Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Transcriptomic and genetic characterization of DLBCL has increased the understanding of its intrinsic pathogenesis and provided potential therapeutic targets. However, the role of the microenvironment in DLBCL biology remains less understood. Here, we performed a transcriptomic analysis of the microenvironment of 4,655 DLBCLs from multiple independent cohorts and described four major lymphoma microenvironment categories that associate with distinct biological aberrations and clinical behavior. We also found evidence of genetic and epigenetic mechanisms deployed by cancer cells to evade microenvironmental constraints of lymphoma growth, supporting the rationale for implementing DNA hypomethylating agents in selected patients with DLBCL. In addition, our work uncovered new therapeutic vulnerabilities in the biochemical composition of the extracellular matrix that were exploited to decrease DLBCL proliferation in preclinical models. This novel classification provides a road map for the biological characterization and therapeutic exploitation of the DLBCL microenvironment.Significance: In a translational relevant transcriptomic-based classification, we characterized the microenvironment as a critical component of the B-cell lymphoma biology and associated it with the DLBCL clinical behavior establishing a novel opportunity for targeting therapies.This article is highlighted in the In This Issue feature, p. 1307

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB^1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial³. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy—a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8⁺ cytotoxic T cells, but relative sparing of CD4⁺ effector subsets. Dual ICB potently engaged CD4⁺ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that—together with CD4⁺ and CD8⁺ T cells—contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Purpose: The response to immune checkpoint inhibitors (ICI) often differs between genders in non–small cell lung cancer (NSCLC), but metanalyses results are controversial, and no clear mechanisms are defined. We aim at clarifying the molecular circuitries explaining the differential gender-related response to anti–PD-1/anti–PD-L1 agents in NSCLC. Experimental Design: We prospectively analyzed a cohort of patients with NSCLC treated with ICI as a first-line approach, and we identified the molecular mechanisms determining the differential efficacy of ICI in 29 NSCLC cell lines of both genders, recapitulating patients’ phenotype. We validated new immunotherapy strategies in mice bearing NSCLC patient-derived xenografts and human reconstituted immune system (“immune-PDXs”). Results: In patients, we found that estrogen receptor α (ERα) was a predictive factor of response to pembrolizumab, stronger than gender and PD-L1 levels, and was directly correlated with PD-L1 expression, particularly in female patients. ERα transcriptionally upregulated CD274/PD-L1 gene, more in females than in males. This axis was activated by 17-β-estradiol, autocrinely produced by intratumor aromatase, and by the EGFR-downstream effectors Akt and ERK1/2 that activated ERα. The efficacy of pembrolizumab in immune-PDXs was significantly improved by the aromatase inhibitor letrozole, which reduced PD-L1 and increased the percentage of antitumor CD8+T-lymphocytes, NK cells, and Vγ9Vδ2 T-lymphocytes, producing durable control and even tumor regression after continuous administration, with maximal benefit in 17-β-estradiol/ERα highfemale immune-xenografts. Conclusions: Our work unveils that 17-β-estradiol/ERα status predicts the response to pembrolizumab in patients with NSCLC. Second, we propose aromatase inhibitors as new gender-tailored immune-adjuvants in NSCLC. See related commentary by Valencia et al., p. 3832

The Italian Register of Actionable Mutations (RATIONAL) is a multicentric, observational study collecting next-generation sequencing (NGS)-based tumour profiling data of patients with advanced solid tumours. The study enrols patients who had available an NGS-based tumour profiling (Pathway-A) or undergo comprehensive genomic profiling (CGP) with FoundationOne CDx assays within the trial (Pathway-B). The primary endpoint was the rate of actionable mutations identified. Sequencing data were available for 738 patients in Pathway-A (218) and -B (520). In Pathway-A, 154/218 (70.6%) tests were performed using NGS panels ≤52 genes, and genomic alterations (GAs) were found in 164/218 (75.2%) patients. In Pathway-B, CGP revealed GAs in 512/520 (98.5%) patients. Levels I/II/III actionable GAs according to the European Society of Medical Oncology Scale for Clinical Actionability of molecular Targets (ESCAT) were identified in 254/554 (45.8%) patients with non-small-cell lung cancer, cholangiocarcinoma, colorectal, gastric, pancreatic and breast cancer. The rate of patients with level I GAs was similar in Pathways A and B (69 versus 102). CGP in Pathway-B revealed a higher number of patients with level II/III GAs (99 versus 20) and potentially germline pathogenic/likely pathogenic variants (58 versus 15) as compared with standard testing in Pathway-A. In patients with cancer of unknown primary, CGP detected OncoKB levels 3B/4 GAs in 31/58 (53.4%) cases. Overall, 67/573 (11.7%) of patients received targeted therapy based on genomic testing. The Italian Register of Actionable Mutations represents the first overview of genomic profiling in Italian current clinical practice and highlights the utility of CGP for identifying therapeutic targets in selected cancer patients.