Genetic Alterations of Gα13 Signaling Pathway with BCL2 over-Expression Confers Lymphoma Dissemination and Inferior Outcome in Germinal Center B Cell Diffuse Large B Cell Lymphoma
Authors: Ennishi Daisuke , Mottok Anja , Shulha Hennady , Farinha Pedro , Chan Fong Chun , Meissner Barbara , Boyle Merrill , Ben-Neriah Susana , Hother Christoffer E , Kridel Robert , Morin Ryan , Marra Marco A , Savage Kerry J. , Sehn Laurie H. , Connors Joseph M. , Steidl Christian , Scott David W , Gascoyne Randy D. 
Published: October 2019

Abstract
Background: Diffuse large B cell lymphoma (DLBCL) is divided into two distinct molecular subtypes, germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. Genetic landscape studies of DLBCL have revealed several GCB-DLBCL specific mutations, including CREBBP, GNA13, EZH2, TNFRSF14, BCL2 and MEF2B. Functional studies have recently shown that the inactivation of Gα13 signaling pathway genes, including GNA13, together with BCL2 over-expression, allows GC B-cells to escape the germinal center niche and widely disseminate. Although these findings revealed a critical role of genetic alterations of Gα13 signaling pathway in GC-driven mouse models of lymphomagenesis, clinical correlation is lacking. Here we analyzed the clinical impact of genetic alterations of Gα13 signaling pathway in a large population-based DLBCL cohort.
Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of GNA13, P2RY8, ARHGEF1, S1PR2 and RHOA was performed using a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Cell-of-origin classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014; 123) in 299 patients and the Hans algorithm (Hans, Blood 2004; 103) in 32 cases with low tumor content (<40%).
Results: Using next generation sequencing, 225 SNVs and 5 Indels were detected in GNA13 (16%), P2RY8 (18%), ARHGEF1 (6%), S1PR2 (3%) and RHOA (6%). SNP 6.0 microarrays revealed heterozygous deletions in GNA13 (2%), ARHGEF1 (1%), S1PR2 (4%) and RHOA (8%), but homozygous deletion was not found in any of these five loci. GNA13, P2RY8 and ARHGEF1 mutations were significantly more frequent in the GCB subtype than ABC subtype (26% vs. 6%; p<.0001, 25% vs 7%; p=.0002, and 8% vs. 5%; p=.008, respectively). 185 GCB-DLBCL cases were further analyzed for clinical correlations.
In the cases with mutations of any of the five Gα13 signaling pathway genes, BCL2 over-expression (cut off; 50%) and translocation was associated with increasing stage (p=.018 and p=.005, respectively), but not in wt cases (p=.53 and p=.63, respectively). Specifically, in the cases with GNA13 and P2RY8 mutations individually, BCL2 over-expression was associated with advanced stage (stage III/IV, p=.018 and p=.037, respectively), but not in wild type (wt) cases. Importantly, BCL2 over-expression in the cases harboring Gα13 pathway mutations was not significantly associated with other poor risk features, including any other IPI factors or bone marrow involvement, indicating that genetic alterations in Gα13 signaling pathway accompanied by BCL2 over-expression might promote lymphoma dissemination into lymph nodes but not extranodal sites.
With a median follow up of 6.5 years for living patients, there was no prognostic impact of harboring any isolated Gα13 pathway mutation in GCB-DLBCL patients. However, in cases with any Gα13 pathway mutations, BCL2 over-expression was significantly associated with an inferior 5y-time to progression (TTP; 90% vs 62%, p=.003) and disease-specific survival (DSS; 90% vs 71%, p=.042), but not in wt cases (Fig 1). In a Cox model of TTP including the IPI, BCL2 over-expression remained prognostic in the cases harboring any Gα13 pathway mutations (HR=4.13 [1.42-12.01], p=.009), but not in wt cases (HR=1.70 [0.62-4.68], p=.31). In cases with any Gα13 pathway alterations including copy number loss, BCL2 over-expression was also significantly associated with an inferior TTP (HR=3.64 [1.39-9.57], p=.009) independent of IPI, but not in the cases without genetic alterations (HR=1.75 [0.57-5.34], p=.33).
Conclusions: Genetic alterations in Gα13 signaling pathway genes cooperate with BCL2 over-expression to promotes lymphoma dissemination to nodal sites and is associated with the poor outcome in GCB-DLBCL
Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP

Disclosures
Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.