Efficacy Of Immune Checkpoint Inhibitors Research Articles

Abstract 4127: Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models

Abstract Despite promising results from recent FDA-approved therapies, many advanced melanoma patients develop resistance to both immunotherapy and targeted therapy. A common resistance mechanism to targeted therapy is upregulation of the PI3K/AKT signaling pathway, which has also been shown to promote the development of melanoma brain metastases. Historically, AKT inhibitors have failed in the clinic due to their limited efficacy or intolerable toxicity. Proteomic analysis comparing non-metastatic vs brain metastatic primary tumors in mice revealed focal adhesion kinase (FAK) as an AKT1 specific effector and a potential alternative therapeutic target. FAK is a non-receptor tyrosine kinase that localizes primarily to focal adhesions to regulate cell migration. To determine whether targeting FAK alone or in combination with the RAF/MEK inhibitor avutometinib reduces brain metastases and prolongs survival, we utilized both autochthonous and syngeneic melanoma mouse models. Mice with either subcutaneous tumors or established brain metastases were treated with FAK inhibitor, RAF/MEK inhibitor, or the combination of FAK and RAF/MEK inhibitors. Each cohort was assessed for tumor onset, growth, metastasis, and survival. Our results show that combined RAF/MEK/FAK inhibition significantly delays tumor onset, causes regression of established tumors, prevents the development of brain metastases, promotes the regression of established brain metastases, and prolongs survival. In addition, patient-derived BRAF V600E melanoma xenograft mouse models resistant to the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, were sensitive to combined RAF/MEK/FAK blockade. The addition of the BRAF inhibitor encorafenib to these models further enhanced the effect on tumor growth. These results support the initiation of a clinical trial evaluating the efficacy of the RAF/MEK inhibitor avutometinib in combination with the FAK inhibitor defactinib in patients with brain metastases from cutaneous melanoma. Additionally, we are assessing non-canonical roles of FAK in modulating the tumor microenvironment to determine whether avutometinib and defactinib also enhance the efficacy of immune checkpoint inhibition in this disease. Citation Format: Karly A. Stanley, Jared D. Almazan, Tursun Turapov, David A. Kircher, Gennie L. Parkman, MiKaela N. Field, Katie M. Culver, Christopher M. Stehn, Silvia Coma, Jonathan A. Pachter, Howard Colman, Sheri L. Holmen. Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models [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 4127.

Abstract 912: CheckpointPx, an interpretable radiology AI tool, predicts checkpoint blockade benefit independent of PDL1 status in non-small cell lung cancers (NSCLC): A multi-institutional validation study

Abstract Background: Immune Checkpoint Inhibitors (ICIs) are a cornerstone in the treatment of cancers such as Non-Small Cell Lung Cancer (NSCLC). Despite the standard use of biomarkers such as PD-L1 expression for selecting ICI regimens, recent studies suggest missed opportunities for benefit in PD-L1 low/negative patients. Therefore, there is an urgent need for new biomarker strategies capable of guiding the use of ICIs in NSCLC. We introduce and validate an AI-powered radiology tool for ICI patient selection that provides interpretable predictions by quantifying aspects of tumor biology on baseline CT scans directly associated with ICI efficacy, such as tumor heterogeneity and twistedness of the tumor-associated vasculature. Methods: We analyzed CT scans of 439 patients treated with ICI at 4 institutions (D1-D4) using the Picture Health Px platform. Experienced radiologists and physicians across institutions delineated target lesions on baseline CT scans. A deep learning model was used to segment pulmonary vessels. CheckpointPx v1.11, an interpretable neural network incorporating quantitative features extracted from within the tumor and its vasculature, was trained to predict ICI response (defined as disease control per RECIST best overall response) on n=247 patients (D1-D3). It was assessed with respect to response and progression free-survival (PFS) on 192 patients (D2-D4), with D4 (n=105) being an external validation set. CheckpointPx was also evaluated within PDL1 subsets among testing set patients where available (n=138). Results: The cohort was predominantly late stage (Stage 3/4) (>85%) and was mixed with respect to ICI line of treatment (1st-4th). 122 of 247 (49%) and 119 of 192 (62%) were responders in training and test cohorts respectively. CheckpointPx included 16 features, such as entropy-based heterogeneity and quantitative vessel tortuosity (twistedness). The model predicted response to ICI with an AUC=0.65 on the test set. The CheckpointPx High Risk group significantly stratified patients by PFS (HR=1.67 [1.22-2.28], p=0.001). This separation remained significant within the subset of PDL1-negative (HR=2.71 [1.35-5.44], p=0.005) and PDL1-positive patients (HR=2.05 [1.22-3.46], p=0.007). Conclusions: From baseline radiology, CheckpointPx was shown to strongly predict ICI outcomes across multiple institutions, NSCLC stages, and lines of ICI. Furthermore, the tool stratified patients by ICI benefit within both PDL1-positive and negative subsets - suggesting the potential of CheckpointPx to address critical gaps in the NSCLC biomarker landscape. CheckpointPx is driven by interpretable imaging biomarkers, and thus its predictions are tied to specific and quantifiable phenotypic attributes of the tumor and its microenvironment. Citation Format: Amogh Hiremath, Seyoung Lee, Jeeyeon Lee, Peter Haseok Kim, Kai Zhang, Salie Lee, Monica Yadav, Maria Jose Aguilera Chuchuca, Taegyu Um, Myungwoo Nam, Liam Il-Young Chung, Hye Sung Kim, Jisang Yu, Trie Arni Djunadi, Leeseul Kim, Youjin Oh, Sungmi Yoon, Zunairah Shah, Yuchan Kim, Ilene Hong, Grace Kang, Jessica Jang, Amy Cho, Soowon Lee, Cecilia Nam, Timothy Hong, Yury S. Velichko, Amit Gupta, Vamsidhar Velcheti, Anant Madabhushi, Nathaniel Braman, Young Kwang Chae. CheckpointPx, an interpretable radiology AI tool, predicts checkpoint blockade benefit independent of PDL1 status in non-small cell lung cancers (NSCLC): A multi-institutional validation study [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 912.

Abstract 3404: Body weight trajectory among patients with non-small-cell lung cancer receiving immune checkpoint inhibitors

Abstract Objectives: Body weight is an important prognostic factor in patients with lung cancer. As immunotherapy involves toxicities, body weight may be affected during the treatment course. The objective of this study is to examine the trajectory of weight changes at 180 and 365 days after the initiation of immune checkpoint inhibitors (ICIs) among patients with NSCLC. Methods: Retrospective chart review and data extraction were performed among patients receiving ICIs at the Ohio State University Comprehensive Cancer Center between 2015 and 2021. Patients with NSCLC were included in this study if their initial treatment entailed a PD-1/PD-L1 ICI), either alone or in combination with chemotherapy/targeted cancer drugs, and if they also had their body weights recorded on ICI initiation and 180 days after (± 30 days) treatment initiation. A subset of these patients who had body weight data on day 365 of ICI initiation were included in additional analysis. Percentages of change in body weight relative to pre-ICI were calculated ([weight at 180 or 360 days minus pre-ICI weight] divided by pre-ICI weight x 100%) and examined by treatment regimen and pre-ICI body mass index (BMI) using t-tests. A sensitivity analysis was performed by excluding 11 patients with cachexia defined by the presence of ICD-9 (799.4) or 10 (R64) codes in their medical records. Results: A total of 554 patients with NSCLC received any ICI; 139 patients met the inclusion criteria, of which 96 had body weight data on day 365. The mean age of diagnosis was 63.8 (SD=10.1); 80% were White and 18.7% were Black. The median time on ICI was 195 days (inter-quartile range, 55-392 days) among patients with ICI only and 255 days (inter-quartile range, 126-479 days) among patients with ICI combined with chemotherapy or targeted cancer drugs. Among patients receiving ICI only, body weight increased among those with normal weight (BMI <25.0; mean change = +7.57%, P=0.013) but decreased among those classified as overweight (BMI = 25.0 to 29.9; −5.2%, P=0.037) or obese (BMI ≥30; −6.5%, P=0.025). Decreases in weight were also observed for patients with obesity and receiving a combined treatment (−9.65%, P=0.043). After one year of treatment, weight changes among patients receiving ICI only remained significant in the normal weight group (+15.6%, P=0.007), but not among patients with other BMIs and treatment regimens. These associations remained similar after excluding the patients with cachexia. Conclusions: Body weight change during ICI therapy may vary by treatment regimen and pre-ICI BMI among patients with NSCLC. Whether the weight changes are related to toxicities and survival warrants further research to shed light on weight management, ICI efficacy, and prevention of treatment-related adverse events. Funding: The study was in part funded by NIH/NCI R37CA248371, OSUCCC CCSG P30CA016058, and Pelotonia Institute for Immuno-Oncology (PIIO) at OSUCCC. Citation Format: Ting-Yuan David Cheng, Ben Kinder, Rand T. Akasheh, Cuthbert Mario Mahenge, Carolyn J. Presley, Peter G. Shields, Xuan Nguyen, Aditi Shendre, Zihai Li, Lang Li. Body weight trajectory among patients with non-small-cell lung cancer receiving immune checkpoint inhibitors [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 3404.

Abstract 3848: Comprehensive protein analysis to identify the proteins responsible for sarcopenia and their impact on antitumor immunotherapy in non-small cell lung cancer

Abstract Background: Previous retrospective studies have shown that sarcopenia (Sar) is associated with poor outcome in cancer patients (pts) receiving immune checkpoint inhibitors (ICIs). We conducted a prospective study to determine whether Sar-related proteins induce Sar and reduce the efficacy of ICI. Method: Non-small cell lung cancer (NSCLC) pts receiving ICI as first-line treatment at Niigata University Medical and Dental Hospital were eligible. Sar was diagnosed with physical performance and skeletal muscle mass. Comprehensive protein analysis of pre-treatment blood samples was performed to identify factors that correlate with muscle loss and ICI effects. Result: 41 patients were enrolled from October 2019 through August 2022. PFS for Sar was 4.6 months, 6.7 months for Pre-Sar, and 16.7 months for Non-Sar (P=0.0028). OS for Sar was not reached, 15.7 months for Pre-Sar, and not reached for Non-Sar (P=0.0014). Leptin, IL-1β, IL-12, and Pentraxin 3 were significantly correlated with loss of muscle mass. In contrast, leptin, insulin, IGF-1, GDF-8, Activin A, IL-2Ra, IL-8, MMP-3, osteopontin, and Pentraxin-3 were significantly associated with poor OS. Pentraxin 3 and leptin were associated with both the effectiveness of immunotherapy and sarcopenia. A multivariate analysis that considered factors commonly associated with the effectiveness of immunotherapy, including age, PD-L1 expression rate, and neutrophil-lymphocyte ratio, Pentraxin 3 was determined to be an independent prognostic factor. Conclusion: This study indicated that Sar-related proteins and inflammatory cytokines are involved in the development of Sar and ICI efficacy. Citation Format: Takaaki Masuda, Satoshi Watanabe, Tomoya Wakabayashi, Susumu Tanaka, Kunihiro Shono, Ryo Suzuki, Tomoki Sekiya, Naohiro Yanagimura, Masashi Arita, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Yu Saida, Satoshi Hokari, Rie Kondo, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Kazuko Sakai, Kazuto Nishio, Motohiko Yamazaki, Hiroyuki Ishikawa, Toshiaki Kikuchi. Comprehensive protein analysis to identify the proteins responsible for sarcopenia and their impact on antitumor immunotherapy in non-small cell lung 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 3848.

Abstract 88: Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development

Abstract Translation of immuno-oncology research into novel therapies relies on animal models to advise target selection and portray mechanism of action (MoA) and pharmacokinetics of lead candidates. Mouse, monkey, and more recently, canine models of human cancers are being utilized to this end. However, one major challenge in developing and utilizing these models is the lack of technology to characterize non-human analogs of therapeutic antibodies. Herein, we report the development of novel cell-based bioassays for mouse, monkey, and canine targets to enable immuno-oncology preclinical studies. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important MoAs for antibody-based cancer immunotherapies. We have developed a suite of cell-based bioassays, each expressing a corresponding animal Fcγ receptor (FcγR) and a luciferase reporter that responds to FcγR activation. These assays exhibit sensitive and quantitative luciferase readouts of FcγR activation. Animal models are further used to provide insight into the potential efficacy of immune checkpoint (IC) inhibitors. Programmed cell death protein 1 (PD-1) is an IC receptor that negatively regulates T cell function. Blockade of PD-1 has demonstrated clinical efficacy, but there remains a significant fraction of patients unresponsive to this therapy. Combination of PD-1 inhibitors with other interventions has been a significant focus for many programs. However, tools for functional validation of non-human PD-1 inhibitors are lacking. We report here the development of two new bioassays for functional characterization of PD-1/PD-L1 inhibitors for mouse and canine targets. Each of these assays consists of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1, and an artificial antigen presenting cell line (aAPC). The bioassays are sensitive, quantitative, and have demonstrated characteristics required for potency assays for validation of antibodies in preclinical combination studies. Citation Format: Jun Wang, Denise Garvin, Aileen Paguio, Elizabeth Perrin, Jim Hartnett, Mei Cong, Jamison Grailer. Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development [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 88.

Abstract 1360: TIGIT blockade combined with local radiotherapy and PD-1 blockade in a murine triple-negative breast cancer model

Abstract Background: Immune checkpoint blockades (ICB) improve outcomes of patients with some solid tumors such has melanoma and lung cancer, however the efficacy of ICB alone showed limited efficacy in many other tumors such as breast cancer. Radiation therapy is a promising combination partner of ICB as a strong immune stimulator so called in situ tumor vaccine, however it also can increase immune suppressive repertoires. TIGIT (T cell Immunoglobulin and ITIM domain) is an inhibitory receptor expressed on activated T cells and NK cells. We evaluated the effects of TIGIT blockade in addition to local RT and PD-1 blockade as a strategy to overcome therapeutic resistance of ICI in a murine breast cancer model. Materials and Methods: 4T1-Luc tumors were treated with various strategies including control, RT, anti-PD-1, anti-TIGIT, RT+anti-PD-1, RT+anti-TIGIT, anti-PD-1+anti-TIGIT, and RT+anti-PD-1+anti-TIGIT. A total of 24 Gy in 3 fractions at 1 week was delivered to the tumor at hindlimb (primary tumor) while the tumor at flank (secondary tumor) was left unirradiated. 10 mg/kg of anti-PD-1 blocking antibodies and 10 mg/kg of anti-TIGIT blocking antibodies were intraperitoneally injected for 6 times for 2 weeks. Flow cytometry, IHC staining, and ELISA were performed to assess the immunologic status after treatments. Results: The triple combination therapy (TCT) group showed the most superior growth delay of primary and secondary tumor growth among treatments. The number of metastatic lung nodule was significantly reduced by TCT as well. Plasma levels of interferon-β and interferon-γ were the highest after TCT. Moreover, TCT significantly increased the proportion of splenic CD8+ dendritic cells among myeloid cells, and effector-memory (CD44+CD62L−) cells among splenic Foxp3− non-regulatory CD4+ and CD8+ T cells. Furthermore, expression of CD226, which is an activating counter-receptor of TIGIT, on splenic CD8+ T cells was elevated by TIGIT blockade, possibly suggesting the activation of systemic immune response by addition of TIGIT blockade to local RT and PD-1 blockade. Meanwhile, TCT decreased splenic Foxp3+ regulatory T cells, as well as the expression of CD39 on splenic Foxp3+ regulatory T cells. The increase of CD8+ T cell infiltration in primary and secondary tumors was most prominent in TCT group. Conclusion: TIGIT blockade elicits systemic immune responses in a murine triple-negative breast cancer model. when combined with local RT and PD-1 blockade, which were correlated with significant delay in the growth of both irradiated and unirradiated tumors. These results suggest that TIGIT blockade could be a viable approach to increase the efficacy of RT and immune checkpoint inhibitors in breast cancer which is a relatively immunologically cold tumor. (Work supported by a grant from National Research Foundation of Korea #2023R1A2C3003782 to In Ah Kim). Citation Format: Seongmin Kim, Seung Hyuck Jeon, In Ah Kim. TIGIT blockade combined with local radiotherapy and PD-1 blockade in a murine triple-negative breast cancer model [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 1360.

Abstract 5080: Antibody drug conjugate trastuzumab deruxtecan suppresses leptomeningeal brain metastasis cells and is potentiated by addition of immune check point inhibitor

Abstract Leptomeningeal disease (LMD) is defined by dissemination of metastatic cancer cells into the subarachnoid space with subsequent involvement of the cerebrospinal fluid and leptomeninges. LMD is an aggressive, late-stage development of cancer, which is increasing in prevalence as improved treatments for systemic malignancies are discovered. It leads to significant neurological morbidity and mortality, and outcomes remain poor despite improving cancer treatment strategies. There is a large population of patients with human epidermal growth factor receptor 2 (HER2) expressing cancers, such as breast and lung cancers, who suffer from leptomeningeal metastasis. There has been evolving research looking at antibody drug conjugates for treatment of these primary pathologies, such as Trastuzumab Deruxtecan (TDxd), which shows improved progression free survival and overall survival in those with HER2 expressing primary cancers, as well as in intraparenchymal brain metastasis. However, as of yet there has been no study of its utility in LMD. Additionally, use of immune check point inhibitors (ICI) for treatment of LMD has been studied, showing some efficacy, but less robust response than that in intraparenchymal brain metastasis. Given the significant population of patients who continue to be affected by leptomeningeal metastasis, there is significant need for an improved treatment regimen. We hypothesize that TDxd will have a killing effect on HER2 expressing LMD cells, which can be further potentiated by the addition of ICIs. To test this hypothesis, we developed breast and lung cancer cell lines (SKBrM-LMD, and H2030BrM-LMD) that express HER2 and preferentially metastasize to leptomeningeal space. These cell lines were also labeled with luciferase. We then treated these cell lines with TDxd and found that TDxd had significant growth inhibitory effect on these cells in vitro when compared to the control. Furthermore, we educated human T Cells against the SkBrM-LMD cells and treated these cells with the aforementioned T cells plus the ICI Anti-PD1. Our results showed that ICI had significant killing effect when compared to the control. We have also created an in vivo LMD model by performing intraventricular injection of HER2 expressing tumor cells into a mouse model, which can be used for testing the efficacy of TDxd and ICI by intrathecal injection. In conclusion, our results indicate that TDxd and ICI have significant killing activity against HER2 expressing LMD cells, and therefore intrathecal administration of these drugs is a promising treatment modality that will be demonstrated using a mouse LMD model. Citation Format: Eleanor C. Smith, Shih-Ying Wu, Ravindra P. Deshpande, Abhishek Tyagi, Kounosuke Watabe. Antibody drug conjugate trastuzumab deruxtecan suppresses leptomeningeal brain metastasis cells and is potentiated by addition of immune check point inhibitor [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 5080.

Abstract 6646: Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is often marked by an immunosuppressive tumor microenvironment, which contributes to the dismal 10-20% response rate to immune checkpoint inhibitor (ICI) therapy in HNSCC patients. Radiotherapy (RT) is a currently standard of care and frontline therapy for HNSCC, yet resistance is common. We sought to examine the combination therapy of RT followed by ICI therapy to test if RT induces an in situ vaccination which enhances the efficacy of subsequent ICI. Methods: We utilized SCC cell lines derived from K15.KrasG12D/Smad4−/− SCCs, which mimic the progression of HPV-unrelated HNSCC, and transplanted SCC cells into immune-competent C57BL/6J mouse recipients. Mice were subjected to RT followed by anti-PD-L1/anti-TGFb therapy. CD8+ splenic T cells of responder or non-responder mice were examined by paired single-cell VDJ sequencing and single cell RNA sequencing (TCR/sc-RNAseq) to look for adaptations of the cytotoxic T cell compartment following combination therapy. Responder and non-responder tumor lines or tumors were treated with RT alone in vitro and in vivo, respectively, to examine major histocompatibility complex I (MHC-I) levels on tumor cells. Results: Responders to this combination therapy exhibited complete tumor eradication while non-responders displayed rapid tumor progression. When re-challenged with same HNSCCs cells, responders rejected implanted tumor cells while naïve recipients rapidly developed HNSCCs. Single-cell TCR sequencing of splenic CD8+ T cells revealed that responders, but not naïve recipients exhibited multiclonal CD8+ T cell expansion specifically of memory cell populations. Responder tumor cells exhibited greater levels of MHC-I protein level presentation than non-responders when stimulated with RT. Conclusions: Our data suggest that 1) elevated MHC-I by SCC tumor cells presentation driven by RT and ICI combination therapy facilitate a systemic expansion of memory CD8+ T cell clonotypes which drive anti-tumor immunity and 2) MHC-I expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and ICI combination therapy in HNSCCs. Citation Format: Hanne Lind, Alexander Strait, Spencer Hall, Jack B. Goon, John D. Aleman, Samantha Chen, Philip Owens, Jing H. Wang, Christian Young, Xiao-Jing Wang. Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models [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 6646.

Abstract 5260: Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling activity. In the previous meeting (AACR2022, 2023), we reported that ERI and liposomal formulation of ERI (ERI-LF) also have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF showed more potent immune modulation and combination activities with anti-PD-1 antibody (PD-1 Ab) than ERI in syngeneic mice models and humanized mice models. Although immune-checkpoint inhibitors (ICI) are using as a key drug for various types of cancers in clinical setting, several clinical questions regarding suitable therapies after ICI therapy and efficacy of ICI rechallenge are remaining. In this study, we evaluated anti-tumor activities of combination of ERI-LF and PD-1 Ab in two syngeneic transplantation models using mouse non-small lung cancer cells, P-glycoprotein-knockout (Pgp-KO) KLN205 cells and Pgp-KO LL2 cells. As a result, combination of 1mg/kg ERI-LF (Q7D×2) with PD-1 Ab (200 ug/head, twice a week ×2 weeks) showed significant stronger antitumor activity compared with each monotherapy in only Pgp-KO KLN205 model, in which ERI-LF increased intratumoral microvascular density as a vascular remodeling activity, not in Pgp-KO LL2 model. We next conducted continuous treatment of PD-1 Ab in Pgp-KO KLN205 model to investigate tumor regrowth during treatment of PD-1 Ab. In this model, tumor growth was inhibited by PD-1 Ab until about two weeks after initiation of the treatment and then this inhibitory activity disappeared after two weeks even by continuous treatment of PD-1 Ab. We performed flow cytometry analysis (FCM) for tumor-infiltrating immune cells and RNAseq analysis using regrowing tumors compared with non-treatment tumors. Expressions of mRNA of several immune inhibitory receptors were upregulated in tumors with regrowth, although CD4+ cells and CD8+ cells were also increased in FCM using tumor samples. Furthermore, we investigated anti-tumor activity of combination of ERI-LF and PD-1 Ab against tumors which changed from inhibitory phase to regrowth phase during PD-1 Ab treatment in Pgp-KO KLN205 model. The combination of ERI-LF at 1mg/kg (Q7D×3) and PD-1 Ab (200 ug/head, twice a week ×3 weeks) also showed potent anti-tumor activity against tumors with regrowth by prior PD-1 Ab treatment as well as PD-1 Ab-naïve tumor, suggesting that this combination therapy might be effective after prior ICI therapy. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers (NCT04078295) is underway. Patients with or without prior ICI therapy were enrolled in a small cell lung cancer cohort of this clinical study. Our preclinical results might provide a scientific rationale for a uniqueness of ERI-LF as a post ICI therapy. Citation Format: Moe Tamura, Yuki Niwa, Taro Semba. Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice [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 5260.

Abstract 7515: Enhancing immune checkpoint inhibitor efficacy with fulvestrant in estrogen receptor-positive breast cancer

Abstract Introduction: Breast cancer (BC) recurs decades after initial treatment that consists majority of the annual 43,000 BC deaths. Thus, Immune Checkpoint Inhibitor (ICI) therapy that bolster anti-cancer immunity is expected to improve long-term survival with reduced recurrence. Neoadjuvant ICI revolutionized management of triple negative breast cancer (TNBC); however, this represents only 5-7% of all BC cases, where estrogen receptor (ER)-positive subtype that consists of 70% of all BCs is not indicated due to less tumor-infiltrating lymphocytes (TILs). Using I-SPY2 trial we aim to investigate the potency of ER-alpha gene (ESR1) expression as a predictive biomarker, and whether the inhibition of the ER with fulvestrant enhances the efficacy of ICIs in ER+ BC. Methods:Total of 1157 BC patients from two independent cohorts (ISPY2 trial; n= 73, TCGA; n = 1084) were included, encompassing both clinical and transcriptomic profiles. The TIL score was determined using the xCell algorithm. Results:Using the transcriptome of the ISPY2 trial, we found that compared to ESR1 low expression patients, ESR1 high patients were associated with significantly lower pCR rates after neoadjuvant immunotherapy with ICI in whole cohort (n=71, pCR rate=30%, p<0.05) and HR+ subtype (n=50, pCR rate=20%, p<0.001), but not in triple-negative breast cancer (TNBC). ESR1 expression outperformed PD-1, PD-L1, and CTLA4 in Area Under the Curve (AUC) of the whole ISPY2 cohort (AUC=0.75, 0.68, 0.48, and 0.62, respectively), and further pronounced in HR+ BC (AUC=0.88, 0.73, 0.55, and 0.65, respectively), suggesting the potency of ESR1 expression as a predictive biomarker for ICI in HR+ BC. In agreement, ESR1 expression inversely correlated with the amount of intratumoral TILs consistently in both ISPY2 and TCGA cohorts in HR+ BC, but only in TCGA in whole cohort (all p<0.05). Among the intratumoral TILs, CD8+ T cell and M1 Macrophages were found to be less infiltrated in HR+ BC in TCGA. Given that ESR1 expression was associated with less intratumoral TILs and a poor response to ICI, we hypothesized that suppression of ER-alpha would enhance the effectiveness of ICI in HR+ BC in vitro. ER-alpha positive MCF7 cells were incubated with activated peripheral blood mononuclear cells with 1:1 ratio, and treated with 0.1 microgram of anti-PDL1, and 1nM of ER antagonist, fulvestrant, which demonstrated minimum cell killing. We found that ICI therapy with anti-PDL1 significantly decreased the cell viability of the MCF7 cells when ER-alpha expression was suppressed by fulvestrant, while activated peripheral blood mononuclear cells or anti-PDL1 alone or the combination did not exhibit any significant cell killing. Conclusion: We found that ESR1 expression is a predictive biomarker of ICI, and ER antagonist fulvestrant enhances the response of HR+ BC to ICI, suggesting that this combination could expand ICI indications to HR+ BC. Citation Format: Jun Arima, Kohei Chida, Masanori Oshi, Arya Mariam Roy, Kohei Taniguchi, Sang-Woong Lee, Kazuaki Takabe. Enhancing immune checkpoint inhibitor efficacy with fulvestrant in estrogen receptor-positive breast 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 7515.

Abstract 3852: Comprehensive genomic profiling of recurrent or metastatic head and neck cancer presenting with combined positive score 90 or more

Abstract Background. Biomarkers that predict response to immune checkpoint inhibitor (ICI) in recurrent metastatic squamous cell carcinoma of the head and neck (RMHNSCC) are not well known. Previously, we have found that Combined positive score (CPS) ≥90 and albumin >3.5 respond well to ICI treatment in patients with RMHNSCC treated with ICI irrespective of addition of chemotherapy. In the present study, we investigated whether RMHNSCC with CPS ≥90 has a characteristic genomic mutational profile. Methods. CPS measurements were performed on a single tumor specimen at the time of diagnosis of RMHNSCC, as approved by Japanese health insurance. Tumor specimens were obtained from the primary tumor as much as possible, and sections that best reflected anti-tumor immunity were decided by a pathologist. We retrospectively reviewed the results obtained from comprehensive genomic profiling (CGP) testing of RMHNSCC cases with CPS ≥90 who were treated by ICI experienced at our institution. Results. There were 117 eligible RMHNSCC cases. The number of primary lesions were oral cavity in 28 cases (23.9%), nasopharynx in 10 cases (8.5%), oropharynx in 21 cases (17.9%), hypopharynx in 33 cases (28.2%), and others in 18 cases (15.4%). Of 117 patients, 28 patients (23.9%) had a CPS ≥90. Of the 28 cases, the number of primary lesions were oral cavity in 12 cases (42.9%), nasopharynx in 4 cases (14.3%), oropharynx in 0 cases (0%), hypopharynx in 8 cases (28.6%), and others in 3 cases (10.7%). When ICI was administered, CPS ≥90 had a statistically significantly better prognosis than CPS <90 for OS and PFS. Of the 28 cases with CPS ≥90, CGP test was performed in 6 cases. The primary lesions in the 6 cases consisted of 3 oral cavity, 2 nasopharynx, 1 hypopharynx, and 1 maxillary sinus. All three oral primary cases had co-mutations in the TERT promoter and HRAS. Two of the nasopharyngeal primary cases had mutations in JAK3 and STAT, respectively. One of the hypopharyngeal primary cases had mutations in FGFR3. Conclusion. RMHNSCC with CPS ≥90 had unique mutations that could account the enhanced PD-L1 expression, such as TERT/HRAS co-mutation, JAK-STAT pathway mutation, and FGFR pathway mutation. The above findings may be useful for predicting the efficacy of ICI for RMHNSCC. Citation Format: Yuki Saito, Kage Hidenori, Kenya Kobayashi, Osamu Fukuoka, Koji Yamamura, Aya Shinozaki-Ushiku, Katsutoshi Oda, Kenji Kondo. Comprehensive genomic profiling of recurrent or metastatic head and neck cancer presenting with combined positive score 90 or more [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 3852.

Abstract 5263: The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation

Abstract Background: To date, there have been no clinical molecules that are selective for KRASG12D mutant tumors. Although immune checkpoint inhibitors (ICIs) have improved the survival for patients with lung cancer, inferior efficacy of ICIs has been reported in KRASG12D mutant NSCLC. MRTX1133 is a selective KRASG12D inhibitor, which has been investigated in early clinical studies. In this study, we aimed to reveal the immune status using preclinical murine model of KRASG12D mutated lung cancer. In addition, we investigated whether MRTX1133 effects the anti-tumor immunity in mice bearing lung cancer with KRASG12D. Method: CCSP-rtTA/TetO-Cre mice were crossed with LSL-KRASG12D knock-in mice to obtain CCSP-rtTA/TetO-Cre/LSL-KRASG12D conditional mutant mice (KRASG12D mice). These mice were fed an autoclaved rodent diet supplemented with 0.0625% doxycycline when the mice were about 8~12 weeks old. Mice were treated with 10 mg/kg MRTX1133 in vehicle once daily by intraperitoneal injection for 4 or 8 days. After administration of MRTX1133, the lungs and spleens were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: More regulatory T cells (Tregs) infiltrated the tumors of lung of mice treated with doxycycline for a longer period of time. The expression of immune checkpoint molecules (ICMs), including PD-L1, PD-1, TIGIT, TIM-3, LAG-3, were also upregulated on CD4+ and CD8+ T cells in the KRASG12D mice. Treatment with MRTX1133 strongly inhibited KRASG12D tumor progression, reduced the percentage of Tregs and downregulated the expression of PD-1 on CD4+ and CD8+ T cells infiltrating in lung cancers. The expression of the other ICMs remained high after MRTX1133 administration. In addition, MRTX1133 tended to increase the percentage of tumor-specific CD8+ effector T cells infiltration in lung tumors. Conclusion: T cells were exhausted in mice with KRASG12D mutant lung cancer. MRTX1133 demonstrates the therapeutic efficacy in preclinical murine KRASG12D mutated lung cancer model. This efficacy is more likely to relate not only signal inhibition but also enhances the anti- tumor immune responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with KRASG12D mutant lung cancer. Citation Format: Kunihiro Shono, Satoshi Watanabe, Takaaki Masuda, Ryo Suzuki, Tomoya Wakabayashi, Tomoki Sekiya, Susumu Tanaka, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Rie Kondo, Yu Saida, Satoshi Hokari, Naohiro Yanagimura, Masashi Arita, Riuko Ohashi, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Toshiaki Kikuchi. The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation [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 5263.

Abstract 233: Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an approximate 10% 5-year survival rate. PDAC patients rarely benefit from treatment with immune checkpoint inhibitors (ICIs) due to a tumor microenvironment (TME) associated with constrained effector T cell infiltration, and the presence of a suppressive myeloid cell population. Objective: To identify a combinatorial therapeutic strategy targeting the myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs) within the PDAC TME to increase the efficacy of ICIs. Methods: C57BL/6 mice were orthotopically transplanted with syngeneic 7940b PDAC cells derived from transgenic KPC mouse. 7 days post-transplantation mice were treated with InVivoPlus anti-mouse PD-1 (CD279), cabozantinib (cabo, tyrosine kinase inhibitor) or a combination of drugs for a further 7 days. NanoString CosMx™ spatial molecular imaging (CosMx™ SMI) was performed using FFPE sections collected from core biopsies and surgically resected tissues. PDAC patient derived organoids (PDOs) were generated from the core biopsies or surgical biospecimens. Cancer-associated fibroblasts (CAFs) were isolated from the organoid cultures. Autologous immune cells (IMM) were cultured from patient peripheral blood mononuclear cells. PDO/CAF/IMM co-cultures were used to predict the patient response to cabo plus pembrolizumab combinatorial therapy. Results: Mice treated with a combination of anti-PD-1 and cabo exhibited significant EpCAM/PD-L1-expressing cell death that correlated with increased CTL proliferation and decreased PMN-MDSC death. Mice that were depleted of CD8+ CTLs using anti-CD8 antibody in vivo did not exhibit tumor cell death in response to combinatorial therapy and thus supporting the role of CTLs in driving effective immunotherapy in combination with cabozantinib treatment. Unexpectedly, a significant decrease in extracellular matrix protein fibronectin was observed. Human PDOs orthotopically transplanted into NSG mice developed pathology consistent with human PDAC with strong expression of CD44v9, IL6, vimentin and αSMA. Using PDAC PDO/CAFs/IMM co-cultures, pembro significantly induced tumor cell death in the presence CTLs, MDSCs significantly inhibited this response. Depletion of MDSCs using cabo in the PDO/CAFs/IMM co-cultures resulted in a significant increase in tumor cell death with a concomitant increase in CTL proliferation. Compared to core biopsies, surgical biospecimens collected post-standard of-care demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) increases MDSCs, 2) change in CAFs phenotype and 3) increased expression of CD44v9. Conclusions: Cabozantinib depletes the PDAC TME of MDCSs leading to sensitization to ICI and tumor cell death and thus offers a potential combinatorial therapeutic approach. Citation Format: Pritha Adhikary, Jayati Chakrabarti, Meenu Priya Resmi, Jiang Wang, Davendra Sohal, Rachna T. Shroff, Syed A. Ahmad, Yana Zavros. Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab [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 233.

Abstract 1353: Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (anti-PDL1 or anti-CTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of anti-PDL1 and anti-CTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of anti-PDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and anti-CTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. mice. Lymph nodes and tumors from mice treated with IR and anti-CTLA4 vs. IR and isotype control demonstrated differential expression of genes in T cell functions and enrichment in both CD4+ and CD8+ T cell populations. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining anti-CTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Linsey Gong, Xiaoyong Huang, Stephanie D. White, Hanz T. Chung, Danny Vesprini, Tera N. Petchiny, Emmanouil Fokas, Hansen He, Robert S. Kerbel, Stanley K. Liu. Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model [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 1353.

Abstract 6118: Sex-related disparity in immune checkpoint inhibitor benefit for patients with esophageal squamous cell carcinoma: A systematic review and meta-analysis

Abstract Sex-related dimorphism in cancer incidence and immune response is widely recognized, yet the impact of an individual's sex on the effectiveness of Immune Checkpoint Inhibitors (ICIs) for patients with advanced esophageal squamous cell carcinoma (ESCC) remains poorly understood. In this study, we conducted a comprehensive meta-analysis to evaluate the heterogeneity of ICIs in patients with advanced ESCC. We collected studies from PubMed, MEDLINE, Embase, and Scopus, spanning from the inception of the databases to September 30, 2023. These studies focused on phase III randomized trials comparing first-line ICIs (PD-1/PD-L1) plus chemotherapy with chemotherapy alone for advanced or metastatic ESCC. Additionally, we reviewed abstracts and presentations from AACR, ASCO, ESMO, and WCLC. Inclusion criteria mandated the reporting of overall survival (OS) data stratified by sex and nonselective baseline PD-L1 expression. The study adhered to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The analysis included 6 randomized clinical trials comprising 3603 ESCC patients, of whom 3090 (85.8%) were male and 513 (14.2%) were female. A significant OS benefit was observed for ICIs plus chemotherapy in all patients (HR, 0.68; 95% CI, 0.62-0.74; P < 0.001). Similarly, ICIs plus chemotherapy correlated with a significantly prolonged OS in males (HR, 0.67; 95% CI, 0.61-0.74, P < 0.001). However, there was no significant OS benefit observed in females with ICIs plus chemotherapy (HR, 0.79; 95% CI, 0.58-1.09; P = 0.148). Females had a tendency toward less benefit than males (pooled HR, 1.18; 95% CI, 0.89-1.57; P = 0.245). Our findings suggest that there is no statistically significant association between patient sex and the efficacy of ICIs for ESCC patients. Importantly, we report, for the first time, a lack of survival benefit with the ICI plus chemotherapy regimen in the first-line setting compared to chemotherapy alone in female ESCC patients. Citation Format: Chaoqi Zhang, Peng Wu, Jingjing Liu, Dongyu Li, Nan Sun, Jie He. Sex-related disparity in immune checkpoint inhibitor benefit for patients with esophageal squamous cell carcinoma: A systematic review and meta-analysis [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 6118.

Abstract 2533: In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events

Abstract Background: The clinical efficacy of immune-checkpoint inhibitors is complicated by the risk for immune-related adverse events (irAEs) that can involve any organ systems. To date, (1) we cannot predict irAE risk reliably at patient level and (2) irAEs remain a diagnosis of exclusion as there are no specific clinical or biological markers. The goal of this study was to elucidate intricate transcriptomic and proteomic signatures of immune cells in patients evolving irAEs. Methods: This pilot trial was conducted as part of a prospective multicenter cohort study (NCT04631731). The study cohort consisted of n=71 patients who were treated with ICIs across Blacktown and Westmead Hospitals, NSW, Australia. Peripheral blood mononuclear cells (PBMCs) were collected from both pre-treatment and after 6-8 weeks post-ICI commencement. Samples were further utilized for RNA sequencing, flow cytometry and Chromium 3’ gene expression analysis (10x Genomics). Results: The median age was 67 years old with the 78% (n=56) of Caucasian origin. The predominant number of patients were treated with dual ICIs. N=21 patients experienced irAEs. Firstly, a differential analysis established a set of genes which were significantly (FDR<0.01) dysregulated between patients with (group A) and without irAEs (group B) and were mostly related to cell cycle and regulation. Notably, phospholipase D signaling pathway was exclusively upregulated in group A and is known to be linked to B cell expansion. Secondly, a cell enrichment analysis within the group A revealed a significant increase of CD4+ and CD8+ T central memory (Tcm) cells upon the toxicity onset as compared to baseline stage. Thirdly, the flow cytometry data established that group A had (1) higher expression of CD27 on CD4+ T cells, a member of Traf-linked tumor necrosis factor receptor family essential for T cells’ maturation and (2) lower expression of inhibitory receptor LAG3 (CD223) which is crucial for maintaining homeostasis of immune system through suppression of T cell proliferation. Finally, we sequenced CD45+ cells from n=4 patients from group A and n=4 from group B at both pre- and post-treatment stages. The unsupervised clustering established 17 different clusters in our single cell dataset with cluster 11 was unique only to patients from group A. The differential analysis established that a set of n=62 genes was significantly upregulated (p<0.05) in cluster 11. Functional annotation established that B cell activation and proliferation pathways were enriched by those genes further stressing B cells’ role in irAEs development. Conclusions: Our real-world cohort study established significant molecular and cellular differences between patients with and without irAEs. Further research in larger cohorts is imperative to validate the established findings and to elucidate the precise mechanisms contributing to the development of irAEs. Citation Format: Dmitrii Shek, Bo Gao, Joey Lai, Brian Gloss, Adnan Nagrial, Ines Pires da Silva, Matteo S. Carlino, Scott A. Read, Golo Ahlenstiel. In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events [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 2533.

Abstract 6868: Exploring myeloid landscapes according to EGFR mutated NSCLC tumor microenvironment

Abstract Introduction: Although the use of immune checkpoint inhibitors (ICIs) has improved the survival rate of some patients, EGFR mutant (MT) NSCLC patients exhibit a poor response to ICIs compared to EGFR wild-type (WT) patients. It has been demonstrated that EGFR MT NSCLC cells contribute to immunosuppressive tumor microenvironments (TMEs) by secreting inhibitory cytokines and metabolites and recruiting pro-tumor immunogenic cells, thereby diminishing ICI efficacy. In particular, myeloid cells represent a major component of the TME and are involved in regulating tumor progression and metastasis, indicating anti- and pro-inflammatory heterogeneous functions. While single-cell based analyses have been conducted in various cell types, studies on the diversity of myeloid cells between EGFR WT and EGFR MT NSCLCs are insufficient. Therefore, we explored to reveal the presence of multifaceted myeloid cell populations and to modulate its functions to augment ICI response in NSCLC patients with EGFR MT using single-cell RNA analysis. Methods: Using the 10x Genomics Chromium system, we have generated single-cell RNA expression of a total of 904,174 cells from primary lung tissues of 95 patients with EGFR-WT (n=35), EGFR-MT TKI naïve (n=55), TKI post (n=5) and matched normal (n=22). Nine distinct cell lineages were annotated with canonical marker gene expression, focusing particularly on the myeloid (n=212,673 cells). Results: We characterized 22 transcriptional states of myeloid cells: 4 clusters of alveolar macrophages (AMs; PPARG+), 4 clusters of interstitial macrophages (IMs; SPP1+, FOLR2+, CCL2+), 4 clusters of conventional DCs (cDCs; CLEC9A+, CD1C+, LAMP3+), 2 clusters of mast cells (KIT+), 1 cluster of plasmacytoid DCs (pDCs; LILRA4+), proliferating cells (MKI67+), monocytes (FCN1+), neutrophil like cells (CSF3R+) and 4 other unidentified clusters. In tumor tissues, IMs constituted a significantly higher proportion compared to normal tissues (p<0.001), with EGFR-WT and EGFR-MT IMs exhibiting distinct characteristics. EGFR-WT IMs had a high proportion of cluster expressing FOLR2, a marker of tissue resident macrophages associated with CD8 T cell infiltration and activation. On the other hand, a major component of IMs from EGFR-MT was a cluster expressing SPP1 and its regulator MIF. SPP1 is well known to be involved in cancer cell progression, epithelial-mesenchymal transition, autophagy, and epigenetic alterations. The abundance of SPP1 IMs in the EGFR-MT TME refers to the formation of an immunosuppressive TME, potentially compromising the effectiveness of ICIs. Conclusions: We found that EGFR-MT TME comprised a high proportion of immunosuppressive IM cluster expressing SPP1, implying that it contributes to an immunosuppressive TME. Our findings suggest the possibility of developing strategies to enhance ICI responses by targeting SPP1-induced immunosuppressive TME in EGFR-mutant NSCLC patients. Citation Format: Eun Ji Lee, Juwon Ahn, You Won Lee, Minyeop Kim, Seung Yeon Oh, Yong Joon Park, Su-Jin Choi, JuHyeon Lee, Kyumin Lim, Kyoung-Ho Pyo, Jae Hwan Kim, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Mi Ran Yun, Byoung Chul Cho. Exploring myeloid landscapes according to EGFR mutated NSCLC tumor microenvironment [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 6868.

Abstract 108: Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model

Abstract Mutations in STK11 that lead to loss of its protein LKB1 occur in 15-20% of non-squamous non-small cell lung cancer (NSCLC) and are frequently co-mutated with oncogenic KRAS mutations. KRAS and STK11 co-mutant NSCLC is associated with poor prognosis, low PD-L1 expression, low T-cell infiltration, and poor response to immune-checkpoint inhibitors (ICI). Loss of LKB1 leads to altered transcriptional programs with the activation of CREB-dependent gene transcription and changes in the tumor microenvironment including recruitment of suppressive myeloid cells. We used the syngeneic Kras mutant murine CMT167 tumor cell line that is wild-type for Tp53 and Stk11 and sensitive to ICI and generated isogeneic Stk11 knockout cell lines (CMT167-Stk11-KO) by CRISPR/Cas-9 deletion. Loss of Stk11 recapitulated LKB1-loss transcriptional signatures and rendered CMT167 tumors resistant to ICI. Single-cell RNA sequencing (10x) of implanted tumors demonstrated significantly less CD8 T-cells and increased neutrophils (PMN) in Stk11-KO versus Stk11-WT tumors. Analysis of differentially expressed genes in the tumor compartment identified increased expression of complement pathway genes including the central mediator C3 in CMT-167-Stk11-KO tumors. Examination of a well characterized clinical cohort of human KRAS-mutant NSCLC patient samples with and without STK11 mutation confirmed the increased expression of complement C3 in STK11-mutant tumors. Analysis of a human NSCLC cell line panel demonstrated that C3 expression was modulated by LKB1. Knockout of C3 in CMT167-Stk11-KO tumor resulted in dramatic inhibition of tumor growth and re-sensitized CMT167-Stk11-KO tumors to ICI treatment in WT mice. Using C3-/- knockout mice, this effect was determined to be reliant on tumor-derived C3 and dependent on adaptive immunity as no difference in growth of CMT167-Stk11-KO tumors occurred in nude mice or in WT mice after CD8 T-cell depletion. We then compared the transcriptome of Stk11-KO tumor cells with and without C3 deletion, and observed that C3 upregulates the expression of Cxcl1, Cxcl2 and Cxcl3 which mediate PMN recruitment and activation. Since these chemokines ligate Cxcr2, we asked whether Cxcr2 mediated tumor growth and ICI resistance in Stk11-KO tumors. While treatment with single-agent Cxcr2 inhibitor and anti-PD-1 therapy had no effect on tumor growth, the combination resulted in significant suppression of tumor growth in vivo. These results support a role for tumor-derived C3 suppressing CD8 T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in patients with STK11-mutant NSCLC. Citation Format: Sora Suzuki, Catrina Ting, Bojidar Kandar, Te-An Chen, Anm Nazmul Khan, Thejaswini Giridharan, Brahm Segal, Edwin H. Yau. Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model [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 108.

A multicenter study evaluating efficacy of immune checkpoint inhibitors in advanced non-colorectal digestive cancers with microsatellite instability

BackgroundOne randomized phase III trial comparing chemotherapy (CT) with immune checkpoint inhibitors (ICI) has demonstrated significant efficacy of ICI in deficient DNA mismatch repair system/microsatellite instability-high (dMMR/MSI-H) metastatic colorectal cancer. However, few studies have compared ICI with CT in other advanced dMMR/MSI-H digestive tumors. MethodsIn this multicenter study, we included patients with advanced dMMR/MSI-H non-colorectal digestive tumors treated with chemotherapy and/or ICIs. Patients were divided retrospectively into two groups, a CT group and an immunotherapy (IO) group. The primary endpoint was progression-free survival (PFS). A propensity score approach using the inverse probability of treatment weighting (IPTW) method was applied to deal with potential differences between the two groups. Results133 patients (45.1/27.1/27.8% with gastric/small bowel/other carcinomas) were included. The majority of patients received ICI in 1st (29.1%) or 2nd line (44.4%). The 24-month PFS rates were 7.9% in the CT group and 71.2% in the IO group. Using the IPTW method, IO treatment was associated with better PFS (HR=0.227; 95% CI 0.147–0.351; p < 0.0001). The overall response rate was 26.3% in the CT group versus 60.7% in the IO group (p < 0.001) with prolonged duration of disease control in the IO group (p < 0.001). In multivariable analysis, predictive factors of PFS for patients treated with IO were good performance status, absence of liver metastasis and prior primary tumor resection, whereas no association was found for the site of the primary tumor. ConclusionsIn the absence of randomized trials, our study highlights the superior efficacy of ICI compared with standard-of-care therapy in patients with unresectable or metastatic dMMR/MSI-H non-colorectal digestive cancer, regardless of tumor type, with acceptable toxicity.

Combinational delivery of TLR4 and TLR7/8 agonist enhanced the therapeutic efficacy of immune checkpoint inhibitors to colon tumor.

Immunotherapy is regarded as a potent cancer treatment, with DC vaccines playing a crucial role. Although clinical trials have demonstrated the safety and efficacy of DC vaccines, loading antigens in vitro is challenging, and their therapeutic effects remain unpredictable. Moreover, the diverse subtypes and maturity states of DCs in the body could induce both immune responses and immune tolerance, potentially affecting the vaccine's efficacy. Hence, the optimization of DC vaccines remains imperative. Our study discovered a new therapeutic strategy by using CT26 and MC38 mouse colon cancer models, as well as LLC mouse lung cancer models. The strategy involved the synergistic activation of DCs through intertumoral administration of TLR4 agonist high-mobility group nucleosome binding protein 1 (HMGN1) and TLR7/8 agonist (R848/resiquimod), combined with intraperitoneal administration of TNFR2 immunosuppressant antibody. The experimental results indicated that the combined use of HMGN1, R848, and α-TNFR2 had no effect on LLC cold tumors. However, it was effective in eradicating CT26 and MC38 colon cancer and inducing long-term immune memory. The combination of these three drugs altered the TME and promoted an increase in anti-tumor immune components. This may provide a promising new treatment strategy for colon cancer.