Diffuse large B cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is a clinically and genetically heterogeneous disease that is further classified into transcriptionally defined activated B cell (ABC) and germinal center B cell (GCB) subtypes. We carried out a comprehensive genetic analysis of 304 primary DLBCLs and identified low-frequency alterations, captured recurrent mutations, somatic copy number alterations, and structural variants, and defined coordinate signatures in patients with available outcome data. We integrated these genetic drivers using consensus clustering and identified five robust DLBCL subsets, including a previously unrecognized group of low-risk ABC-DLBCLs of extrafollicular/marginal zone origin; two distinct subsets of GCB-DLBCLs with different outcomes and targetable alterations; and an ABC/GCB-independent group with biallelic inactivation of TP53, CDKN2A loss, and associated genomic instability. The genetic features of the newly characterized subsets, their mutational signatures, and the temporal ordering of identified alterations provide new insights into DLBCL pathogenesis. The coordinate genetic signatures also predict outcome independent of the clinical International Prognostic Index and suggest new combination treatment strategies. More broadly, our results provide a roadmap for an actionable DLBCL classification.
CD19-directed chimeric antigen receptor (CAR-19) T cells are groundbreaking immunotherapies approved for use against large B-cell lymphomas. Although host inflammatory and tumor microenvironmental markers associate with efficacy and resistance, the tumor-intrinsic alterations underlying these phenomena remain undefined. CD19 mutations associate with resistance but are uncommon, and most patients with relapsed disease retain expression of the wild-type receptor, implicating other genomic mechanisms. We therefore leveraged the comprehensive resolution of whole-genome sequencing to assess 51 tumor samples from 49 patients with CAR-19–treated large B-cell lymphoma. We found that the pretreatment presence of complex structural variants, APOBEC mutational signatures, and genomic damage from reactive oxygen species predict CAR-19 resistance. In addition, the recurrent 3p21.31 chromosomal deletion containing the RHOA tumor suppressor was strongly enriched in patients for whom CAR T-cell therapy failed. Pretreatment reduced expression or monoallelic loss of CD19 did not affect responses, suggesting CAR-19 therapy success and resistance are related to multiple mechanisms. Our study showed that tumor-intrinsic genomic alterations are key among the complex interplay of factors that underlie CAR-19 efficacy and resistance for large B-cell lymphomas.
In the version of this article originally published, an asterisk was omitted from Fig. 1a. The asterisk has been added to the figure.Additionally, a "NOTCH2" label was erroneously included in Fig. 4a. The label has been removed.
Highlights d Rocaglates both inhibit and activate different protein synthesis factors d Downstream translatome remodeling includes substantial translational activation d Rocaglates induce eEF1ε1-dependent proapoptotic GEF-H1/ RHOA/JNK signaling in cancer cells d Cytotoxicity not replicated by eIF4A silencing urges reevaluation of drug mechanisms Authors
Chimeric antigen receptor-reprogrammed autologous T cells directed to CD19 are breakthrough immunotherapies for heavily pretreated patients with aggressive B-cell lymphomas but still fail to cure most patients. Host inflammatory and tumor microenvironmental factors associate with CAR-19 resistance, but the tumor-intrinsic factors underlying these phenomena remain undefined. To characterize genomic drivers of resistance, we interrogated whole genome sequencing of 30 tumor samples from 28 uniformly CAR-19-treated large-cell lymphoma patients. We reveal that patterns of genomic complexity (i.e., chromothripsis and APOBEC mutational activity), and distinct genomic alterations (deletions of RB1 or RHOA) associate with more exhausted immune microenvironments and poor outcome after CAR-19 therapy. Strikingly, pretreatment reduced expression or sub-clonal mutation of CD19 did not affect responses, suggesting CAR-19 therapy successes are due not only to direct antigen-dependent cytotoxicity but require surmounting immune exhaustion in tumor microenvironments to permit broader host responses that eliminate tumors
Introduction: Anti-CD-19 chimeric antigen receptor-reprogrammed autologous T cells are breakthrough immunotherapies for heavily pretreated patients with aggressive B-cell lymphomas; however, across CAR-19 products, ~60% of patients do not achieve remission or relapse and unfortunately typically progress and rapidly die. Factors associated with impaired response to CAR-19 include inflammatory markers such as interferon signaling and clinical factors such as the need for bridging therapy and high pre-CAR-19 tumor burden, but cell-intrinsic driver of CAR-19 resistance remain largely undefined. Methods: To characterize the genomic mechanisms involved in diffuse large B cell lymphoma (DLBCL) resistance to CAR-19, we interrogated whole genome sequencing (WGS) from 28 relapsed/refractory (r/r) aggressive lymphoma patients treated with axicabtagene ciloleucel (axi-cel). The median coverage was 44.8X. To increase statistical power of analyses, we included also 50 newly diagnosed DLBCL patients from the Pan-Cancer Analysis of Whole Genomes (PCAWG). Results: As reported in other series, neither double hit cytogenetics nor MYC-BCL2 double expression associated with CAR-19 resistance, despite their negative predictive power for newly diagnosed DLBCL patients. Chapuy and LymphGen classification algorithms also demonstrated no prognostic significance. Among known mutated driver genes, only TP53 was significantly enriched in our cohort in comparison to the PCAWG cohort (p=0.002), but it did not predict poor CAR-19 outcome. Among other genes known to be involved in DLBCL pathogenesis, only mutations in NFKBIA or MYC, associated with worse PFS (p=0.04, p=0.025 respectively). Next, we identified 12 single base substitution (SBS) mutational signatures detected in our cohort of r/r lymphomas, four of which are caused by exposure to distinct chemotherapies (Landau et al., 2020, Nat Comm). The melphalan-related signature (SBS-MM1) was identified in 4 out 5 patients who received high dose melphalan followed by autologous stem cell transplant, and 75% of patients exposed to platinum had evidence of one of the three known platinum signatures. Across different SBS signatures, only presence of APOBEC (SBS2 and SBS13) associated with worse PFS with 4/5 patients progressing (p=0.03). We compared newly diagnosed and r/r DLBCL by GISTIC2.0 copy number variation (CNV) analysis, revealing three gene deletions significantly enriched in our r/r cohort: TP53, RHOA and RB1. Interestingly, the deletions involving RHOA and RB1 both independently predicted poor outcome (p=0.0007 and p=0.05 respectively) with 5/5 and 6/8 patients progressing respectively. The third, involving TP53 (46.4% of patients), had no prognostic impact but reflected the high-risk nature of the heavily pretreated tumors. WGS allows detailed identification of structural variants and complex events. Indeed, we found evidence of chromothripsis, a catastrophic event in which one or more chromosomes are shattered and aberrantly reassembles generating multiple aneuploidies, in 39.3% of r/r DLBCL. This strongly associated with poor CAR-19 outcome, with 9/11 affected cases experiencing early progression (p=0.041). Finally, reduced expression (n=3) or genomic alteration (n=3) of CD19 did not associate with poor outcome. We found one case, with durable response, containing a sub-clonal mutation in CD19 (L174V) at baseline, previously reported as associated with CAR-19 resistance. In line with recent evidence, these findings indicate that antigen-mediated tumor killing is not the only mechanism of tumor eradication, and CD19-independent resistance mechanisms predominate. Conclusions: Leveraging the high resolution of WGS, we observed that markers of genomic complexity (chromothripsis and APOBEC) and specific genomic alterations (RHOA and RB1 deletion) associate with resistance to CAR-19 immunotherapy for aggressive B-cell lymphomas. Fifteen out of sixteen patients (93.8%) who relapsed on CAR-19 contained at least one of the described genomic alterations. Recent data demonstrate that an immunosuppressed TME leads to CAR-19 failure in patients, and animal studies show activation of host T cells by CAR-T cells. Combining these findings with these genomics findings, successful CAR-19 therapy must overcome the immune-exhausted tumor microenvironment to mobilize the host immune system and eliminate the tumor. Figure 1 Figure 1. Disclosures Jain: Takeda: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria. Faramand: Novartis: Research Funding; Kite/Gilead: Research Funding. Landgren: Amgen: Research Funding; Janssen: Research Funding; Amgen: Honoraria; Celgene: Research Funding; Janssen: Other: IDMC; Janssen: Honoraria; Takeda: Other: IDMC; GSK: Honoraria. Locke: Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Gerson Lehrman Group: Consultancy; Calibr: Consultancy, Other: Scientific Advisory Role; Janssen: Consultancy, Other: Scientific Advisory Role; Umoja: Consultancy, Other; Novartis: Consultancy, Other, Research Funding; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Takeda: Consultancy, Other; Emerging Therapy Solutions: Consultancy; EcoR1: Consultancy; Cowen: Consultancy; Wugen: Consultancy, Other; Legend Biotech: Consultancy, Other; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy. Maura: Medscape: Consultancy, Honoraria; OncLive: Honoraria. Davila: Precigen: Research Funding.
Chimeric antigen receptor T (CAR-T) cells are an emerging approach for the treatment of hematologic and solid tumor malignancies. Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel were the first FDA-approved CAR-T therapies targeting CD19 for patients with relapsed/refractory (r/r) large B-cell lymphoma. Pivotal studies showed complete response (CR) rates of 58% and 40%, respectively, and we sought to investigate if the data are similar to our single-center results. We carried out a retrospective analysis of patients diagnosed with r/r diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, or mantle cell lymphoma who were treated with axi-cel or tisagenlecleucel at Sylvester Comprehensive Cancer Center in Miami, FL between January 2016 and October 2019. Primary objectives were to identify clinical characteristics associated with improved overall and progression-free survival (OS and PFS). Secondary analyses included incidence of post-CAR-T toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Our analysis included 44 patients: 32 received FDA-approved commercial product and 12 received axi-cel through clinical trials. Median age at time of CAR-T therapy was 62 years, and 70% of patients were male. Median number of prior treatments was 4, and 14 patients had undergone prior hematopoietic stem cell transplantation (12 autologous, 2 allogeneic). By day-30 post-treatment PET scan, 25 patients (57%) achieved CR or partial response (PR), while 16 (36%) had progressive (PD) or stable disease (SD). The remaining 3 patients decompensated rapidly post-infusion. Overall, patients with a CR or PR at 30 days had significantly improved OS and PFS compared to patients with PD or SD (OS p = 0.009, PFS p < 0.001). Univariate analyses showed patients requiring aggressive supportive measures in the post-infusion period had decreased OS compared to those who did not: requirement for ICU care (p = 0.018), vasopressor use (p = 0.01), and steroid treatment (p = 0.018) were all associated with inferior survival. There was no survival difference in DLBCL patients classified as double expressor or double hit; however, patients with germinal center B-cell (GCB) DLBCL trended strongly towards improved OS (p = 0.073) compared to non-GCB patients. CRS affected 35 patients (80%), while 24 patients (55%) experienced ICANS. Incidence of toxicities did not vary significantly in patients who received CAR-T commercially or in clinical trials. Patients who did not experience CRS had improved OS (p=0.061), and of patients who had CRS or ICANS, SD/PD patients had significantly worse PFS (p= <0.001, p= 0.024). This single-center retrospective analysis of patients receiving CAR-T therapy for r/r large B-cell lymphoma showed that incidence and management of toxicities and factors such as tumor subtype associate with treatment response. Further investigations into these factors may provide more insight into optimal management of patients undergoing CAR-T therapy. Citation Format: Fahmin Basher, Caroline A. Coughlin, Deukwoo Kwon, Lazaros Lekakis, Jonathan Schatz. Single-center experience of chimeric antigen receptor T-cell (CAR-T) immunotherapy in relapsed/refractory large B-cell lymphoma identifies association of acute toxicities with inferior disease outcomes [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-55.
In the version of this article originally published, some text above the "Tri-nucleotide sequence motifs" label in Fig. 2a appeared incorrectly. The text was garbled and should have appeared as nucleotide codes.Additionally, the labels on the bars in Fig. 2c were not italicized in the original publication. These are gene symbols, and they should have been italicized.The colored labels above the graphs in Fig. 4b were also erroneously not italicized. These labels represent gene names and loci, and they should have been italicized.
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