FAS Mutations in Follicular Lymphoma Are Rare but Associated with Aggressive Clinical Behavior.

Follicular lymphoma (FL) is considered an indolent but incurable lymphoma. Treatment with cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) provides complete or partial responses in most patients. The BCL2 translocation t(14;18) is present in 85% of the cases, but additional genomic alterations must occur to induce overt FL. We have sequenced the genome and transcriptome of a cytogenetically normal FL sample taken from a patient (pt 1) that had an unusually aggressive clinical course with the aim of identifying genomic alterations that could contribute to FL pathogenesis. We identified a mutation in FAS/CD95, a key component of the extrinsic apoptotic pathway. We hypothesized that FAS mutations may contribute to treatment resistance in FL by inhibiting apoptosis. This study investigates the prevalence and clinical outcome of patients with FL harbouring mutations in the exons coding for the functional “death domain” of the FAS gene.

The initial FL sample was subjected to cytogenetic analysis and whole genome tiling array comparative genomic hybridization followed by next-generation sequencing of the tumour genome and tumor transcriptome following the manufacturer's protocol (Illumina). FAS emerged as a potential candidate that could explain the unusually aggressive FL. We performed PCR amplification of exons 7,8,9 and the 3'UTR of the FAS gene with universal M13F(-21) and M13 primer extensions on pre-treatment FL samples derived from 214 patients including 33 diffuse large B cell lymphoma samples that had evolved from a prior FL (i.e. paired FL and DLBCL). PCR products were purified using AMPpure magnetic beads and bi-directionally sequenced using BigDye® Terminator v3.1 and an ABI 3730 XL sequencer. Analysis was performed using Mutation Surveyor. Mutations were considered present if they were observed in both forward and reverse reads.

Patient 1 had a t(14;18) negative, grade 1 FL, that progressed rapidly despite CVP-R and second line chemotherapy but is now in complete remission following an allogeneic stem cell transplant. The FL immunophenotype was CD19⁺, CD10⁺, BCL2⁺, BCL6⁺ and lambda clonal. Minimal genomic gains and losses were observed by aCGH. After filtering known single nucleotide polymorphisms (SNPs), a total of 320 candidate novel protein-altering changes were identified (affecting 298 genes) in the tumor genome and transcriptome sequence data. Validation of the mutated genes by Sanger sequencing revealed a novel, somatic and coding mutation in FAS at genomic position chr10:90764005, changing C to T, resulting in a premature truncation of the protein. We then sequenced exons 7, 8, 9 and the 3'UTR of the FAS gene from 214 FL patients. Ten novel FAS mutations were detected, of these six were coding, three of which produced a truncated protein. Of the six coding mutations, two were observed in the transformed DLBCL sample. Coding mutations in FAS appeared to be associated with an aggressive clinical course (median time to progression=12 months for coding mutations (n=6) vs 34 months for non-coding or wild type (n=208), P=0.06). 2 of the 6 patients developed early transformation to DLBCL, 2 had treatment resistant FL that required allogeneic bone marrow transplant and 2 have already died due to progressive treatment refractory FL.

Next generation sequencing technology revealed novel somatic mutations in a cytogenetically normal FL sample, one of which was a mutation in the FAS gene. Sanger sequencing of a large cohort of FL samples revealed that 5% of cases harboured novel mutations in the death domain of FAS (2% coding, 3% non-coding). Coding mutations were rare but when present were associated with atypically aggressive disease.

Connors:Roche Canada: Research Funding. Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.