Overview Of The Genomic Landscape Of High Risk Diffuse Large B-Cell Lymphoma Using Targeted DNA and RNA Sequencing

The implementation of chemoimmunotherapy (R-CHOP) has dramatically improved the outcome of B-cell NHL particularly DLBCL (> 50% OS at 5 years with very few late relapses). Most failures to R-CHOP occur early (80% in the 1^st 18 months), are not easily salvageable even with high dose therapy-ASCT and are not reliably predicted with current available prognostic models. There is a growing awareness of the molecular heterogeneity of DLBCL beyond well-established germinal center (GC) and non-GC subtypes. Additional genomic alterations include structural alterations (such as in the “double-hit” lymphoma (DHL) (harboring rearrangements or additional copies of MYC and BCL2 or less commonly BCL6); predominantly deregulated or activated driving pathways (ex NF-κB particularly associated with mutations in genes such as CARD11, CD79B or MYD88 in ABC subtype). The recent use of high-resolution technologies has revealed a much higher complexity of the mutational landscape in NHL.

Here, we studied 35 DLBCL primary tumors from very poor outcome DLBCL patients with a median OS of 8.4 months (range .3-44.5 months). Targeted DNA and RNA sequencing using a NGS based genomic profiling assay was performed in a CLIA certified laboratory (Foundation Medicine) and genomic alterations were compared to those previously published for DLBCL: Genomic DNA and total RNA was isolated from 40µm of tissue from archived clinical formalin fixed and paraffin-embedded (FFPE) blocks. Illumina adaptor-ligated sequencing libraries were created and captured by solution hybridization using two custom DNA oligonucleotide baitsets targeting all coding exons of 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >810X unique coverage for DNA and >20,000,000 total pairs for RNA, to enable accurate detection of genomic alterations.

We identified 214 genomic alterations, an average of 6.1 per sample, in 71 genes: 87 substitutions/indels, 85 nonsense or frameshifting mutations resulting in truncation of known tumor suppressor genes, 4 gene amplifications, 15 homozygous gene deletions, and 25 translocations. In the 35 samples, 12 harbored immunoglobulin (Ig) translocations with MYC, BCL2 or BCL6 (2 MYC/IgH, 7 BCL2/IgH, and 6 BCL6/IgH or IgL) and RNA-seq allowed detection of 4 non-Ig partner BCL6 fusions, including RHOH-BCL6, ST6GAL1-BCL6, and GAPDH-BCL6. Alterations aggregated in pathways previously implicated in DLBCL pathogenesis, including chromatin modification (18%) and NF-κB signaling (16%). More than 50% of ABC-DLBCL and 27% of GCB-DLBCL carry somatic mutations in multiple genes, regulators of NF-κB signaling. Intriguingly, alterations in TP53 and MLL2 occurred at approximately double the frequency previously reported for DLBCL overall (32% vs. 17% and 47% vs 24%, respectively, Pasqualucci et al, Nature Genetics, 2011), reflecting likely the poor outcome population of our cohort.

We demonstrate technical feasibility of comprehensive genomic profiling of DLBCL from clinical FFPE specimens, with a low overall failure rate (1/36 samples for DNA, 2/36 for RNA), deep unique coverage and simultaneous detection of multiple rearrangements (BCL2, BCL6, Myc) using a combination of DNA and RNA sequencing. We additionally observe a potentially interesting enrichment of alterations in P53 and MLL2 in aggressive disease, with cohort expansion and more detailed genotype/phenotype correlative analysis ongoing.

Wang:Foundation Medicine: Employment. Nahas:Foundation Medicine: Employment. Donahue:Foundation Medicine: Employment. He:Foundation Medicine: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medincine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine: Employment.