VEGF-A drives T cell exhaustion via TOX-dependent transcriptional programming in microsatellite stable colorectal cancers.

Sequence alterations in microsatellites and an elevated mutational burden are observed in 20% of gastric cancers and associated with clinical response to anti–PD-1 antibodies. However, 50% of microsatellite instability–high (MSI-H) cancers are intrinsically resistant to PD-1 therapies. We conducted a phase II trial of pembrolizumab in patients with advanced MSI-H gastric cancer and included serial and multi-region tissue samples in addition to serial peripheral blood analyses. The number of whole-exome sequencing (WES)–derived nonsynonymous mutations correlated with antitumor activity and prolonged progression-free survival (PFS). Coupling WES to single-cell RNA sequencing, we identified dynamic tumor evolution with greater on-treatment collapse of mutational architecture in responders. Diverse T-cell receptor repertoire was associated with longer PFS to pembrolizumab. In addition, an increase in PD-1+ CD8+ T cells correlated with durable clinical benefit. Our findings highlight the genomic, immunologic, and clinical outcome heterogeneity within MSI-H gastric cancer and may inform development of strategies to enhance responsiveness. Significance: This study highlights response heterogeneity within MSI-H gastric cancer treated with pembrolizumab monotherapy and underscores the potential for extended baseline and early on-treatment biomarker analyses to identify responders. The observed markers of intrinsic resistance have implications for patient stratification to inform novel combinations among patients with intrinsically resistant features. See related commentary by Fontana and Smyth, p. 2126. This article is highlighted in the In This Issue feature, p. 2113

Gastric cancer (GC) patients develop malignant ascites as the disease progresses owing to peritoneal metastasis. GC patients with malignant ascites have a rapidly deteriorating clinical course with short survival following the onset of malignant ascites. Better optimized treatment strategies for this subset of patients are needed. To define the cellular characteristics of malignant ascites of GC, we used single-cell RNA sequencing to characterize tumor cells and tumor-associated macrophages (TAMs) from four samples of malignant ascites and one sample of cerebrospinal fluid. Reference transcriptomes for M1 and M2 macrophages were generated by in vitro differentiation of healthy blood-derived monocytes and applied to assess the inflammatory properties of TAMs. We analyzed 180 cells, including tumor cells, macrophages, and mesothelial cells. Dynamic exchange of tumor-promoting signals, including the CCL3–CCR1 or IL1B–IL1R2 interactions, suggests macrophage recruitment and anti-inflammatory tuning by tumor cells. By comparing these data with reference transcriptomes for M1-type and M2-type macrophages, we found noninflammatory characteristics in macrophages recovered from the malignant ascites of GC. Using public datasets, we demonstrated that the single-cell transcriptome-driven M2-specific signature was associated with poor prognosis in GC. Our data indicate that the anti-inflammatory characteristics of TAMs are controlled by tumor cells and present implications for treatment strategies for GC patients in which combination treatment targeting cancer cells and macrophages may have a reciprocal synergistic effect.

Purpose: Immune checkpoint inhibitors (ICIs) are used for the treatment of various cancers, but clinical trials of anti-PD-1 with recurrent glioblastoma patients have failed to show clinical benefits. In this study, we examined the differentiation status of CD8⁺ tumor-infiltrating lymphocytes (TILs) from primary glioblastoma patients and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in glioblastoma. Methods: We isolated TILs from 98 patients with newly diagnosed glioblastoma and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-CTLA-4 and their proliferation assessed. Results: CD8⁺ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared to peripheral blood CD8⁺ T cells. Among CD8⁺ TILs, PD-1⁺ cells exhibited more terminally differentiated phenotypes (i.e., Eomes^hiT-bet^lo) than PD-1^- cells. These data were confirmed by analyzing NY-ESO-1₁₅₇-specific CD8⁺ TILs. Evaluating the proliferation of CD8⁺ TILs after ex vivo stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of Eomes^hiT-bet^lo cells among PD-1⁺CD8⁺ TILs. When anti-CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8⁺ TIL proliferation was observed in patients with low percentages of Eomes^hiT-bet^lo CD8⁺ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of Eomes^hiT-bet^lo CD8⁺ TILs, who did not respond to anti-PD-1. Conclusions: In primary glioblastoma, the differentiation status of CD8⁺ TILs determines their reinvigoration ability upon ICI treatment.

Purpose: Immune checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma (MM). Experimental Design: Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed MM patients. We examined the expression of immune checkpoint receptors in BM CD8⁺ T cells and their functional restoration by ex vivo treatment with anti-PD-1 and TGF-b inhibitors. Results: We confirmed the upregulation of PD-1 and PD-L1 expression in CD8⁺ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8⁺ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8⁺ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8⁺ T cells from MM patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti-PD-1 did not increase the proliferation of BM CD8⁺ T cells from MM patients, indicating that T-cell exhaustion in MM is hardly reversed by PD-1 blockade alone. Intriguingly, anti-PD-1 significantly increased the proliferation of BM CD8⁺ T cells from MM patients in the presence of inhibitors of TGF-b, which was overexpressed by myeloma cells. Conclusions: Our findings indicate that combined blockade of PD-1 and TGF-b may be useful for the treatment of MM.

Background: Patients with ALK‐rearranged non‐small cell lung cancer (ALK+ NSCLC) inevitably acquire resistance to ALK inhibitors. Longitudinal monitoring of cell‐free plasma DNA (cfDNA) next‐generation sequencing (NGS) could predict the response and resistance to tyrosine kinase inhibitor (TKI) therapy in ALK+ NSCLC.Methods: Patients with ALK+ NSCLC determined by standard tissue testing and planned to undergo TKI therapy were prospectively recruited. Plasma was collected at pretreatment, 2 months‐post therapy, and at progression for cfDNA‐NGS analysis, Guardant 360.Results: Among 92 patients enrolled, circulating tumor DNA (ctDNA) was detected in 69 baseline samples (75%): 43 ALK fusions (62.3%) and two ALK mutations without fusion (2.8%). Two patients showed ALK‐resistance mutations after ceritinib; G1202R, and co‐occurring G1202R and T1151R. Eight patients developed ALK resistance mutations after crizotinib therapy; L1196M (n = 5), G1269A (n = 1), G1202R (n = 1), and co‐occurring F1174L, G1202R, and G1269A (n = 1). Absence of ctDNA at baseline was significantly associated with longer progression‐free survival (PFS; median 36.1 vs. 11.4 months, p = 0.0049) and overall survival (OS; not reached vs. 29.3 months, p = 0.0200). ctDNA clearance at 2 months (n = 29) was associated with significantly longer PFS (25.4 vs. 11.6 months, p = 0.0012) and OS (not reached vs. 26.1 months, p = 0.0307) than those without clearance (n = 22). Patients with co‐occurring TP53 alterations and ALK fusions at baseline (n = 16) showed significantly shorter PFS (7.28 vs. 13.0 months, p = 0.0307) than those without TP53 alterations (n = 25).Conclusions: cfDNA‐NGS facilitates detection of ALK fusions and resistance mutations, assessment of prognosis, and monitoring dynamic changes of genomic alterations in ALK+ NSCLC treated with ALK‐TKI.