only intronic mutations, all six with concomitant MYC rearrangements. Additionally, a high rate of these mutations involved activated-induced
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