Correlative Biomarkers for CART19 Response in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Introduction: CD19 CAR T cell (CART19) therapy has changed the paradigms for managing relapsed diffuse large B cell lymphoma (DLBCL) however there remain challenges to achieving long term durable responses for majority of patients. Studies of lymphoma microenvironment (LME) hypothesize LME may affect CART19 response (Kotlov et al, Cancer Discov, 2021). In this study, we assessed the somatic mutation and LME profile through whole exome (WES) and RNA-sequencing (RNA-Seq) of patients treated with CART19 therapy to identify biomarkers of response.

Methods: Patients from our center treated with commercial CART19 cells and biospecimen consents were identified for tumor biopsy samples pre and post-CART19 therapy. Matched normal were collected when available. WES and RNA-Seq were performed on tumor samples. Cell of origin (COO), double hit gene signatures (DHITs), and lymphoma microenvironment (LME) subtypes were calculated as described in Kotlov et al (Cancer Discov, 2021). The primary outcome endpoint was persistent response 6 months (mo) post CART19 infusion. Progression free (PFS) and overall survival (OS) was assessed by Kaplan Meier method and curves compared using log rank test.

Results: WES and RNA-Seq were performed on 54 tumor samples from 49 patients with R/R DLBCL receiving CART19, either axicabtagene ciloleucel (n = 30), tisagenlecleucel (n = 18), or lisocabtagene maraleucel (n = 1). Samples were collected before (n=41) and after (n=13) CART19 therapy, and of these there are 5 patients with paired pre and post-CART samples. WES was successful in 49 samples from 44 patients, while RNA-Seq was successful in 51 samples from 47 patients. In 49 patients, overall response at 3 mo was 77.6%, (38/49) (59.2% CR, 18.4% PR), and PFS at 6 mo was 49% (24/49). Transformed DLBCL comprised 37% (18/49) of patients with overall response at 3 mo 89% (16/18) (61% CR, 28% PR), and PFS at 6 mo was 61% (11/18). We confirmed prognostic biomarkers to CAR-T therapy, such as increased lactate dehydrogenase (LDH, p = 0.01), standard uptake values (SUV, p = 0.02), and metabolic tumor volume (MTV, p = 0.02). Genomic features added further nuance into the understanding of CART19 outcomes (Figure 1).

Single feature analysis revealed PIK3CA amplification was associated with improved PFS (p = 0.03), and MHCII RNA expression correlated with response to CART19 at 6 mo in GCB DLBCL (p = 0.03). Upregulation of MHCII is likely associated with the centrocyte-like phenotype, which was confirmed by a higher centrocyte B-cell associated gene signature in GCB in responders versus non-responders (p = 0.01) (Dybkær et al, JCO, 2015). Differential gene expression (DEG) analysis also revealed higher expression of MHCII genes in GCB-like responders and patients with transformed FL. In DLBCL, TGF-β pathway is inactivated via SMAD1 whose methylation suppress gene expression and inhibit chemo-sensitization. SMAD1 suppression is also associated with a depleted LME (Clozel et al, Cancer Discov, 2013; Stelling et al, Blood, 2018). High expression of SMAD1 was associated with being progression free at 6 mo in both activated B cell-like (ABC) (p = 0.003) and GCB (p = 0.06) DLBCL.

The cohort was classified into favorable (germinal center-like and mesenchymal) and unfavorable (inflamed and depleted) LME subtypes (Figure 2). Favorable LME subtypes demonstrated superior OS relative to unfavorable subtypes (p = 0.02). Longitudinal analysis of one patient with paired pre- and post- CART19 tumor samples detected a potential mechanism of CAR-T relapse through B2M loss of function mutations in ABC DLBCL. Interestingly, the LME also switched from mesenchymal to depleted between the pre- and post-CART19 samples. Other paired samples will be further analyzed.

Conclusion: Integrated molecular analysis of the LME and genomic alterations provides valuable information about correlative biomarkers for response to CART19 immunotherapy in DLBCL patients. With this cohort, PIK3CA amplification corresponded to improved PFS in DLBCL. We observed increased MHCII expression and centrocyte-like cells in GCB DLBCL CART19 responders, while SMAD1 was higher in ABC responders. Germinal center-like and mesenchymal LME exhibited increased OS, while inflamed and depleted LME subtypes had decreased OS. These genomic biomarkers for CART19 treatment response may help guide clinical treatment plans, minimize adverse events, or develop new therapeutic approaches.

Batlevi:Dava Oncology: Other: Provision of Services; ADC Therapeutics: Other: Provision of Services; Bristol-Myers Squibb: Other: Ownership / Equity Interests; Provision of Services; GLG Pharma: Consultancy; Seattle Genetics: Consultancy; Life Sciences: Consultancy; Roche/Genentech: Research Funding; Xynomic: Research Funding; Juno/Celgene: Consultancy; Kite Pharma: Consultancy; Autolus: Research Funding; Bayer: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Epizyme: Research Funding. Nos:BostonGene Corp.: Ended employment in the past 24 months, Patents & Royalties. Kotlov:BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Sorokina:BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Bagaev:BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Shah:Amgen: Research Funding; Beyond Spring: Research Funding; Janssen: Research Funding. Shouval:MyBiotics: Consultancy; Medexus: Consultancy, Ended employment in the past 24 months. Ewalt:Acceleron Pharm: Consultancy; Loxo Oncology at Lilly: Consultancy. Belova:BostonGene Corp.: Ended employment in the past 24 months. Degryse:BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Tazearslan:BostonGene Corp.: Consultancy, Current Employment, Current holder of stock options in a privately-held company, Honoraria. Podsvirova:BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Love:BostonGene Corp: Current Employment. Postovalova:BostonGene Corp.: Current holder of stock options in a privately-held company, Patents & Royalties. Fowler:BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; CelGene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Perales:Orca Bio: Consultancy; VectivBio AG: Honoraria; Vor Biopharma: Honoraria; Cidara Therapeutics: Consultancy; Sellas Life Sciences: Consultancy; Abbvie: Honoraria; Astellas: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria; MorphoSys: Consultancy, Honoraria; Takeda: Honoraria; Medigene: Consultancy; Servier: Consultancy; Bellicum: Honoraria; DSMB: Other; Omeros: Consultancy; Nektar Therapeutics: Consultancy, Honoraria; Novartis: Honoraria; Miltenyi Biotec: Consultancy, Honoraria; Merck: Consultancy; Kite, a Gilead Company: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Bristol-Mysers Squibb: Honoraria. Dogan:Loxo: Consultancy; Roche: Other: Research Funding; EUSA Pharma: Consultancy; Seattle Genetics: Consultancy; Peer View: Honoraria; Takeda: Other: Research Funding; Physicians’ Education Resource: Consultancy, Honoraria; Incyte: Consultancy. Arcila:Physicians’ Education Resource: Consultancy; Clinical Care Options: Consultancy; Invivoscribe: Consultancy; Janssen Global Services: Consultancy; PeerView Institute for Medical Education: Consultancy; AstraZeneca: Consultancy; Biocartis US, Inc: Consultancy; Bristol-Myers Squibb: Consultancy. Salles:Roche/Genentech, Gilead Sciences, Janssen, Celgene, Novartis, MorphoSys AG, Epizyme, Alimera Sciences, Genmab, Debiopharm Group, Velosbio, Bristol-Myers Squibb, BeiGene, Incyte, Miltenyi Biotec, Ipsen, Kite, a Gilead Company, Loxo, Rapt: Consultancy; AbbVie, BeiGene, Bristol Myers Squibb, Celgene, Debiopharm, Epizyme, Genentech/Roche, Genmab, Incyte, Kite, a Gilead Company, Miltenyi, MorphoSys, Takeda, and VelosBio: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech, Janssen, Celgene, Gilead Sciences, Novartis, AbbVie, MorphoSys AG, Amgen, Bayer, Epizyme, Regeneron, Kite, a Gilead Company: Honoraria. Sauter:Gamida Cell: Consultancy; Kite Pharma Inc.: Consultancy; Karyopharm Therapeutics Inc.: Consultancy; Precision Biosciences: Other: PI; Genzyme/Sanofi: Other: PI; BMS: Other: PI; Ono Pharmaceuticals: Consultancy; CSL Behring: Consultancy.