Purpose: Only a minority of patients with advanced non–small cell lung cancer (NSCLC) truly benefits from single-agent PD-1 checkpoint blockade, and more robust predictive biomarkers are needed. Experimental Design: We assessed tumor samples from 67 immunotherapy-treated NSCLC cases represented in a tissue microarray, 53 of whom had pretreatment samples and received monotherapy. Using GeoMx Digital Spatial Profiling System (NanoString Technologies), we quantified 39 immune parameters simultaneously in four tissue compartments defined by fluorescence colocalization [tumor (panCK+), leucocytes (CD45+), macrophages (CD68+), and nonimmune stroma]. Results: A total of 156 protein variables were generated per case. In the univariate unadjusted analysis, we found 18 markers associated with outcome in spatial context, five of which remained significant after multiplicity adjustment. In the multivariate analysis, high levels of CD56 and CD4 measured in the CD45 compartment were the only markers that were predictive for all clinical outcomes, including progression-free survival (PFS, HR: 0.24, P = 0.006; and HR: 0.31, P = 0.011, respectively), and overall survival (OS, HR: 0.26, P = 0.014; and HR: 0.23, P = 0.007, respectively). Then, using an orthogonal method based on multiplex immunofluorescence and cell counting (inForm), we validated that high CD56+ immune cell counts in the stroma were associated with PFS and OS in the same cohort. Conclusions: This pilot scale discovery study shows the potential of the digital spatial profiling technology in the identification of spatially informed biomarkers of response to PD-1 checkpoint blockade in NSCLC. We identified a number of relevant candidate immune predictors in spatial context that deserve validation in larger independent cohorts.

Immunotherapy resistance in non–small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti–PD-1), highly metastatic and fibrotic, and secreted TGFβ/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFβ antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically. Significance: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti–PD-1 therapy, which can be overcome by targeting PTEN loss–mediated immunosuppression.

Background Most patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC. Methods We initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively. Results Using continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK–/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted p<0.05) upregulation of PD-L1, TIM-3, ICOS, and CD40 in two independent cohorts. Conclusions This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC.

The new IASLC/ATS/ERS lung adenocarcinoma classification from a clinical perspective: current concepts and future prospects

Platinum-based chemotherapy plus PD1 axis blockade is the standard of care in the first-line treatment of extensive-stage small cell lung cancer (SCLC). Despite the robust and consistent increase in long-term survival with PD1 axis inhibition, the magnitude of the benefit from immunotherapy seems lower than that for other solid tumors. Several immune evasive mechanisms have been shown to be prominently altered in human SCLC, including T-cell exclusion, downregulation of components of the MHC class I antigen processing and presentation machinery, or upregulation of macrophage inhibitory checkpoints, among others. New immunotherapies aiming to target some of these dominant immune suppressive features are being intensively evaluated preclinically and clinically in SCLC. They include strategies to enhance the efficacy and/or reverse features that promote intrinsic resistance to PD1 axis inhibition (e.g., restoring MHC class I deficiency and targeting DNA damage response) and novel immunomodulatory agents beyond T-cell checkpoint blockers (e.g., T cell–redirecting strategies, antibody–drug conjugates, or macrophage checkpoint blockers). Among them, delta-like ligand 3–targeted bispecific T-cell engagers have shown the most compelling preliminary evidence of clinical efficacy and hold promise as therapies that might contribute to further improve patient outcomes in this disease. In this study, we first provide a brief overview of key tumor microenvironment features of human SCLC. Then, we update the current clinical evidence with immune checkpoint blockade and review other emerging immunotherapy strategies that are gaining increasing attention in SCLC. We finally summarize our future perspective on immunotherapy and precision oncology for this disease.

Purpose: Transcriptomic subtyping holds promise for personalized therapy in extensive-stage small cell lung cancer (ES-SCLC). In this study, we aimed to assess intratumoral transcriptomic subtype diversity and to identify biomarkers of long-term chemoimmunotherapy benefit in human ES-SCLC.Experimental Design: We analyzed tumor samples from 58 patients with ES-SCLC enrolled in two multicenter single-arm phase IIIb studies evaluating frontline chemoimmunotherapy in Spain: n = 32 from the IMfirst trial and n = 26 from the CANTABRICO trial. We used the GeoMx Digital Spatial Profiler system to perform multi-region transcriptomic analysis. For subtype classification, we performed hierarchical clustering using the relative expression of ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), and YAP1 (SCLC-Y).Results: Subtype distribution was found to be similar between bothcohorts, except for SCLC-P, which was not identified in the CANTABRICO_DSP cohort. A total of 44% of the patients in both cohorts had tumors with multiple coexisting transcriptional subtypes. Transcriptional subtypes or subtype heterogeneity was not associated with outcomes. Most potential targets did not show subtype-specific expression. Consistently in both cohorts, tumors from patients with long-term benefit (time to progression ≥12 months) contained an IFNγ-dominated mRNA profile, including enhanced capacity for antigen presentation. Hypoxia and glycolytic pathways were associated with resistance to chemoimmunotherapy.Conclusions: This work suggests that intratumoral heterogeneity, inconsistent association with outcome, and unclear subtype-specific target expression might be significant challenges for subtype-based precision oncology in SCLC. Preexisting IFNγ-driven immunity and mitochondrial metabolism seem to be correlates of long-term efficacy in this study, although the absence of a chemotherapy control arm precludes concluding that these are predictive features specific for immunotherapy.