Limited Response Rates Research Articles

Abstract IA02: Preserving tumor-draining lymph nodes in the era of immuno-oncology: Opportunities for precision chemo-radio-immunotherapy

Abstract The advent of the current cancer immunotherapy era based on the success of immune checkpoint blockade (ICB) inhibiting programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) has revolutionized the standard of care for cancer treatment. However, the limited response rates to ICB across multiple cancer types suggest that new approaches and treatment strategies are needed in order to achieve durable cancer remission. This includes re-examining the possibility that standard of care treatments, including surgery and chemo-radiation, may compromise the tumor response when delivered in concert with ICB. In particular, many cancer types, including head and neck squamous cell carcinoma (HNSCC), have a propensity to metastasize to locoregional lymph nodes, and standard oncologic management often involves elective ablation of tumor draining lymph nodes (tdLN). In a recent study, we have used tobacco-signature HSNCC syngeneic mouse models and showed that tdLN ablation by surgery or radiation therapy limits the tumor ICB response, thereby worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, we provided evidence that cDC1 dendritic cells in the tumor lesions and tdLN are required for an effective anti-tumor immune response. Recently, we have cross-integrated large single-cell RNA-sequencing datasets from intratumoral immune cells and identified a novel mechanism by which antigen-specific CD8+ T cells promote intratumoral cDC1 accumulation and T cell persistence, leading to improved overall survival. As a multipronged approach, we have also built on this computational pipeline to identify druggable oncogenic signaling networks that override cancer immune surveillance. Evidence will be presented that disruption of specific HNSCC driving signaling axes is sufficient to sculp the tumor immune microenvironment and disable their immune evasive mechanisms, thereby providing opportunities for the development of new precision therapies increasing the response ICB. The emerging information also provided a rationale for investigating the use of a new generation of radiation-sensitizing antibody drug conjugates targeting cancer-specific oncosignaling drivers, in combination with radiotherapy and ICB in the neoadjuvant setting, when tdLN are still intact. Ultimately, evidence will be presented supporting the future evaluation of spatially precise radiosensitizing antibody drug conjugate-directed chemo-radio-immunotherapy for precision cancer care in HNSCC and other malignancies. Citation Format: J. Silvio Gutkind. Preserving tumor-draining lymph nodes in the era of immuno-oncology: Opportunities for precision chemo-radio-immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr IA02

Combination Immunotherapy With Radiotherapy in Non-Small Cell Lung Cancer: A Review of Evidence.

Radiotherapy plays a fundamental role in the treatment of patients with all stages of non-small-cell lung cancer (NSCLC). The emergence of immune checkpoint inhibitors (ICIs) has transformed the standard of care in these patients. The use of ICIs is increasingly utilized in the definitive setting as an adjunct to chemoradiotherapy or surgery and remains a vital component in the treatment of metastatic disease. Despite improvements in patient survival, the use of immunotherapy as monotherapy has shown limited overall response rates with susceptibility to resistance. Radiotherapy has been identified as a viable option to enhance the response rate to ICI and improve outcomes in NSCLC. We queried the English PubMed database utilizing variably combined search items including "radiation," "chemoradiation," "immune checkpoint," "immunotherapy," "stereotactic body radiotherapy," and "non-small-cell lung". We additionally searched various acceptable alternative terms for similar keywords such as "radiotherapy" in place of "radiation." These results were subsequently curated for relevance and impact on current treatment paradigms. In this review, we discuss preclinical and clinical studies relating to combinatorial use of immunotherapy and radiation in NSCLC. These studies are presented in the context of early-stage, operable stage III, unresectable stage III, and metastatic disease. The majority of the data illustrate promising results regarding the additive or synergistic effects of radiation and immunotherapy with a suggestion that the timing of these treatment modalities is crucial to optimizing outcomes. While there is now evidence regarding the favorable interplay between radiation and immunotherapy in NSCLC, there remain multiple unanswered questions which are expected to be addressed in ongoing clinical trials.

Landscape of targeted therapies for lung squamous cell carcinoma.

Lung cancer, a common type of malignant neoplasm, has seen significant advancements in the treatment of lung adenocarcinoma (LUAD). However, the management of lung squamous cell carcinoma (LSCC) continues to pose challenges. Traditional treatment methods for LSCC encompass surgical resection, chemotherapy, and radiotherapy. The introduction of targeted therapy and immunotherapy has greatly benefited LSCC patients, but issues such as limited immune response rates and adverse reactions persist. Therefore, gaining a deeper comprehension of the underlying mechanisms holds immense importance. This review provides an in-depth overview of classical signaling pathways and therapeutic targets, including the PI3K signaling pathway, CDK4/6 pathway, FGFR1 pathway and EGFR pathway. Additionally, we delve into alternative signaling pathways and potential targets that could offer new therapeutic avenues for LSCC. Lastly, we summarize the latest advancements in targeted therapy combined with immune checkpoint blockade (ICB) therapy for LSCC and discuss the prospects and challenges in this field.

Targeting VPS18 hampers retromer trafficking of PD-L1 and augments immunotherapy.

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have achieved impressive antitumor clinical outcomes. However, the limited response rates suggest the incomplete understanding of PD-L1 regulation. Here, we demonstrate that vacuole protein sorting 11 and 18 (VPS11/18), two key players in vesicular trafficking, positively regulate PD-L1 and confer resistance to immune checkpoint blockade therapy. VPS11/18 interact with PD-L1 in endosome recycling accompanied by promoting PD-L1 glycosylation and protein stability. VPS18 deficiency enhances antitumor immune response. Pharmacological inhibition by VPS18 inhibitor RDN impaired PD-L1 member trafficking and protein stability. Combination treatment of RDN and anti-cytotoxic T lymphocyte-associated antigen 4 synergistically enhances antitumor efficacy in aggressive and drug-resistant tumors. RDN exerted lung-preferred distribution and good bioavailability, suggesting a favorable drug efficacy. Together, our study links VPS18/11-mediated trans-Golgi network recycling of PD-L1 and points to a promising treatment strategy for the enhancement of antitumor immunity.

Single-cell RNA sequencing reveals microenvironmental infiltration in non-small cell lung cancer with different responses to immunotherapy.

Immunotherapy represents a groundbreaking and monumental achievement in the field of cancer therapy, marking a significant advancement in fighting against this devastating disease. Lung cancer has showed consistent clinical improvements in response to immunotherapy treatments, yet, it is undeniable that challenges such as limited response rates acquire resistance, and the unclear fundamental mechanisms were inevitable problems. The cellular composition was defined and distinguished through single-cell RNA sequencing (scRNA-seq) analysis of MPR (major pathologic response) and NMPR (non-major pathologic response) samples in GSE207422, including four primary MPR samples and eight primary NMPR samples. We found obvious difference in CD8+ T cell population between MPR and NMPR samples, with high expression of TYMS, RRM2, and BIRC5 in NPMR samples. Meanwhile, the proportion of macrophages and tumor epithelial cells infiltration increased in the NMPR samples. We discovered biomarkers (ACTN4, ATF3, BRD2, CDKN1A, and CHMP4B) in epithelial cells which were potentially represented worse outcomes. By exploring the difference of tumor microenvironment (TME) in samples with different corresponding degrees of neoadjuvant immunotherapy, this research introduces a number of novel biomarkers for predicting the response of treatment and a theoretical basis for overcoming immunotherapy resistance.

Regulation of Tumor Microenvironment through YAP/TAZ under Tumor Hypoxia.

In solid tumors such as hepatocellular carcinoma (HCC), hypoxia is one of the important mechanisms of cancer development that closely influences cancer development, survival, and metastasis. The development of treatments for cancer was temporarily revolutionized by immunotherapy but continues to be constrained by limited response rates and the resistance and high costs required for the development of new and innovative strategies. In particular, solid tumors, including HCC, a multi-vascular tumor type, are sensitive to hypoxia and generate many blood vessels for metastasis and development, making it difficult to treat HCC, not only with immunotherapy but also with drugs targeting blood vessels. Therefore, in order to develop a treatment strategy for hypoxic tumors, various mechanisms must be explored and analyzed to treat these impregnable solid tumors. To date, tumor growth mechanisms linked to hypoxia are known to be complex and coexist with various signal pathways, but recently, mechanisms related to the Hippo signal pathway are emerging. Interestingly, Hippo YAP/TAZ, which appear during early tumor and normal tumor growth, and YAP/TAZ, which appear during hypoxia, help tumor growth and proliferation in different directions. Peculiarly, YAP/TAZ, which have different phosphorylation directions in the hypoxic environment of tumors, are involved in cancer proliferation and metastasis in various carcinomas, including HCC. Analyzing the mechanisms that regulate the function and expression of YAP in addition to HIF in the complex hypoxic environment of tumors may lead to a variety of anti-cancer strategies and combining HIF and YAP/TAZ may develop the potential to change the landscape of cancer treatment.

CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity.

Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic for intratumoral (IT) injection that co-delivers IL2 and IL12 on a single molecule in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of IL2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL12. LAIR2 and HSA function to retain CLN-617 in the treated tumor by binding collagen and increasing molecular weight, respectively. We found that IT administration of a murine surrogate of CLN-617, mCLN-617, eradicated established treated and untreated tumors in syngeneic models, significantly improved response to anti-PD1 checkpoint therapy, and generated a robust abscopal response dependent on cellular immunity and antigen cross-presentation. CLN-617 is being evaluated in a clinical trial in patients with advanced solid tumors (NCT06035744).

EBF1-COX4I2 signaling axis promotes a myofibroblast-like phenotype in cancer-associated fibroblasts (CAFs) and is associated with an immunosuppressive microenvironment

Immunotherapy has limited response rates in colorectal cancer (CRC) due to an immunosuppressive tumor microenvironment (TME). Combining transcriptome sequencing, clinical specimens, and functional experiments, we identified a unique group of CAF subpopulations (COX4I2 + ) with inhibited mitochondrial respiration and enhanced glycolysis. Through bioinformatics predictions and luciferase reporter assays, we determined that EBF1 can upstreamly regulate COX4I2 transcription. COX4I2 + CAFs functionally and phenotypically resemble myofibroblasts, are important for the formation of the fibrotic TME, and are capable of activating the M2 phenotype of macrophages. In vitro experiments demonstrated that COX4I2 + CAFs promote immunosuppressive TME by blocking CD8 + T cell infiltration and inducing CD8 + T cell dysfunction. Using multiple independent cohorts, we also found a strong correlation between the immunotherapy response rate of CRC patients and COX4I2 expression in their tumors. Our results identify a CAF subpopulation characterized by activation of the EBF1-COX4I2 axis, and this group of CAFs can be targeted to improve cancer immunotherapy outcomes.

Exploiting bacteria for cancer immunotherapy.

Immunotherapy has revolutionized the treatment of cancer but continues to be constrained by limited response rates, acquired resistance, toxicities and high costs, which necessitates the development of new, innovative strategies. The discovery of a connection between the human microbiota and cancer dates back 4,000 years, when local infection was observed to result in tumour eradication in some individuals. However, the true oncological relevance of the intratumoural microbiota was not recognized until the turn of the twentieth century. The intratumoural microbiota can have pivotal roles in both the pathogenesis and treatment of cancer. In particular, intratumoural bacteria can either promote or inhibit cancer growth via remodelling of the tumour microenvironment. Over the past two decades, remarkable progress has been made preclinically in engineering bacteria as agents for cancer immunotherapy; some of these bacterial products have successfully reached the clinical stages of development. In this Review, we discuss the characteristics of intratumoural bacteria and their intricate interactions with the tumour microenvironment. We also describe the many strategies used to engineer bacteria for use in the treatment of cancer, summarizing contemporary data from completed and ongoing clinical trials. The work described herein highlights the potential of bacteria to transform the landscape of cancer therapy, bridging ancient wisdom with modern scientific innovation.

Preliminary results from a phase 1 study of AC699, an orally bioavailable chimeric estrogen receptor degrader, in patients with advanced or metastatic breast cancer.

3074 Background: Endocrine therapy is a critical component of treatment for over 200,000 patients diagnosed yearly with estrogen receptor positive (ER+)/HER2- breast cancer. Although selective ER degraders (SERDs) have improved progression-free survival in this patient population, as monotherapy they have shown very limited overall response rates (ORR). AC699 is a novel chimeric degrader that binds, ubiquitinates and degrades ERα by bringing it into close proximity with an E3 ligase. This mechanism of action may result in greater specificity and more complete target blockade compared to SERDs. Initial results from the ongoing first-in-human dose escalation study of AC699 (NCT05654532) are presented here. Methods: The study employed a 3+3 design, with the option of adding backfill patients to each cohort cleared and deemed safe. Patients must have had locally advanced or metastatic ER+/HER2- breast cancer and received at least 2 prior lines of endocrine treatment, or at least 1 prior line if combined with a CDK4/6 inhibitor. AC699 was administered orally once daily in 28-day cycles. Tumor responses were assessed every 2 cycles using RECIST v1.1 criteria. The relationship between ESR1 mutational status and anti-tumor activity was investigated as an exploratory analysis. Results: A total of 22 patients had received AC699 in 3 dose cohorts (100, 200 and 300 mg) at the time of data cutoff. The median age was 61 years and the median lines of prior therapy was 5 (range 2-10). Prior treatments included CDK4/6 inhibitor (100%), aromatase inhibitor (91%), fulvestrant (82%), novel oral SERD or covalent antagonist (SERCA) (23%), and ER chimeric degrader (14%). Eighteen patients had measurable disease while 4 had bone lesions only. There were no dose-limiting toxicities, dose reductions or discontinuations for treatment-related AEs, and the maximum tolerated dose was not reached. The most common treatment-emergent AEs were fatigue (23%), dehydration (18%) and nausea (18%). All treatment-related AEs were Grade 1 or 2, including nausea (18%) and hot flushes (14%). Fifteen patients had at least 1 scan before the data cutoff date and of these, 3 were not evaluable for ORR due to having bone lesions only. Among the 12 evaluable patients, 4 (33%) achieved partial response: 3 confirmed and one unconfirmed. The clinical benefit rate (CBR) which also includes patients with stable disease ≥24 weeks was 42% (5/12). In the subgroup of evaluable patients harboring a confirmed ESR1 mutation, the ORR was 67% (4/6), the CBR was 83% (5/6) and the median time on treatment was 168 days (range 56-336), with 4 of 7 patients still receiving AC699. Conclusions: Preliminary data from the ongoing phase 1 trial evaluating AC699 indicate promising safety, tolerability, and anti-tumor activity, at doses up to 300 mg orally once daily. A phase 2 study will begin enrolling in early 2024. Clinical trial information: NCT05654532 .

Real-world treatment patterns and outcomes of patients with advanced melanoma treated with nivolumab plus relatimab.

e21501 Background: Programmed death-1 (PD-1) receptor inhibitors significantly increase survival rates for advanced melanoma, yet first-line PD-1 inhibitor therapy shows limited overall response rates (32.6%) and progression-free survival (4.6 months). Relatimab (RELA) was recently approved for the treatment of metastatic and unresectable stage III to IV melanoma in combination with nivolumab (NIVO). RELATIVITY-047 showed an ORR of 43% as a first-line treatment while RELATIVITY 020 showed an ORR of 9-12% in patients progressed on anti-PD-1 therapy. Although clinical trials have demonstrated NIVO-RELA provides benefit for patients with advanced melanoma, little is known about real-world treatment patterns and outcomes. Methods: We performed a retrospective review of all patients with stage III and IV melanoma treated with NIVO-RELA as first-(1L) or second-line or beyond (2L+) therapy at Inova Schar Cancer Institute between September 2021 to September 2023. Primary endpoints were overall response rate (ORR), complete response (CR), partial response (PR), stable disease (SD), and progression of disease (PD) which were evaluated using the RECIST v1.1 criteria. The Kaplan–Meier method was used to generate overall survival (OS) and progression free survival (PFS) median values. Results: Eighty-eight patients were followed for an average of 6.5 months following NIVO-RELA initiation. For 1L treated patients, ORR was 58% (21% PR, 37% CR). Median PFS was 11 months (95% CI 3.1-11). For 2L+ treated patients, ORR was 33% (14% PR, 19% CR). Median PFS was 9.8 months (95% CI 4.7-17.2). Subgroup analyses observed significantly different ORR between patients previously treated with versus without anti-CTLA4 therapy (29% v 44%; p = 0.017), patients treated with NIVO-RELA more than vs within 6 months from their last other therapy (50% v 27%; p = 0.004), and among patients with stage III vs stage IV disease (67% v 30%; p = 0.031) (Table 1). Conclusions: These real-world results support the use of NIVO + RELA in patients with advanced melanoma and emphasize the impressive efficacy of the drug combination in the IL and 2L+ settings compared to RELATIVITY-020 and 047. PFS and OS for 1L and 2L+ therapy with NIVO + RELA in this study aligned with or superposed results from previous trials. Further analyses with increased sample sizes and longer follow-up is warranted. [Table: see text]

Evaluation of the efficacy, safety, and pharmacokinetics of nanodispersible cannabidiol oral solution (150 mg/mL) versus placebo in mild to moderate anxiety subjects: A double blind multicenter randomized clinical trial

BackgroundAnxiety disorders, an increasingly prevalent global mental health illness, affected approximately 301 million individuals worldwide in 2019. There is an unmet need for the treatment of anxiety disorders, as current therapies are associated with limited response rates, residual symptoms, and adverse effects. ObjectivesTo evaluate the efficacy, safety, and pharmacokinetics of nanodispersible cannabidiol (CBD) oral solution versus placebo for the treatment of mild to moderate anxiety disorders. MethodsThis phase 3 prospective, randomized, double blind, parallel group, placebo-controlled, 15-week cohort study took place at multiple sites across India. Eligible participants were randomly assigned to one of the two treatment arms (CBD or placebo) in a 1:1 ratio. Results178 participants were randomized to receive CBD (n=89) or placebo (n=89). The study met both primary (GAD-7 and HAM-A scores) and secondary outcomes (CGI-I, CGI-S, PHQ-9 and PSQI scores). The GAD-7 score difference between the end of treatment and baseline for the CBD versus the placebo was −7.02 (S.E: 0.25, 95% CI −7.52; −6.52), p<0.0001. Similarly, the HAM-A score difference at the end of treatment compared to baseline for the CBD versus the placebo was −11.9 (S.E: 0.33, 95% CI −12.6; −11.3), p<0.0001. ConclusionsNanodispersible CBD was therapeutically safe with no serious adverse events, well tolerated, and effective for the treatment of mild to moderate anxiety disorders, as well as associated depression and sleep quality disturbances. These results pave way for probable prospective use of nanodispersible CBD formulation for various psychiatry disorders alone or in conjunction with other drugs.

Abstract LB273: Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes

Abstract Introduction: Inhibition of poly(ADP-ribose) polymerase (PARP) induces synthetic lethality in cells exhibiting defects in homologous repair (HR) pathways, such as BRCA1 and BRCA2-mutated breast or ovarian cancers, and 4 different PARP inhibitors (PARPi) are currently approved by the FDA. While HR-deficient cancers usually respond well to single agent PARPi, HR-proficient cancers are generally less sensitive to the treatment. Acquired resistance to PARPi due to activating mutations in BRCA genes are also not uncommon. In this work, we have examined strategies for enhancing PARPi activity in a HR-proficient context and identified histone deacetylases (HDACs) as targets for pharmacological synthetic lethality in the context of PARPi. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 were determined using CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by immunoblotting and immunofluorescence using gamma-H2AX antibodies, and comet assays for confirmation of double-strand breaks. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module. In vivo metastasis formations were examined using the pulmonary metastasis assay (PuMA). Results and discussion: Combinations of PARP and HDAC inhibitors in HR-proficient tumor cells demonstrated that HDACi could enhance the efficacy of PARPi in vitro. This was associated with increased acute and sustained DNA-damage distinct cell cycle profiles showing activity in both S- and G2/M-phases. Importantly, dual single-molecule PARP-HDAC inhibitors showed potent inhibition of PARP1 and PARP2 activity and PAR synthesis, comparable to olaparib, as well as inhibition of HDAC with IC50 values in the low µM range. Dual PARP-HDAC inhibition decreased survival of HR-proficient cells in both 2D cultures and 3D spheroid assays with lower EC50 values than olaparib or vorinostat alone. Importantly, lung metastasis formation was significantly impaired by dual PARP-HDAC inhibition. Conclusion: Combination of PARP inhibition and HDAC inhibition showed increased efficacy compared to single agent PARP inhibition in HR-proficient cells. Development of bifunctional inhibitors may provide a novel therapeutic opportunity for tumors with limited response rates to single agent PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Fariba Ghaidi, Mona Marzban, Hans Adomat, Xiaoqi Chen, John Langlands, Dennis Brown, Jeffrey Bacha, Colin Collins, Poul H. Sorensen, Wang Shen, Mads Daugaard. Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB273.

Abstract 5141: Autoantibody profiling for predictive biomarkers for immune-related adverse events and clinical benefit in rare tumors treated with anti-PD-1 therapy

Abstract Background: Immune checkpoint inhibitors (ICIs) have changed the cancer treatment paradigm; however, with high frequency of immune-related adverse events (irAEs) and limited response rates in select patients. This underscores the unmet need for the development of biomarkers predictive for the development of irAEs and ICI response. Herein, we conducted autoantibody (AAb) profiling to assess baseline (BL) AAb associations with clinical benefit (CB) and irAEs in patients (pts) with rare tumors (RT) treated with programmed cell death protein 1 (anti-PD-1) therapy. Methods: AAbs against 1168 antigens were analyzed using the SeroTag antibody discovery technology. The study included 41 pts with RT who received anti-PD-1 therapy. We conducted a Significance Analysis of Microarray (SAM) to analyze the association between BL AAbs with irAEs and CB (defined as pts achieving complete response (CR), partial response (PR), or stable disease (SD) for ≥4 months by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1). AAb biomaker candidates were discovered based on filtering for SAM hits with p &amp;lt; 0.05 and a delta score |D| &amp;gt;2. Results: Table 1 outlines patients’ demographics. CB and irAEs were observed in 34% and 39% of patients, respectively. Eighteen BL AAbs were significantly associated with CB such as DNA ligase III (LIG3) and small nuclear ribonucleoprotein polypeptide (SNRPA) (p = 0.002 and p = 0.003, respectively). Likewise, 13 BL AAbs showed significant associations with any-grade irAEs, particularly AAbs to breast cancer 2 (BRCA2) DNA repair associated antigen and chromatin-modifying protein 4A (CHMP4A) (p = 0.001 and p = 0.003, respectively). Conclusion: The predictive potential of autoantibodies as biomarkers is promising. Further evaluation in larger cohorts is needed for validation of our findings and understanding the underlying mechanism. Table 1. Patients’ demographics Total patients n=41 Median age in years (range) 66 (35-84) Male n=19 Female n=22 Tumor type Carcinoma of unknown primary n=13 Squamous cell carcinoma of the skin n=9 Small cell malignancies of non-pulmonary origin n=7 Adrenocortical carcinoma n=6 Vascular sarcoma n=5 Other rare histology n=1 Clinical benefit CR, PR, or SD ≥ 4 months n=14 SD &amp;lt;4 months or progressive disease (PD) n=23 Not evaluable n=4 irAEs Yes n=16 No n=25 Citation Format: Mohamed H. Derbala, Joud Hajjar, Bettzy Stephen, Serdar A. Gurses, Evan Kwiatkowski, Petra Budde, Hans-Dieter Zucht, Manuel Bräutigam, Ann-Sophie Schubert, Behnaz Ahangarianabhari, Enedelia Rodriguez, Mohamed Gouda, Lilibeth Castillo, Abdulrazzak Zarifa, Jeffrey A. How, Justin T. Moyers, David S. Hong, Funda Meric-Bernstam, Aung Naing. Autoantibody profiling for predictive biomarkers for immune-related adverse events and clinical benefit in rare tumors treated with anti-PD-1 therapy [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 5141.

Abstract 298: Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer

Abstract Patients with multifocal, locally advanced prostate cancer are at the highest risk of recurrence and death. Neoadjuvant therapies have limited response rates in this patient cohort and there remains a critical need to develop curative treatments. Prostate cancer (PC) shows marked heterogeneity, which is not limited to tumor cells but extends across immune and stromal compartments and has been linked to metastatic disease and therapeutic resistance. Cancer associated fibroblasts (CAFs) have been proposed to play a critical role in PC progression and invasion, but it is still unclear how CAFs interact with tumor cells. We evaluated CAFs from prostatectomy specimens as drivers of resistance to neoadjuvant androgen receptor signaling inhibitor (ARSi) and chemotherapy and assessed treatment response in co-culture systems in the context of CAF presence. We have utilized a novel radio-pathology tool that integrates PSMA PET/MRI scans to identify regions of interest that associate with heterogenous treatment response. MRI scans were utilized to print patient-specific 3D molds to micro dissect live tissue for subsequent cell sorting and molecular analyses. We have established a multi-parameter flow cytometry panel to characterize primary prostate fibroblasts from tumor foci and adjacent normal prostate tissue. In addition, co-culture assays were performed including immortalized CAF (hPrCSC-44) and MSC-derived fibroblast. To examine the tumor-promoting role of CAFs, fibroblasts were co-cultured with androgen-sensitive 3D tumor PC spheroids (LNCaP, C42B and LAPC4) in the integrated microfluidic STACKs platform that allows for the assessment of spatio-temporal interaction in a variety of culture microenvironments to model complex interactions. Cytotoxicity in 3D PC spheroids was assessed after Apalutamide, Darolutamide, or Docetaxel treatment by confocal microscopy. Integrated DNA, RNA, and radiology analysis of tumor biopsies have identified gene signatures associated with early biochemical recurrence. Most biopsies had low tumor purity, evidence of the cytotoxic impact of chemohormonal therapy while high tumor purity predicted high tumor expression. Poor clinical outcome was significantly associated with elevated stromal scores while epithelial and stromal enrichment were inversely correlated (rho= -0.80, p&amp;lt; 5 × 10−16). The presence of CAFs significantly decreased Docetaxel (C42B only vs C42B+CAF, 86.07% vs. 44.57%, respectively, p&amp;lt;0.001; LAPC4 only vs LAPC4+CAF, 70.98% vs 26.64%, respectively, p&amp;lt;0.001) and Darolutamide-induced (C42B only vs C42B+CAF, 52.59% vs 37.87%, respectively, p=0.0407). In conclusion, recurrent PC is linked to increased stromal content and the presence of CAFs supported tumor survival in response to ARSi and Docetaxel treatment. The molecular drivers of this phenomenon are currently being investigated. Citation Format: Nikolett Lupsa, Erika Heninger, Adeline Ding, Shannon R. Reese, Xavier T. Hazelberg, Aaron M. LeBeau, Brian P. Johnson, Peter P. Geiger, David J. Beebe, David A. Quigley, Joshua M. Lang. Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate 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 298.

Abstract 5936: A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models

Abstract The introduction of targeted immunomodulating agents has transformed cancer treatment over the last decade by demonstrating unprecedented efficacy in patients who respond. However, limited clinical response rates as well as adverse events underline the need to identify additional novel modalities in cancer immunotherapy. Here we report on the discovery of a novel class of low molecular weight compounds for oral application that selectively enhance tumor-reactive T cell cytotoxicity. Hit-to-lead development of compound hit series A was performed based on medicinal chemistry to investigate structure-activity-relations. A-306, a representative of the A compound class, induced increased T cell proliferation, elevated levels of the activation markers CD25 and CD69 as well as enhanced secretion of IFN-γ, IL-2 and TNF-α upon anti-CD3/CD28 stimulation. A-306 treatment without T cell receptor ligation had no effect on T cell activation, suggesting selective activation potential only in the context of antigen encounter. Furthermore, A-306 strengthened T-cell mediated killing of M21 melanoma cells and enhanced T cell function in response to viral antigens by showing a Th1 signature. Medicinal chemistry efforts resulted in front runner compounds A-481 and A-687 that show a similar profile and potencies down to nanomolar level. In a murine B16-SIY melanoma and EO771 breast cancer model oral single-agent administration with front runner compounds was well tolerated, showed good bioavailability in lymphoid organs and plasma and resulted in significant tumor growth inhibition beyond the end of treatment (D21). T cells from tumor draining lymph nodes of long-term surviving mice in both cancer models showed a distinct activation pattern indicative of anti-tumor immunity. Furthermore, oral application of A-481 in combination with intra peritoneal administration of anti-PD1 antibodies eradicated B16-SIY melanoma cells in a synergistic manner. Taken together, we report on the identification of a novel class of small molecules possessing high potential for selective anti-tumor activation of the immune system upon oral administration. Front runner compounds A-481 and A-687, which significantly inhibit tumor growth in a B16-SIY melanoma and EO771 breast cancer mouse model, lead to prolonged survival beyond the end of treatment and display a good safety profile. The final candidate compound will undergo testing in toxicity studies and enter a first-in-human trial in selected solid tumors in 2025. Citation Format: Bianca Gapp, Gunther Zischinsky, Maria Urban, Nora Birnbacher, Alice Schwarzböck, Sabine Grünaug, Tommaso Gastaldi, Anton Stütz, Peter Nussbaumer, Stefan Stranner, Julia Buchberger, Andreas Birbach, Daniela Schögl, Mario Kuttke, Alexander Dohnal, Romana Gugenberger. A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse 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 5936.

Abstract 5132: Multi-omics analysis uncovers predictive biomarkers for the efficacy and outcomes of immune checkpoint inhibitor in combination with chemotherapy in advanced unresectable biliary tract cancers

Abstract Background: Biliary tract cancer, typically diagnosed at advanced stages with a poor prognosis, has witnessed a glimmer of hope in the progress of immune checkpoint inhibitors (ICIs). Yet, their limited response rates necessitate the search for effective biomarkers to refine patient selection. Methods: A total of 125 patients with a confirmed histological diagnosis of unresectable advanced or metastatic biliary tract cancers (BTC) who received first-line ICIs in combination with chemotherapy (chemoimmunotherapy) were prospectively enrolled. All baseline samples from 125 patients underwent targeted DNA sequencing, with an additional 62 patients undergoing RNA sequencing, and 85 patients had accessible mIHC data. The associations between molecular characteristics and the response to chemoimmunotherapy, progression-free survival (PFS) and overall survival (OS) were evaluated. Results: The cohort had a median age of 63 years (range from 34 to 82) and 52.8% (66/125) were male, including 54 with gallbladder cancer, 57 with intrahepatic cholangiocarcinoma, and 14 with extrahepatic cholangiocarcinoma. The median duration of follow-up was 14.8 months for the entire cohort, with the median PFS and OS of 6.9 months (95%CI: 6.2-7.9) and 11.8 months (95%CI: 10.3-14.8), respectively. The most mutated genes were TP53 (64/125, 51.2%), KRAS (34/125, 27.2%), ERBB2 (18/125, 14.4%), and ARID1A (17/125, 13.6%). Mutations of TP53 (51.2%, p = 0.042), BRCA2 (4.8%, p = 0.002), cytokine genes (6.4%, p = 0.004), and high tumor mutation burden (p = 0.072) demonstrated significant correlation with chemoimmunotherapy response. KRAS G12D mutations (PFS: P &amp;lt; 0.001; OS: P = 0.034) and ARID1A loss-of-function mutations (PFS: P = 0.009; OS: P = 0.012) were adverse survival factors, while high CXCL9 or CTLA4 expression was associated with response (CXCL9, P = 0.014; CTLA4, P = 0.067), improved PFS (CXCL9, P = 0.018; CTLA4, P = 0.008), and longer OS (CXCL9, P = 0.010; CTLA4, P = 0.008) under chemoimmunotherapy. Patients were classified into three subtypes using the identified survival biomarkers. Among them, Type I patients, characterized by the absence of KRAS G12D or ARID1A mutations but expressing high levels of CTLA4 or CXCL9, demonstrated the best outcomes under chemoimmunotherapy. Interestingly, further RNA analysis suggested that elevated CXCL9 expression correlated with heightened immune checkpoint expressions, including CTLA4, PD-L1, and PD-1 (all P &amp;lt; 0.001), as well as increased tumor microenvironment immune activity, findings validated in two additional independent patient cohorts both internal and external. Conclusions: Our study revealed predictive biomarkers relevant to the response and efficacy of immune checkpoint inhibitors in combination with chemotherapy in advanced biliary tract cancer. Citation Format: Jieer Ying, Qi Xu, Jiaojiao Ni, Hanlin Chen, Chaoqun Li, Yanru Xie, Qinhong Zheng, Jianying Jin, Junrong Yan, Xiaoying Wu, Qiuxiang Ou, Li Yuan, Wei Zhuo, Haimeng Tang. Multi-omics analysis uncovers predictive biomarkers for the efficacy and outcomes of immune checkpoint inhibitor in combination with chemotherapy in advanced unresectable biliary tract cancers [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 5132.

Spiky metal-organic framework nanosystem for enhanced cuproptosis-mediated cancer immunotherapy

Immunotherapy, particularly αPD-1 therapy, has been hailed as a significant breakthrough in cancer treatment. However, the limited response rates and unknown mechanisms hinder the clinical application of αPD-1 blockade boosting with hybrid nanoparticles. Herein, we introduce a strategy to enhance cancer immunotherapy by leveraging cuproptosis, initiated by the spiky copper-based metal-organic framework (S@Cu-MOF) loaded with polyphyllin I (PPI) and synergized with αPD-1 therapy. Interestingly, the unique spiky structure of S@Cu-MOF not only significantly enhances tumor cell uptake but also leads to pronounced DNA damage. The application of S@Cu-MOF/PPI with αPD-1 therapy led to obvious tumor regression and prolonged survival due to cuproptosis, which involved substantial mitochondrial damage. This process amplifies the cGAS/STING immune pathway, enhancing the immune response against cancer. Additionally, this strategy promoted immunogenic cell death (ICD), recruitment of mature dendritic cells (DCs), T cell-mediated immunity, and effective memory CD8+ T cells, resulting in significant abscopal tumor suppression and lung metastasis reduction. Our study highlights the potential of S@Cu-MOF/PPI as a sensitizer for αPD-1 with the mechanism of cuproptosis and the cGAS/STING pathway, offering a promising strategy for the next generation of immunotherapy.

BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma

Interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells allows tumor cells to evade T cell-mediated immune surveillance. Strategies targeting PD-1/PD-L1 have shown clinical benefits in a variety of cancers. However, limited response rates in hepatocellular carcinoma (HCC) have prompted us to investigate the molecular regulation of PD-L1. Here, we identify B cell lymphoma-2-associated transcription factor 1 (BCLAF1) as a key PD-L1 regulator in HCC. Specifically, BCLAF1 interacts with SPOP, an E3 ligase that mediates the ubiquitination and degradation of PD-L1, thereby competitively inhibiting SPOP-PD-L1 interaction and subsequent ubiquitination and degradation of PD-L1. Furthermore, we determined an SPOP-binding consensus (SBC) motif mediating the BCLAF1-SPOP interaction on BCLAF1 protein and mutation of BCLAF1-SBC motif disrupts the regulation of the SPOP-PD-L1 axis. In addition, BCLAF1 expression was positively correlated with PD-L1 expression and negatively correlated with biomarkers of T cell activation, including CD3 and CD8, as well as with the level of immune cell infiltration in HCC tissues. Besides, BCLAF1 depletion leads to a significant reduction of PD-L1 expression in vitro, and this reduction of PD-L1 promoted T cell-mediated cytotoxicity. Notably, overexpression of BCLAF1 sensitized tumor cells to checkpoint therapy in an in vitro HCC cells-Jurkat cells co-culture model, whereas BCLAF1-SBC mutant decreased tumor cell sensitivity to checkpoint therapy, suggesting that BCLAF1 and its SBC motif serve as a novel therapeutic target for enhancing anti-tumor immunity in HCC.

(080) Outcomes After Isolated Epididymal Pain

Abstract Introduction Chronic epididymitis imposes significant physical and psychosocial distress on affected patients. Despite being a commonly encountered urologic condition, there remains a paucity of understanding and literature surrounding the management and natural history of isolated epididymal pain. Typically, patients who do not respond to conservative management undergo an epididymectomy. However, the literature on its efficacy is also scarce, with success rates varying widely from 10-90% in existing studies. Objective To better describe the etiology, pathogenesis and natural history of isolated epididymal pain. Furthermore, we aimed to describe the rates of success associated with epididymectomy. Methods A retrospective cohort study was conducted at the Manitoba Men’s Health Clinic, with approved by the University of Manitoba REB. All patients presenting with chronic epididymitis, defined as discomfort or pain localized to the epididymis for at least three months, were identified. Information regarding patient demographics, past medical and surgical history, duration of pain, localization of pain, findings on previous ultrasounds, STI testing results, prior conservative therapies trialed and response rates, as well as response rates to surgical therapy were collected. Results From April 2022 to 2023, a total of 274 patients with chronic orchialgia were identified, and among them, 74 patients specifically presented with chronic isolated epididymal pain. The average duration of symptoms was as follows: 21.6% (n=16) of patients experienced symptoms for 3-6 months, 9.5% (n=7) for 6-12 months, and 63.5% (n=47) for over 12 months. 13.5% (n=10) had associated ejaculatory pain, 8.1% (n=6) had lower urinary tract symptoms, and 4.1% (n=3) had erectile dysfunction. Ultrasound findings were observed in 68.9% of patients, with 31.1% having an epididymal cyst, 27.1% having a varicocele, 5.4% having a spermatocele, and 4.1% having a hydrocele. 49.4% (n=40) did not undergo any intervention prior to referral. After referral, 58.8% (n=43) of patients had undergone antibiotic treatment, 12.2% (n=9) had tried an NSAID, and one patient was referred for pelvic floor physical therapy. Among those who underwent conservative therapy, only 36.2% of patients reported a positive response. Surgical intervention was performed on 23 patients, including 16 who underwent an epididymectomy, three who underwent cord denervation, and two who underwent vasovasostomy and spermatocelectomy each. 81.3% of patients (n=13) who underwent an epididymectomy had a positive response to the surgical intervention, defined as satisfactory improvement of pain, while all patients undergoing other surgical interventions experienced a positive response. Conclusions Chronic epididymal pain is a condition with limited data surrounding its management. Prior to referral, a large proportion of patients did not undergo any conservative treatment, and of those that did, there was limited response rates. For those who underwent surgical intervention, all were pain free on follow up, except three patients who underwent and epididymectomy. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Boston Scientific.