Sustained improved survival of patients with metastatic melanoma after the introduction of anti-PD-1-based therapies.

BACKGROUND: The introduction of modern therapies improved the median survival of patients with metastatic melanoma (MM). Here, we determined the real-world impact of modern treatments on the long-term survival of MM. METHODS: In a population-based study, we extracted all cases of MM diagnosed in four non-consecutive years marked by major changes in available 1st line treatments (2012, 2014, 2016, and 2018) from the Danish MM Database. Patients were grouped into "trial-like" and "trial-excluded" based on common trial eligibility criteria. RESULTS: We observed a sustained improved survival of "trial-like" patients diagnosed in 2016 or in 2018, compared to 2012 or 2014, but no major differences in 2018 versus 2016. In contrast, while survival of "trial-excluded" patients in 2016 was better compared to 2014 and 2012, survival in 2018 was improved over all previous years. We then developed a prognostic model based on multivariable stratified Cox regression, to predict the survival of newly diagnosed MM patients. Internal validation showed excellent discrimination and calibration, with a time-area-under-the-curve above 0.79 at multiple time horizons, for up to four years after diagnosis. CONCLUSIONS: The introduction of modern treatments such as anti-PD-1 has led to a sustained, improved survival of real-world patients with MM, regardless of their eligibility for clinical trials. We provide an updateable prognostic model that can be used to improve patient information. Overall, these data highlight a positive population-based impact of modern treatments and can help health technology assessment agencies worldwide to evaluate the appropriateness of drug pricing based on known cost-benefit data.

Intratumoral T-cell and B-cell receptor architecture associates with distinct immune tumor microenvironment features and clinical outcomes of anti-PD-1/L1 immunotherapy.

BACKGROUND: Effective cooperation between B-cells and T-cells within the tumor microenvironment may lead to the regression of established tumors. B-cells and T-cells can recognize tumor antigens with exquisite specificity via their receptor complexes. Nevertheless, whether a diverse intratumoral B-cells and T-cell receptor (BCR, TCR) repertoire affects the tumor immune microenvironment (TIME) and clinical outcomes in patients treated with immunotherapy is unclear. METHODS: We extracted information on BCR and TCR repertoire diversity from large clinical datasets and measured the association between immune receptor diversity features, the TIME, and clinical outcomes of patients treated with anti-PD-1/PD-L1 immunotherapy. RESULTS: In multiple tumor types, an increasingly diverse TCR repertoire was strongly associated with a highly activated TIME, while BCR diversity was more associated with antibody responses but not with the overall B-cell infiltration nor with measures related to intratumoral CD8+T cell activity. Neither TCR nor BCR diversity was independent prognostic biomarkers of survival across multiple cancer types. However, both TCR and BCR diversity improved the performance of predictive models combined with established biomarkers of response to immunotherapy. CONCLUSION: Overall, these data indicate a currently unexplored immunological role of intratumoral B-cells associated with BCR diversity and antibody responses but independent of classical anticancer T-cells intratumoral activities.

Seasonal variation in effect of anti-PD-1 initiation on overall survival among patients with advanced melanoma.

Melanoma is a highly immunogenic cancer, and circannual rhythms influence the activity of the immune system. We retrospectively collected information on all cases with metastatic melanoma (ocular melanoma excluded) that initiated treatment with BRAF-inhibitor-based therapy (BRAFi) or anti-PD-1 monotherapy (PD-1). Cases were divided in two groups based on treatment initiation in the summer half-year (April to September) or winter half-year (October to March). We collected a total of 1054 (BRAF-i) and 1205 (PD-1) patient cases. Median follow-up was 39.7 (BRAFi) and 47.5 (PD-1) months. We did not observe differences in outcomes across patients who were treated in summer versus winter in the BRAFi cohort. Furthermore, we did not observe significant differences in ORR, CRR, and PFS in the PD-1 cohort. However, in patients with BRAF wild-type disease of the PD-1 cohort, treatment initiation in summer was associated with an improved OS (mOS 39.7 months [summer] versus 21.3 months [winter]; HR 0.70, 95% CI 0.57-0.86, p = .0007). This result remained robust to multivariable proportional hazards adjustment (HR 0.70, 95% CI 0.57-0.87, p = .001). Initiation of immunotherapy in summer is associated with prolonged survival in patients with BRAF wild-type melanoma living in Denmark.

Dissecting tumor microenvironment heterogeneity in syngeneic mouse models: insights on cancer-associated fibroblast phenotypes shaped by infiltrating T cells.

Murine syngeneic tumor models have been used extensively for cancer research for several decades and have been instrumental in driving the discovery and development of cancer immunotherapies. These tumor models are very simplistic cancer models, but recent reports have, however, indicated that the different inoculated cancer cell lines can lead to the formation of unique tumor microenvironments (TMEs). To gain more knowledge from studies based on syngeneic tumor models, it is essential to obtain an in-depth understanding of the cellular and molecular composition of the TME in the different models. Additionally, other parameters that are important for cancer progression, such as collagen content and mechanical tissue stiffness across syngeneic tumor models have not previously been reported. Here, we compare the TME of tumors derived from six common syngeneic tumor models. Using flow cytometry and transcriptomic analyses, we show that strikingly unique TMEs are formed by the different cancer cell lines. The differences are reflected as changes in abundance and phenotype of myeloid, lymphoid, and stromal cells in the tumors. Gene expression analyses support the different cellular composition of the TMEs and indicate that distinct immunosuppressive mechanisms are employed depending on the tumor model. Cancer-associated fibroblasts (CAFs) also acquire very different phenotypes across the tumor models. These differences include differential expression of genes encoding extracellular matrix (ECM) proteins, matrix metalloproteinases (MMPs), and immunosuppressive factors. The gene expression profiles suggest that CAFs can contribute to the formation of an immunosuppressive TME, and flow cytometry analyses show increased PD-L1 expression by CAFs in the immunogenic tumor models, MC38 and CT26. Comparison with CAF subsets identified in other studies shows that CAFs are skewed towards specific subsets depending on the model. In athymic mice lacking tumor-infiltrating cytotoxic T cells, CAFs express lower levels of PD-L1 and lower levels of fibroblast activation markers. Our data underscores that CAFs can be involved in the formation of an immunosuppressive TME.

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells.

BACKGROUND: High expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor microenvironment (TME). Depletion of L-Arg has detrimental consequences for T cells and leads to T-cell dysfunction and suppression of anticancer immune responses. Previous work from our group has demonstrated the presence of proinflammatory ARG2-specific CD4 T cells that inhibited tumor growth in murine models on activation with ARG2-derived peptides. In this study, we investigated the natural occurrence of ARG2-specific CD8 T cells in both healthy donors (HDs) and patients with cancer, along with their immunomodulatory capabilities in the context of the TME. MATERIALS AND METHODS: A library of 15 major histocompatibility complex (MHC) class I-restricted ARG2-derived peptides were screened in HD peripheral blood mononuclear cells using interferon gamma (IFN-γ) ELISPOT. ARG2-specific CD8 T-cell responses were identified using intracellular cytokine staining and ARG2-specific CD8 T-cell cultures were established by enrichment and rapid expansion following in vitro peptide stimulation. The reactivity of the cultures toward ARG2-expressing cells, including cancer cell lines and activated regulatory T cells (Tregs), was assessed using IFN-γ ELISPOT and a chromium release assay. The Treg signature was validated based on proliferation suppression assays, flow cytometry and quantitative reverse transcription PCR (RT-qPCR). In addition, vaccinations with ARG2-derived epitopes were performed in the murine Pan02 tumor model, and induction of ARG2-specific T-cell responses was evaluated with IFN-γ ELISPOT. RNAseq and subsequent GO-term and ImmuCC analysis was performed on the tumor tissue. RESULTS: We describe the existence of ARG2-specific CD8+ T cells and demonstrate these CD8+ T-cell responses in both HDs and patients with cancer. ARG2-specific T cells recognize and react to an ARG2-derived peptide presented in the context of HLA-B8 and exert their cytotoxic function against cancer cells with endogenous ARG2 expression. We demonstrate that ARG2-specific T cells can specifically recognize and react to activated Tregs with high ARG2 expression. Finally, we observe tumor growth suppression and antitumorigenic immunomodulation following ARG2 vaccination in an in vivo setting. CONCLUSION: These findings highlight the ability of ARG2-specific T cells to modulate the immunosuppressive TME and suggest that ARG2-based immunomodulatory vaccines may be an interesting option for cancer immunotherapy.

Coexisting Alterations of MHC Class I Antigen Presentation and IFNγ Signaling Mediate Acquired Resistance of Melanoma to Post-PD-1 Immunotherapy.

Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti-programmed death 1 (anti-PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti-PD-1 plus anti-lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti-PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.

Clinical value of routine [18F]2-fluoro-2-deoxy-d-glucose positron emission tomography scans as a decision tool for early immunotherapy discontinuation in advanced melanoma.

Routine [18F]2-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) may help predict clinical outcomes after response to immunotherapy. With a European Medicines Agency-recommended treatment length until disease progression or unacceptable toxicity, the optimal duration of immunotherapy remains to be defined. In a retrospective study, we retrieved from the Danish Metastatic Melanoma Database (DAMMED), all patients that were annotated as a partial or complete response based on the computed tomography (CT) of serial FDG-PET-CT scans. Patients treated with an anti-Programmed Death (PD)-1-containing regimen for <18 months, and ≥4 months without disease progression after halting anti-PD-1 were included. Cases were divided into an "elective" and a "toxicity" group based on the reason for treatment discontinuation. A total of 140 patients were included. At 29.3 months of median follow-up, a higher proportion of patients remained alive in the "elective" group (93% vs 75%, P = .0031) with an improved melanoma-specific (HR 0.07, 95% CI 0.02-0.32, P = .0041) survival (MSS). Patients without FDG-avid lesions at the time of treatment discontinuation had an improved MSS (HR 0.03, 95% CI 0.01-0.17, P = .0002), and the absence of FDG-avid lesions was the only independent predictive feature of improved MSS in multivariate analysis. In conclusion, patients with metastatic melanoma who obtain an early response and early discontinue immunotherapy have an excellent prognosis, especially in the absence of FDG-PET avid lesions when discontinuing treatment. These data support the option of early discontinuation, limiting possible overtreatment and thereby toxicity, health and economic expenses and improving logistics.

Transcriptomic signatures of tumors undergoing T cell attack.

BACKGROUND: Studying tumor cell-T cell interactions in the tumor microenvironment (TME) can elucidate tumor immune escape mechanisms and help predict responses to cancer immunotherapy. METHODS: We selected 14 pairs of highly tumor-reactive tumor-infiltrating lymphocytes (TILs) and autologous short-term cultured cell lines, covering four distinct tumor types, and co-cultured TILs and tumors at sub-lethal ratios in vitro to mimic the interactions occurring in the TME. We extracted gene signatures associated with a tumor-directed T cell attack based on transcriptomic data of tumor cells. RESULTS: An autologous T cell attack induced pronounced transcriptomic changes in the attacked tumor cells, partially independent of IFN-γ signaling. Transcriptomic changes were mostly independent of the tumor histological type and allowed identifying common gene expression changes, including a shared gene set of 55 transcripts influenced by T cell recognition (Tumors undergoing T cell attack, or TuTack, focused gene set). TuTack scores, calculated from tumor biopsies, predicted the clinical outcome after anti-PD-1/anti-PD-L1 therapy in multiple tumor histologies. Notably, the TuTack scores did not correlate to the tumor mutational burden, indicating that these two biomarkers measure distinct biological phenomena. CONCLUSIONS: The TuTack scores measure the effects on tumor cells of an anti-tumor immune response and represent a comprehensive method to identify immunologically responsive tumors. Our findings suggest that TuTack may allow patient selection in immunotherapy clinical trials and warrant its application in multimodal biomarker strategies.

TGFß-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The tumor microenvironment (TME) in PDAC is highly immunosuppressive and desmoplastic, which could explain the limited therapeutic effect of immunotherapy in PDAC. One of the key molecules that contributes to immunosuppression and fibrosis is transforming growth factor-ß (TGFß). The aim of this study was to target the immunosuppressive and fibrotic TME in PDAC using a novel immune modulatory vaccine with TGFß-derived peptides in a murine model of pancreatic cancer. METHODS: C57BL/6 mice were subcutaneously inoculated with Pan02 PDAC cells. Mice were treated with TGFß1-derived peptides (major histocompatibility complex (MHC)-I and MHC-II-restricted) adjuvanted with Montanide ISA 51VG. The presence of treatment-induced TGFß-specific T cells was assessed by ELISpot (enzyme-linked immunospot). Changes in the immune infiltration and gene expression profile in tumor samples were characterized by flow cytometry, reverse transcription-quantitative PCR (RT-qPCR), and bulk RNA sequencing. RESULTS: Treatment with immunogenic TGFß-derived peptides was safe and controlled tumor growth in Pan02 tumor-bearing mice. Enlargement of tumor-draining lymph nodes in vaccinated mice positively correlated to the control of tumor growth. Analysis of immune infiltration and gene expression in Pan02 tumors revealed that TGFß-derived peptide vaccine increased the infiltration of CD8+ T cells and the intratumoral M1/M2 macrophage ratio, it increased the expression of genes involved in immune activation and immune response to tumors, and it reduced the expression of myofibroblast-like cancer-associated fibroblast (CAF)-related genes and genes encoding fibroblast-derived collagens. Finally, we confirmed that TGFß-derived peptide vaccine actively modulated the TME, as the ability of T cells to proliferate was restored when exposed to tumor-conditioned media from vaccinated mice compared with media from untreated mice. CONCLUSION: This study demonstrates the antitumor activity of TGFß-derived multipeptide vaccination in a murine tumor model of PDAC. The data suggest that the vaccine targets immunosuppression and fibrosis in the TME by polarizing the cellular composition towards a more pro-inflammatory phenotype. Our findings support the feasibility and potential of TGFß-derived peptide vaccination as a novel immunotherapeutic approach to target immunosuppression in the TME.

Qualitative Analysis of Tumor-Infiltrating Lymphocytes across Human Tumor Types Reveals a Higher Proportion of Bystander CD8+ T Cells in Non-Melanoma Cancers Compared to Melanoma.

Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8+ and CD4+ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8+ and CD4+ TIL responses were detected in over half of the patients in vitro, and greater CD8+ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4+ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8+ and CD4+ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation.