Exome Sequencing of Hodgkin's and Non-Hodgkin Composite Lymphomas Identifies Shared Somatic Mutations Indicative of Common Founding Precursors

Background: Composite lymphomas (CLs) describe the rare situation when two morphologically distinct lymphomas occur in the same patient. Prior studies have supported a shared origin of CLs based on shared chromosomal translocations and/or the presence of shared immunoglobulin rearrangements. We used exome sequencing to examine somatic events in two cases of CLs. We hypothesized that each lymphoma will have shared and unique mutations, consistent with the presence of a common lymphoma precursor. Further, we postulated that the shared mutations represent potential initiating events in lymphomagenesis. Finally, because each case includes a Hodgkin (HL) and non-Hodgkin lymphoma (NHL), we hypothesize that the somatic mutations found in HL may offer insight into genomic drivers of HL.

Clinical Synopsis:Case 1 was a 57-year-old man who presented with abdominal pain associated with a small bowel obstruction (SBO) and lymphadenopathy (LAD). Pathology showed DLBCL (diffuse large B-cell lymphoma, non-germinal center subtype; stage IV). He was treated with 6 cycles of R-CHOP with complete response. Seven months after the completion of R-CHOP, he presented with left neck LAD. An excisional biopsy revealed classical HL (stage II). Hodgkin and Reed-Sternberg (HRS) cells comprised 10% of the sample. He was treated with 3 cycles of ABVD and involved-field radiation therapy. Case 2 was a 66-year-old man with a history of solitary LAD who presented with increasing LAD, night sweats, and a chest wall mass. An excisional biopsy revealed both follicular lymphoma (FL; grade I-II) and classical HL in distinct areas of the lymph node. HRS cells comprised 30% of the sample. FISH studies were positive for t(14;18). He was treated with ABVD for 6 cycles with partial response. Upon progression, a repeat lymph node biopsy showed persistent HL. He was treated with bendamustine and rituximab for 4 cycles with progressive disease.

Methods: We performed whole exome sequencing on formalin fixed paraffin embedded tumor samples and matched normal skin samples. We used our standard somatic variant calling pipeline to call somatic variants. SNV (single nucleotide variant) and indel calls were filtered for basic quality metrics and, using in-house software, were processed through a Bayesian classifier to remove false positive somatic events. Filtered variants were manually reviewed to further verify somatic status. Somatic variants were validated using Ampliseq in Case 1.

Results: Sequencing resulted in >90% of the target regions with at least 20x in all samples. The mean depth of the NHL and normal samples in Case 1 was >65x. To account for the low malignant tumor cellularity of HL, the HL sample of Case 1 was sequenced to a mean depth of 310x. The mean depth for all samples in Case 2 was >100x (FL = 188x; normal = 104x; HL = 215x). After filtering, we identified 60 and 133 variants in 57 and 101 genes in Case 1 and Case 2, respectively. In Case 1, we identified three sites inTP53, TNFRSF14, and RASAL2 that were shared. We also identified variants in HIST1H2AG, KMT2D, and STAT3 unique to the DLBCL sample, and two mutations in PTPRT unique to the HL sample of Case 1 (Figure 1). In Case 2, we identified shared variants in TNFRSF14 and HIST1H2BF. We also identified two distinct BCL10 mutations in the FL and HL samples of Case 2 (Figure 1).

Conclusions: From the shared somatic mutations identified, we infer that a shared lymphoma precursor for each case is likely, and the shared mutations may be early initiating events. In both cases, a shared nonsense mutation was found at TNFRSF14. TNFRSF14 is recurrently mutated in NHL, and recurrent deletions involving TNFRSF14 have been described in HL. Further work is needed to understand how TNFRSF14 alterations drive HL vs. NHL. Finally, it is intriguing that the HL samples contain independent mutations in PTPRT and BCL10. PTPRT is a known cancer gene, and BCL10 has been shown to be associated with the pathogenesis of B-cell NHLs. These data provide new hypotheses for loci involved in HL pathogenesis.

Figure 1

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Variant allele frequencies (VAFs) in CL samples from Case 1 (A) and Case 2 (B). (A) shows the VAFs of confirmed variants in the DLBCL vs. HL. (B) shows VAFs of FL vs. HL. Variants of note are highlighted in black. The lack of separation of sites in Case 2 may be the result of some admixture of the two lymphomas, which were sequenced from cores taken from the same lymph node.

Figure 1

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Variant allele frequencies (VAFs) in CL samples from Case 1 (A) and Case 2 (B). (A) shows the VAFs of confirmed variants in the DLBCL vs. HL. (B) shows VAFs of FL vs. HL. Variants of note are highlighted in black. The lack of separation of sites in Case 2 may be the result of some admixture of the two lymphomas, which were sequenced from cores taken from the same lymph node.

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