Immune Checkpoint Blockade In Patients Research Articles

Abstract RF01-06: Efficacy and safety of toripalimab plus metronomic chemotherapy in HER2 negative metastatic breast cancer: a multicenter phase II trial based on a Bayesian adaptive randomized design

Abstract Background: Combining Immune checkpoint blockade (ICB) with chemotherapy may significantly improve efficacy in patients with breast cancer. Metronomic chemotherapy is based on more frequent and low-dose drug administrations compared with conventional chemotherapy. Some clinical studies with small samples have tried the combination of metronomic chemotherapy and ICB in patients with advanced tumors. However, whether metronomic chemotherapy is more suitable for ICB than conventional chemotherapy is still unclear. Thus, we performed the first clinical trial, to our knowledge, of metronomic chemotherapy combined with PD-1 blockade comparing with the efficacy of combined conventional chemotherapy and ICB. Methods: This study is a multicenter randomized phase 2 trial using a multi-arm design with Bayesian adaptive randomization and efficacy monitoring. Eligible patients have advanced HER2 negative breast cancer, with no more than one prior line of standard chemotherapy. Patients were randomized to 5 groups: ① metronomic vinorelbine (NVB, 40 mg/day, TIW) monotherapy (the control cohort); ② NVB + Toripalimab (anti-PD1 antibody, 240 mg Q3W); ③Bevacizumab (5 mg/kg Q3W) + NVB+ Toripalimab (the BEV cohort); ④ Cisplatin (50mg/m Q3W) + NVB + Toripalimab (the DDP cohort), ⑤Cyclophosphamide (50mg/day, QD) + Capecitabine (500 mg, TID) + NVB+ Toripalimab (the VEX cohort). The primary endpoint is disease control rate (DCR), and secondary endpoints are objective response rate (ORR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Mass cytometry time-of-flight (CyTOF) analyses of paired blood samples were performed to demonstrate dynamic changes in systemic immune profile. Results: A total of 103 patients were randomized. Characteristics were as expected for advanced breast cancer and balanced between cohorts. Adverse events of any grade occurred in 64 patients, with 5.2% grade >3. The rate of nausea was significantly higher in the cisplatin cohort than in the others (P< 0.001). Toripalimab was not associated with any previously unreported toxicity. Among the five treatment cohorts, the VEX cohort and the cisplatin cohort had the highest DCR, 69.7% (95% CI 51.7–85.9%) and 73.7% (95% CI 56.1–88.7%), respectively. Followed by the bevacizumab cohort, DCR is 55.7% (95% CI 37.4%–73.6%). Most objective responses were observed in the VEX cohort (ORR: 18.5%; 95% CI 5.6–34.1%), followed by cisplatin (ORR 14.5%; 95% CI 3.3–29.0%). It is worth noting that the PFS of patients in the VEX cohort was the longest, reaching 6.6 months (95% CI 4.0-5.9), followed by 4.0 months (95% CI 2.4-10.4) in patients of the bevacizumab cohort. The PFS of patients receiving the cisplatin regimen was relatively short, only 3.5 months (95% CI 2.2-5.3). Similarly, patients in the cisplatin cohort had shorter OS than those in the VEX and bevacizumab cohorts. In the TNBC subgroup, again, patients in the VEX cohort had the highest DCR (74.1%, 95%CI 47.9%-95.4%) and longest PFS (9.8 months, 95%CI 3.8-21.9). We obtained over 63 million cells in total and clustered CD45+ immune cells into 32 clusters. The systemic immune profile of patients changed dramatically over 2 cycles of PD-1 blockade. However, only the change of cluster 30 differed between responders and non-responders. This is a group of intermediate monocytes with a high expression of CD38, which decreased in responders but significantly increased in non-responders. CD38-NAD+ axis regulates the PD1+ exhausted T cell function. Meanwhile, the overall expression of CD38 in monocytes was significantly increased by DDP and BEV treatment compared with baseline, but not in the VEX groups. DDP significantly increased the expression level of CD38 on NK cells, while BEV or VEX treatment did not affect it. Conclusions: These data suggest promising clinical efficacy and evidence of cooperativity between metronomic VEX chemotherapy and PD-1 blockade. Citation Format: Hongnan Mo, Xiaoying Sun, Jingtong Zhai, Jiashu Han, Hewei Ge, Yuhan Wei, Xiuwen Guan, Haili Qian, Fei Ma. Efficacy and safety of toripalimab plus metronomic chemotherapy in HER2 negative metastatic breast cancer: a multicenter phase II trial based on a Bayesian adaptive randomized design [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-06.

High-Grade Pleomorphic Sarcomas Treated with Immune Checkpoint Blockade: The MD Anderson Cancer Center Experience.

Undifferentiated pleomorphic sarcomas (UPSs) are amongst the most common subtypes of soft-tissue sarcomas. Few real-world data on the use of immune checkpoint blockade (ICB) in UPS patients and other high-grade pleomorphic STS patients are available. The purpose of our study is to describe the efficacy and toxicity of ICB in patients with advanced UPSs and other high-grade pleomorphic sarcomas treated at our institution. This is a retrospective, observational study of all patients with metastatic high-grade pleomorphic sarcomas treated with FDA-approved ICB at MD Anderson Cancer Center between 1 January 2015 and 1 January 2023. Patients included in trials for which results are not yet published were excluded. Thirty-six patients with advanced/metastatic pleomorphic sarcomas were included. The median age was 52 years. A total of 26 patients (72%) had UPSs and 10 patients (28%) had other high-grade pleomorphic sarcomas. The median follow-up time was 8.8 months. The median PFS was 2.9 months. The 3-month PFS and 6-month PFS were 46% and 32%, respectively. The median OS was 12.9 months. The 12-month OS and 24-month OS were 53% and 29%, respectively. The best response, previous RT, and type of ICB treatment were significantly and independently associated with shorter PFS (p = 0.0012, p = 0.0019 and p = 0.036, respectively). No new safety signal was identified, and the toxicity was overall manageable with no toxic deaths and only four patients (11%) stopping treatment due to toxicity. Real-world retrospective data are consistent with the published literature, with a promising 6-month PFS of 32%. Partial or stable responders to ICB treatment have significantly improved PFS compared to progressors.

CRISPR-Cas9 screening identifies KRAS-induced COX-2 as a driver of immunotherapy resistance in lung cancer.

Oncogenic KRAS impairs anti-tumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of how oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive cyclooxygenase-2 (COX-2) in cancer cells. Oncogenic KRAS potently induced COX-2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX-2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX-2/PGE2 remodeled the tumor microenvironment by inducing pro-inflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX-2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX-2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer.

Deubiquitinase USP4 suppresses antitumor immunity by inhibiting IRF3 activation and tumor cell-intrinsic interferon response in colorectal cancer

Despite the approval of immune checkpoint blockade (ICB) therapy for various tumor types, its effectiveness is limited to only approximately 15% of patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC). Approximately 80%–85% of CRC patients have a microsatellite stability (MSS) phenotype, which features a rare T-cell infiltration. Thus, elucidating the mechanisms underlying resistance to ICB in patients with MSS CRC is imperative. In this study, we demonstrate that ubiquitin-specific peptidase 4 (USP4) is upregulated in MSS CRC tumors and negatively regulates the immune response against tumors in CRC. Additionally, USP4 represses the cellular interferon (IFN) response and antigen presentation and impairs PRR signaling-mediated cell death. Mechanistically, USP4 impedes the nuclear localization of interferon regulator Factor 3 (IRF3) by deubiquitinating the K63-polyubiquitin chain of TRAF6 and IRF3. Knockdown of USP4 enhances the infiltration of T cells in CRC tumors and overcomes ICB resistance in an MC38 syngeneic mouse model. Moreover, published datasets revealed that patients showing higher USP4 expression exhibited decreased responsiveness to anti-PD-L1 therapy. These findings highlight an essential role of USP4 in the suppression of antitumor immunity in CRC.

Abstract 6200: Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer

Abstract Background: Gastric cancer remains the leading cause of cancer-related deaths worldwide, ranking fifth for incidence and fourth for mortality. Combining neoadjuvant therapy with immune checkpoint inhibitors has demonstrated improved outcomes for patients with gastric cancer. However, responses exhibit heterogeneity, and the understanding of the mediators influencing therapy response is limited. Methods: In this study, we collected 80 blood and tumor samples from 22 gastric cancer patients treated with XELOX/SOX alone or its combination with Nivolumab (anti-PD-1). Paired single-cell RNA-, TCR-, and BCR-sequencing were performed on pre- and post-treatment samples to investigate the dynamics of immune and tumor cells during the development of response/resistance to therapy. Results: Our findings revealed that elevated levels of baseline CD4 and CD8+ tissue-resident memory T (TRM) cells, in activated and stressed states, are associated with a superior response to combined therapy, and treatment further amplifies this cell population in responsive patients. Tracking T cell receptor clones across pre-/post-treated blood and tumor samples disclosed that the expanded clonotypes of TRM cells are exclusively detected in tumors, and considerable preexisting clones contribute to the expansion of these T cell populations following treatment. The simultaneous presence of TRM, B, and dendritic cells in pre-treated tumors predicted a favorable response, and they concertedly expanded in tumors responsive to anti-PD-1 therapy. Conclusions: Our findings highlight the significance of TRM cells in effective responses to anti-PD-1 therapies and indicate that their cellular communication with B and dendritic cells play a pivotal role in shaping the antitumor immunity elicited by combination therapy. Together, this study illuminates vital immune cell subsets during immune checkpoint blockade treatment, provides biological insights into the mechanism of successful combination therapy, and unravels potential targets for improving treatment efficacy in gastric cancer. Citation Format: Enyu Dai, Jiang-Jiang Qin, Can Hu, Zhiyuan Xu, Guangchun Han, Yanshuo Chu, Ruiping Wang, Minghao Dang, Yunhe Liu, Guangsheng Pei, Jiahui Jiang, Fuduan Peng, Yuanyuan Zhang, Andre Luis Faustino, Jaffer A. Ajani, Linghua Wang, Xiangdong Cheng. Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6200.

Tumor immune microenvironment in odontogenic carcinomas: Evaluation of the therapeutic potential of immune checkpoint blockade.

Despite recent advances in the use of immune checkpoint blockade (ICB) across various cancer types, its efficacy in odontogenic carcinomas remains unexplored. This study aims to investigate PD-L1 expression and the tumor immune microenvironment (TIME) in odontogenic carcinomas to determine the therapeutic potential of ICB and the significance of immune markers. The expressions of PD-L1 and T cell markers (CD3, CD8, and FOXP3) were visualized by immunohistochemistry in 21 tissue samples of odontogenic carcinomas. Tumoral PD-L1 expression and the density and spatial distribution of T cell subsets were evaluated, from which TIME was determined. The associations of the variables with clinicopathological and prognostic factors were statistically analyzed. PD-L1 was positively expressed in 52.4% (11/21) of the cases studied. Among tumor types, ameloblastic carcinoma showed significantly higher PD-L1 expression (p = 0.016). TIME based on the intratumoral and stromal T cell distribution was immune-inflamed in 61.9% (13/21) and immune-excluded in 38.1% (8/21), with no immune-desert cases. PD-L1 expression was associated with the densities of all intratumoral T cell subsets (p = 0.03 for CD3, p = 0.03 for CD8, and p = 0.008 for FOXP3) but not with those of stromal T cells. High PD-L1 expression was associated with larger tumor size (p = 0.021), while the intratumoral CD8/CD3 ratio was inversely correlated with tumor size (p = 0.048). These findings indicate the involvement of adaptive immune resistance in a subset of odontogenic carcinomas and support the therapeutic potential of ICB in patients with these rare malignancies.

Abstract B026: Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement

Abstract Background: Mismatch repair deficient (MMRd) endometrial cancer (EC) represents one third of all ECs. Identifying patients with MMRd EC enables testing for Lynch Syndrome (LS) and access to FDA-approved immune checkpoint blockade (ICB) therapy. Recent data have highlighted the diversity within MMRd tumors, suggesting worse outcomes and lower response to ICB in patients with MLH1 loss. Our aim was to characterize a cohort of MMRd ECs to elucidate if clinically meaningful substratification of this molecular subtype (MLH1 loss vs. other MMRd) could be achieved. Methods: MMRd ECs were identified from retrospective institutional and population-based cohorts (1994-2016) with clinicopathologic data, immunohistochemistry (IHC) assessment of estrogen receptor (ER) and L1CAM and CTNNB1 mutation status recorded. Multiplex IHC for immune markers (CD3, CD8, CD79a, CD138, PD-1, PD-L1, FoxP3, IDO-1) was assessed and compared in patients with MLH1 loss or PMS2/MLH1 loss (when 2 antibody MMRd testing performed) vs. isolated MSH2, MSH6, PMS2 or MSH2/MSH6 loss (remainder of MMRd). Results: 655 MMRd ECs were identified, 52% of cases assessed with 4 antibody IHC (MLH1, PMS2, MSH2, MSH6) and 48% with 2 (PMS2 and MSH6). This included 32 (5%) patients with MLH1 loss and 488 (75%) with PMS2/MLH1 loss (together 80% of MMRd cohort), 1% (n= 9) MSH2, 11% (n=75) MSH6, 4% (n=24) PMS2, and 4% (n=24) with loss of MSH2/MSH6). 76 cases were confirmed to have MLH1 hypermethylation but 67% of cases with loss of MLH1 or PMS2/MLH1 did not have methylation testing performed. The majority MMRd ECs were FIGO stage I (75%) and endometrioid histotype (90%). Patients with loss of MLH1 or PMS2/MLH1 were older (p<0.001), had higher BMI (p<0.001) and had tumors with more LVI (p=0.031), deep myoinvasion (p<0.001) and grade 3 (p=0.046) compared to remainder of MMRd EC. 9% of the MMRd cohort were ER negative, 8% with loss of MLH1 or PMS2/MLH1 and 16% in remainder of MMRd. Inferior outcomes were observed for progression-free survival and overall survival in patients with MLH1 or PMS2/MLH1 loss compared with the remainder of MMRd. ER, L1CAM and CTNNB1 status were not associated with clinical outcomes across the total MMRd cohort, nor within MLH1 or PMS2/MLH1 loss. There was diversity in the immune landscape within MMRd EC with significantly lower CD8 levels in MLH1 or PMS2/MLH1 loss compared to the remainder of MMRd EC. Furthermore, 25% of the MLH1 or PMS2/MLH1 loss group were tumor infiltrating lymphocyte (TIL) ‘low’/immune cold by cluster analysis compared to 11% in the remainder of MMRd EC. Only 16 patients (2.4%) were identified as having LS but 82% of these MMRd patients had not been tested (31% non-LS testing attributable to identification of hypermethylation of MLH1). Conclusion: Substratification within MMRd ECs can provide prognostic and predictive information, with loss of MLH1 and/or its dimer partner PMS2 identifying a subset of MMRd EC with inferior outcomes that may be attributed to lower TIL. ER, L1CAM, and CTNNB1 mutation status do not add prognostic refinement within MMRd EC. Citation Format: Amy Jamieson, Jennifer Pors, Samuel Leung, Derek Chiu, Stefan Kommoss, Aline Talhouk, David G. Huntsman, Naveena Singh, Blake Gilks, Jessica N. McAlpine. Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B026.

Moving toward improved immune checkpoint immunotherapy for advanced prostate cancer.

Human prostate cancer is a heterogenous malignancy that responds poorly to immunotherapy targeting immune checkpoints. The immunosuppressive tumor microenvironment that is typical of human prostate cancer has been the main obstacle to these treatments. The effectiveness of these therapies is also hindered by acquired resistance, leading to slow progress in prostate cancer immunotherapy. Results from the highly anticipated late-stage clinical trials of PD-1/PD-L1 immune checkpoint blockade in patients with advanced prostate cancer have highlighted some of the obstacles to immunotherapy. Despite the setbacks, there is much that has been learned about the mechanisms that drive resistance, and new strategies are being developed and tested. Here, we review the status of immune checkpoint blockade and the immunosuppressive tumor microenvironment and discuss factors contributing to innate and adaptive resistance to immune checkpoint blockade within the context of prostate cancer. We then examine current strategies aiming to overcome these challenges as well as prospects.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma

ABSTRACT PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

Combined PD-L1/TGFβ blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Liver-directed treatment is associated with improved survival and increased response to immune checkpoint blockade in metastatic uveal melanoma: results from a retrospective multicenter trial

Metastases of uveal melanoma (UM) spread predominantly to the liver. Due to low response rates to systemic therapies, liver-directed therapies (LDT) are commonly used for tumor control. The impact of LDT on the response to systemic treatment is unknown. A total of 182 patients with metastatic UM treated with immune checkpoint blockade (ICB) were included in this analysis. Patients were recruited from prospective skin cancer centers and the German national skin cancer registry (ADOReg) of the German Dermatologic Cooperative Oncology Group (DeCOG). Two cohorts were compared: patients with LDT (cohort A, n = 78) versus those without LDT (cohort B, n = 104). Data were analyzed for response to treatment, progression-free survival (PFS), and overall survival (OS). The median OS was significantly longer in cohort A than in cohort B (20.1 vs. 13.8 months; P = 0.0016) and a trend towards improved PFS was observed for cohort A (3.0 vs. 2.5 months; P = 0.054). The objective response rate to any ICB (16.7% vs. 3.8%, P = 0.0073) and combined ICB (14.1% vs. 4.5%, P = 0.017) was more favorable in cohort A. Our data suggest that the combination of LDT with ICB may be associated with a survival benefit and higher treatment response to ICB in patients with metastatic UM.

Safety and efficacy of immune checkpoint blockade in patients with advanced nonsmall cell lung cancer and brain metastasis.

The presence of brain metastases (BM) is a negative prognostic factor for patients with advanced nonsmall cell lung cancer (NSCLC). Their incidence seems to be higher in patients with oncogene-driven tumours, especially those with EGFR-mutated or ALK-rearranged tumours. Although targeted treatments demonstrate significant efficacy regarding BM, they only apply to a minority of NSCLC patients. On the other hand, systemic therapies for nononcogenic-driven NSCLC with BM have shown limited clinical benefit. In recent years, immunotherapy alone or combined with chemotherapy has been adopted as a new standard of care in first-line therapy. This approach seems to be beneficial to patients with BM in terms of efficacy and toxicity. Combined immune checkpoint inhibition as well as the combination of immunotherapy and radiation therapy show promising results with significant, but overall acceptable toxicity. A pragmatic approach of allowing enrolment of patients with untreated or symptomatic BM in randomised trials evaluating immune checkpoint inhibitors strategies, possibly coupled with central nervous system-related endpoints may be needed to generate data to refine treatment for this patient population.

Prediction of response to immune checkpoint blockade in patients with metastatic colorectal cancer with microsatellite instability

Mismatch repair-deficient (dMMR) tumors displaying microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). However, a proportion of patients with dMMR/MSI mCRC exhibit resistance to ICI. Identification of tools predicting MSI mCRC patient response to ICI is required for the design of future strategies further improving this therapy. We combined high-throughput DNA and RNA sequencing of tumors from 116 patients with MSI mCRC treated with anti-programmed cell death protein 1 ± anti-cytotoxic T-lymphocyte-associated protein 4 of the NIPICOL phase II trial (C1, NCT03350126, discovery set) and the ImmunoMSI prospective cohort (C2, validation set). The DNA/RNA predictors whose status was significantly associated with ICI status of response in C1 were subsequently validated in C2. Primary endpoint was progression-free survival by immune RECIST (iRECIST) (iPFS). Analyses showed no impact of previously suggested DNA/RNA indicators of resistance to ICI, e.g. MSIsensor score, tumor mutational burden, or specific cellular and molecular tumoral contingents. By contrast, iPFS under ICI was shown in C1 and C2 to depend both on a multiplex MSI signature involving the mutations of 19 microsatellites hazard ratio cohort C2 (HRC2) = 3.63; 95% confidence interval (CI) 1.65-7.99; P= 1.4× 10-3] and the expression of a set of 182 RNA markers with a non-epithelial transforming growth factor beta (TGFB)-related desmoplastic orientation (HRC2= 1.75; 95% CI 1.03-2.98; P= 0.035). Both DNA and RNA signatures were independently predictive of iPFS. iPFS in patients with MSI mCRC can be predicted by simply analyzing the mutational status of DNA microsatellite-containing genes in epithelial tumor cells together with non-epithelial TGFB-related desmoplastic RNA markers.

Type 2 Diabetes Mellitus and Efficacy Outcomes from Immune Checkpoint Blockade in Patients with Cancer.

No evidence exists as to whether type 2 diabetes mellitus (T2DM) impairs clinical outcome from immune checkpoint inhibitors (ICI) in patients with solid tumors. In a large cohort of ICI recipients treated at 21 institutions from June 2014 to June 2020, we studied whether patients on glucose-lowering medications (GLM) for T2DM had shorter overall survival (OS) and progression-free survival (PFS). We used targeted transcriptomics in a subset of patients to explore differences in the tumor microenvironment (TME) of patients with or without diabetes. A total of 1,395 patients were included. Primary tumors included non-small cell lung cancer (NSCLC; 54.7%), melanoma (24.7%), renal cell (15.0%), and other carcinomas (5.6%). After multivariable analysis, patients on GLM (n = 226, 16.2%) displayed an increased risk of death [HR, 1.29; 95% confidence interval (CI),1.07-1.56] and disease progression/death (HR, 1.21; 95% CI, 1.03-1.43) independent of number of GLM received. We matched 92 metformin-exposed patients with 363 controls and 78 patients on other oral GLM or insulin with 299 control patients. Exposure to metformin, but not other GLM, was associated with an increased risk of death (HR, 1.53; 95% CI, 1.16-2.03) and disease progression/death (HR, 1.34; 95% CI, 1.04-1.72). Patients with T2DM with higher pretreatment glycemia had higher neutrophil-to-lymphocyte ratio (P = 0.04), while exploratory tumoral transcriptomic profiling in a subset of patients (n = 22) revealed differential regulation of innate and adaptive immune pathways in patients with T2DM. In this study, patients on GLM experienced worse outcomes from immunotherapy, independent of baseline features. Prospective studies are warranted to clarify the relative impact of metformin over a preexisting diagnosis of T2DM in influencing poorer outcomes in this population.

Single-cell RNA and T cell receptor (TCR) sequencing in patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) after induction CTLA4 and PD-1 immune checkpoint blockade (ICB).

6056 Background: Combination CTLA-4 and PD-1 ICB results in durable responses in patients with recurrent and metastatic HNSCC and therefore justifies further investigation in the induction or neoadjuvant setting. Methods: We investigated features of the tumor immune microenvironment (TIME) associated with MHR after induction CTLA-4 and PD-1 ICB in patients with newly diagnosed HPV-positive HNSCC. Tumor samples were collected at baseline and on treatment from 31 patients enrolled in a phase 2 trial of a 6-week cycle of CTLA-4 and PD-1 ICB induction followed by dose/volume-adapted IMRT (50-66Gy) concurrent with cycle 2. Tumors were evaluated for histological response and single-cell RNA sequencing (scRNA-seq) and scTCR-seq were performed on the 10X genomics platform. After stringent clustering and annotation of scRNA-seq data, 76,319 CD8+ and 78,622 CD4+ T cells with paired TCR sequence data were evaluable for transcriptomic, TCR repertoire and clonotype differentiation analysis. Results: Percent reduction in tumor viability on histology correlated significantly with reduction in tumor cell proportion in scRNA-seq data (R2= 0.7). Thirteen (48%) of 27 patients with paired samples (missing data due to COVID) had MHR, defined as ≤10% residual tumor viability on ICB treatment. Through TCR tracing, over 100 T cell clonotypes that expanded significantly (p < 0.05, Fisher’s exact) post-ICB were identified. Highly expanded clonotypes resided mainly in exhausted CD8+ T cell clusters and exhibited a tissue resident memory (TRM) phenotype. Examination of their functional states revealed a variety of T cell modulatory trajectories, including reverse transition from exhausted to less exhausted states and from memory to functional states. When compared to patients without MHR, those with MHR had significantly higher ICB-induced TCR clonotype expansion (p = 0.025, Wilcoxon), reductions of activated Tregs (p = 0.003, Wilcoxon) and terminally exhausted CD8+ T cells (p = 0.003, Wilcoxon), and increase of polyfunctional CD8+ T cells (p = 0.014, Wilcoxon). A lasso regression model associated T cell expansion, basal 7-gene TRM and 5-gene effector scores, and on-treatment proliferation score and effector cell proportion with percent reduction in tumor viability (R2 = 0.66). Conclusions: In HPV-HNSCC, induction CTLA4 and PD1 ICB lead to extensive reinvigoration of CD8+ T cell clonotypes with both an exhausted and TRM phenotype, revealing a high degree of plasticity of distinct T cell clones. Paired RNA and TCR profiling facilitated identification of potentially tumor reactive and ICB-responsive T cells and clonotypes and identified several TIME features associated with tumor cell death. Clinical trial information: NCT03799445 .

Abstract ND12: TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers

Abstract STK11 loss of function mutations occur in 12-15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade in patients and preclinical models. STK11-deficient syngeneic mouse tumor models reflect this biology and are insensitive to anti-PD1 treatment. Informed by in vivo CRISPR-based screens, histone deacetylase 1 (HDAC1) was identified as a target gene, which when knocked out in tumor cells, reverses anti-PD1 resistance driven by STK11 tumor suppressor gene loss. Here, we describe the discovery and development of TNG260 as a potent and selective inhibitor of the HDAC1-containing complex CoREST. TNG260 inhibits CoREST deacetylase activity with 500-fold selectivity over the other HDAC1-containing complexes, NuRD and Sin3, and the HDAC3-containing complex NCoR. In preclinical studies, selective CoREST inhibition by TNG260 results in transcriptional reprogramming of STK11-mutant tumor cells, altering tumor cell cytokine secretion and markedly reducing recruitment of suppressive Treg cells to STK11-mutant tumors. Moreover, TNG260, in combination with anti-PD1 treatment, drives durable tumor regressions in multiple syngeneic STK11-mutant xenograft models. TNG260 has no anti-tumor efficacy in athymic mice, indicating the responses with TNG260 are mediated by T cells. Unlike previously developed pan-HDAC inhibitors, which are directly cytotoxic to cancer (and immune) cells, IND-enabling toxicology studies in rat and dog showed that TNG260 was well-tolerated at exposures predicted to be efficacious in humans, with bone marrow suppression only detectable at doses where TNG260 is no longer selective for CoREST inhibition. TNG260 clinical development will be among the first to combine the power of genetic patient selection and immunotherapy, evaluating patients with STK11 mutant cancers in a trial combining TNG260 and a checkpoint inhibitor. Citation Format: Leanne G. Ahronian, Minjie Zhang, Chengyin Min, Alice W. Tsai, Jacques Ermolieff, Patrick McCarren, Margaret Wyman, David Guerin, Ye Wang, Alborz Bejnood, Kenjie Amemiya, Brian McMillan, Nikitha Das, Preksha Shahagadkar, Brian Doyon, Andre Mignault, Colin Liang, Vassil Elitzin, Samuel R. Meier, Ashley Choi, Yi Yu, John P. Maxwell, Brian B. Haines, Jannik N. Andersen, Heather DiBenedetto, Aaron Weitzman, Alan Huang, Xinyuan Wu. TNG260: A novel, orally active, CoREST-selective deacetylase inhibitor for the treatment of STK11-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND12.

Subsets of IFN Signaling Predict Response to Immune Checkpoint Blockade in Patients with Melanoma.

IFN signaling in the tumor microenvironment is a critical determinant of both response and resistance of cancer to immune checkpoint inhibitors (ICI). We hypothesized that distinct patterns of IFN signaling in melanoma are associated with clinical response or resistance to ICIs. Two tissue microarrays containing samples from 97 patients with metastatic melanoma who received nivolumab, pembrolizumab, or a combination of ipilimumab and nivolumab at Yale New Haven Hospital between 2011 and 2017 were randomized into discovery and validation cohorts. Samples were stained and visualized using multiplexed immunofluorescence microscopy for STAT1, STAT1 phosphorylated at Y701 (pSTAT1Y701), and PD-L1, and signals were quantified using the automated quantitative analysis method of quantitative immunofluorescence. Treatment response was assessed using RECIST, and overall survival was analyzed. For in vitro studies, human melanoma cell lines were stimulated with IFNγ and IFNβ, and Western blotting was performed. Pretreatment STAT1 levels were higher in responders to ICIs [complete response/partial response/stable disease (SD) for > 6 months] than in nonresponders (SD < 6 months/progressive disease). Higher pretreatment STAT1 levels were associated with improved survival after ICIs in both the discovery and validation cohorts. Western blot analysis of human melanoma cell lines stimulated with IFN demonstrated distinct patterns of upregulation of STAT1 compared with pSTAT1Y701 and PD-L1. When combining STAT1 and PD-L1 markers, patients with STAT1highPD-L1low tumors had improved survival compared with those with STAT1lowPD-L1high tumors. STAT1 may better predict melanoma response to ICIs than current strategies, and combined STAT1 and PD-L1 biomarkers may provide insight into IFN-responsive versus IFN-resistant states.

Impact of endogenous glucocorticoid on response to immune checkpoint blockade in patients with advanced cancer.

Previous studies indicate that exogenous use of glucocorticoid (GC) affects immune checkpoint inhibitor (ICI) efficacy. However, there is a paucity of clinical data evaluating the direct impact of endogenous GC on the efficacy for cancer patients with immune checkpoint blockade. We first compared the endogenous circulating GC levels in healthy individuals and patients with cancer. We next retrospectively reviewed patients with advanced cancer with PD-1/PD-L1 inhibitor alone or combination therapy in a single center. The effects of baseline circulating GC levels on objective response rate (ORR), durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS) were analyzed. The association of the endogenous GC levels with circulating lymphocytes, cytokines levels, and neutrophil to lymphocyte ratio, and tumor infiltrating immune cells, were systematically analyzed. The endogenous GC levels in advanced cancer patients were higher than those in early-stage cancer patients as well as healthy people. In the advanced cancer cohort with immune checkpoint blockade (n=130), patients with high baseline endogenous GC levels (n=80) had a significantly reduced ORR (10.0% vs 40.0%; p<0.0001) and DCB (35.0% vs 73.5%, p=0.001) compared to those with low endogenous GC levels (n=50). The increased GC levels was significantly associated with reduced PFS (HR 2.023; p=0.0008) and OS (HR 2.809; p=0.0005). Moreover, statistically significant differences regarding PFS, and OS were also detected after propensity score matching. In a multivariable model, the endogenous GC was identified as an independent indicator for predicting PFS (HR 1.779; p=0.012) and OS (HR 2.468; p=0.013). High endogenous GC levels were significantly associated with reduced lymphocytes (p=0.019), increased neutrophil to lymphocyte ratio (p=0.0009), and increased interleukin-6 levels (p=0.025). Patients with high levels of endogenous GC had low numbers of tumor infiltrating CD3+ (p=0.001), CD8+ T (p=0.059), and CD4+ T (p=0.002) cells, and the numbers of circulating PD-1+ NK cells (p=0.012), and the ratio of CD8+PD-1+ to CD4+PD-1+ (p=0.031) were higher in patients with high levels of endogenous GC compared to low levels of endogenous GC. Baseline endogenous GC increase executes a comprehensive negative effect on immunosurveillance and response to immunotherapy in real-world cancer patients accompanied with cancer progression.

Abstract 1100: 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor

Abstract Background. Ionizing radiation (IR) is a double-edge sword for immunotherapy as it may cause both immunosuppressive and immunostimulatory effects. The interactions of IR with the tumor microenvironment (TME) is a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of CAFs in many cancer types and its presence is associated with poor immune response to immune checkpoint blockade in patients. We hypothesize that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic FAP-41BBL bispecific antibody-like fusion protein could enhance the immune-mediated antitumoral effect of these treatments given in combination. Methods: Murine transplantable tumor cells lines (TSA and MC38) were used to investigate increases in FAP expression on CAFs in tumors under irradiation using IHQ and qPCR. Established orthotopic transplanted models were used. We treated bilateral tumor-bearing mice in which only one of the lesions was locally irradiated (2×6Gy) given alone or in combination with a systemic administration of the FAP-41BBL agonistic bispecific construct. Tumor sizes were followed over time and in the cellular composition microenvironment (TME) was assessed by immunochemistry and multiplex tissue immunofluorescence. Selective depletions of immune cell populations were used to delineate the immune system requirements for efficacy.We measured the changes in FAP expression following radiotherapy in engrafted syngeneic TSA and MC38 models using immunohistochemistry (IHC) and qPCR. TSA cells and CAFs were coinjected orthotopically creating bilateral tumor-bearing models, which were irradiated (2x6Gy) only inone of the lesions, either as a single treatment intervention or in combination with systemic administrations of FAP-41BBL. Treatment efficacy was evaluated measuring tumor volume and changes in the TME. Anti -D8 and a neutralizing anti-type-I IFN mAbs were used to define requirements for the efficacy of the radioimmunotherapy combination. Results. Irradiation of TSA-breast cancer tumors showed clear increases of FAP expression levels after local irradiation. The suboptimal radiotherapy regimen chosen failed to control TSA-derived tumors in in which TSA-breast cancer cells had been co-engrafted with CAF. The combination of irradiation and FAP-41BBL resulted in more prominent increases of FAP expression after local irradiation and, importantly, induced durable complete responses in more than 50% of the mice treated with the combination. A role in the therapeutic effect for CD8+ T cells and type-I IFN was uncovered. Robust immune memory was observed in re-challenge experiments. Conclusion. Our data provides a proof-of-concept and mechanistic insights pertaining the therapeutic efficacy of the bispecific FAP-41BBL fusion protein combined with local radiotherapy. Citation Format: Eneko Garate-Soraluze, Irantzu Serrano-Mendioroz, Carlos E de Andrea, Toni Rullan, Christina Claus, Pablo Umana, Christian klein, Ignacio Melero, Maria E. Rodriguez-Ruiz. 4-1BBL agonist targeted to fibroblast activation protein α synergizes with radiotherapy in murine breast tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1100.

TCR Diversity Underpins Immunotherapy Success

A diverse repertoire of T cells directed against a small number of cancer-specific targets is key to the success of immune checkpoint blockade in patients with metastatic melanoma, a finding that could shape the design of neoantigen-specific vaccines and adoptive cell therapies.