Integrating Genomic Alterations in Diffuse Large B-Cell Lymphoma Identifies New Relevant Pathways and Potential Therapeutic Targets
Authors: Dlouhy Ivan , Karube Kennosuke , Enjuanes Anna , Martín-García David , Nadeu Ferran , Ordoñez Gonzalo R , Rovira Jordina , Royo Cristina , Navarro Alba , Gonzalez Blanca , Castellano Giancarlo , Rubio-Perez Carlota , Tamborero David , Briones Javier , Salar Antonio , Sancho Juan-Manuel , Mercadal Santiago , Gonzalez Barca Eva , Escoda Lourdes , Hiroaki Miyoshi , Ohshima Koichi , Kato Koji , Akashi Koichi , Mozos Anna , Colomo Lluis , Alcoceba Miguel , Valera Alexandra , Carrio Anna , Lopez-Bigas Nuria , Schmitz Roland , Staudt Louis , Jares Pedro , Salaverria Itziar , López-Guillermo Armando , Campo Elias 
Published: October 2019

Abstract
Introduction: DLBCL is biological and clinically highly heterogeneous. Although different genetic aberrations, including recurrent somatic mutations, have been described in this tumor, their clinical impact remains to be clarified. The aim of the present study was to determine somatic mutations and copy number alterations of a selected group of genes in patients with DLBCL, in order to assess their prognostic importance and to identify potential personalized targeted drugs for these patients.
Methods: 150 patients (78M/72F; median age, 66 years) diagnosed with de novo DLBCL no otherwise specified at Hospital Clínic and other institutions of the GELCAB, treated with immunochemotherapy, were included in the study. An independent series of 111 patients (54M/57F; median age, 63 years), diagnosed at different Japanese and Spanish institutions, was used to validate the significant findings. Targeted next generation sequencing (NGS) of 106 representative genes related with DLBCL and Copy Number Alterations (CNA) assessment were performed. Ten functional pathways were pre-defined, including NOTCH, tumor suppressor genes, JAK/STAT, epigenome/chromatic modifier, BCR signaling, PI3K-AKT-mTOR, MAP-kinase, B-cell differentiation, immune surveillance and cell cycle alterations. Cell of origin (COO) of the tumors was established using gene expression or the Lymph2Cx assay. Genomic-guided potential therapeutic opportunities for each patient were identified in silico by a Cancer Genome Interpreter platform.
Results: A total of 765 potential driver mutations were identified in 89 of the 106 genes with a slightly higher number in germinal center B-cell like (GCB) than activated B-cell-like (ABC) DLBCL subtype. The most frequently mutated genes found in >15% of the cases were KMT2D (MLL2), MYD88, CREBBP and TP53, with other 27 genes being mutated in >5% of the cases. Several genes were differentially mutated in GCB DLBCL subtype (KMT2D, CREBBP, TNFRSF14, B2M, EZH2, GNA13, FOXO1, ACTB and SOCS1) or ABC subtype (MYD88, PIM1, CD79B and PRDM1). No relevant differences were observed in the clinical features according to individual mutations or CNA. No single gene mutation predicted response to therapy. Genetic alterations in KLHL6, ETV6, SGK1, L8q12.1, CD79B, PIM1 and TP53 predicted poor OS, whereas mutations of SOCS1 were associated with better outcome. Alterations in NOTCH pathway and tumor suppressor pathway were associated with poor outcome, whereas those of JAK/STAT pathway showed favorable prognosis (see table for detailed data). NOTCH pathway (HR 2.8; p=0.006) and tumor suppressor pathway (HR 2.4; p=0.005) maintained independent significance for OS along with R-IPI (H 4.0; p=0.006) in a multivariate analysis that also included COO and beta2-microglobulin. In addition, the prognostic value of NOTCH and tumor suppressor pathways was confirmed in the independent validation series. Finally, we identified 69 cases (46%) carrying at least one genomic alteration in 9 genes considered a biomarker of drug response supported by data of early clinical trials or pre-clinical assays; tumors of additional 26 patients (17%) had at least one gene alteration that could be exploited by a drug repurposing strategy.
Conclusions: Integrating the deep sequencing analysis of a panel of selected genes and CNA, we have recognized novel target genes and defined the clinical relevance of alterations of NOTCH and tumor suppressor pathways in DLBCL. Using an in silico prescription pipeline we have also identified a number of candidate drugs with potential therapeutic interactions with driver oncogenic proteins. All these findings may orient future preclinical and clinical intervention strategies in DLBCL.
Table Initial features, response to therapy and outcome according to pathways´ status Table. Initial features, response to therapy and outcome according to pathways´ status

Disclosures
Sancho: Celltrion, Inc: Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gonzalez Barca:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Gilead: Speakers Bureau. Ohshima:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau. Akashi:Sunitomo Dainippon Pharma: Consultancy; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Bristol Meyers Squibb: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Shionogi & Co., Ltd: Research Funding; Astellas Pharma: Research Funding.