Overview Of The Genomic Landscape Of High Risk Diffuse Large B-Cell Lymphoma Using Targeted DNA and RNA Sequencing
Authors: Wang Kai , Nahas Michelle , Mato Anthony R , Donahue Amy L , He Jie , Otto Geoff A , Lipson Doron , Yelensky Roman , Ross Jeff , Stephens Phil , Chow Kar Fai , Zielonka Tania , Bhattacharyya Pritish K. , Protomastro Ewelina , Feldman Tatyana , Pecora Andrew L. , Miller Vincent , Goy Andre 
Published: October 2019

Abstract

Background
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 1st 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.

Methods
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.

Results
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.

Conclusions
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.

Disclosures:
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.