Exome Sequencing Of African American Siblings With Activated, B-Cell Like Diffuse Large B-Cell Lymphoma

One of the major challenges of using published genomic studies of diffuse large B-cell lymphoma (DLBCL) is that the majority of these data were generated from DLBCL patients of European American descent. Genes that were differentially mutated between activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL in these populations may differ from the gene mutations associated with the increased frequency of ABC DLBCL in patients of African ancestry. We have shown that African-American (AA) DLBCL patients more commonly present with the ABC subtype (Flowers ASH 2012). Winship Cancer Institute provides the ideal patient population to investigate racial differences in lymphoma at the genome level. We have identified AA half-siblings who presented as new consults in the lymphoma clinic, both with ABC DLBCL. The female sibling had progressive disease after R-CHOP therapy, while the male sibling presented with de novo DLBCL. This observation of siblings of African descent with ABC DLBCL, one of whom had already demonstrated a poor clinical outcome with standard therapy, afforded the additional unique opportunity to examine differential prognosis among AA DLBCL patients using exome sequence.

A tumor sample from a paraffin embedded tissue block was obtained at initial presentation from each sibling, with an additional fresh frozen lymph node obtained from the male subject. Peripheral blood samples were also obtained from each subject to assess DNA from the normal tissue. The NimbleGen SeqCap EZ v3 capture kit was used for whole exome library preparations according to established standard Illumina TruSeq protocols for FFPE samples. Exome sequencing data was generated using the 100x100 paired-end (PE) sequencing protocol for the Illumina HiSeq2000. The PE reads were mapped to Human Reference Genome NCBI36.1/hg19 using Burrow-Wheeler Aligner. VarScan 2 was used for detection of variants (germline and somatic), LOH, and copy number alterations (CNAs) using exome sequence data from the matched tumor and normal samples. Variants were further filtered according to sequencing- or alignment-related artifacts that include among other things, read position, strand bias, variant frequency, distance to 3′, homopolymer, and mapping quality, and visually confirmed by IGV.

Among the genes with variants identified, EP300, MTOR, MYOM2, and PIK3CD, all previously reported as associated with differentiating ABC from GCB DLBCL, were shown to have germline variants in common between the siblings. A single somatic variant in the EP300 gene was shown specific to the sister. In terms of novel genetic variants, post-filtering, we identified the following possibly damaging (non-benign) germline variants: 4 genes with variants in common between siblings, 5 genes with variants specific to the brother (common to FFPE and Fresh): 19 genes with variants specific to the sister. Novel somatic variants included the following: 1 gene variant specific to the brother (common to FFPE and Fresh), 8 genes with variants specific to the sister. The novel somatic variant specific to the brother also had a reported copy loss that was brother-specific. Exploration of publicly available expression data on ABC DLBCL patients with R-CHOP regimens showed significant associations with overall survival for the genes. We are further exploring haplotype block analysis to infer variant blocks shared by the half-siblings from the common parent to integrate with these results.

We provide novel protein-altering variant results for AA half-siblings of differential prognosis that show to be promising novel markers for ABC DLBCL. The question of whether such markers are specific to AA remains yet to be investigated.

No relevant conflicts of interest to declare.