Advanced Melanoma Patients Research Articles

Abstract 1253: Dietary tryptophan catabolite released by intratumoralLactobacillus reuterifacilitates aPD-L1 therapy

Abstract The use of probiotics by cancer patients is increasing, including amongst those undergoing immune checkpoint inhibitor (ICI) therapy. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances spontaneous antitumor immunity and facilitates ICI therapy in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (L.reuteri) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby facilitating ICI therapy efficacy. Moreover, L.reuteri secreted I3A was both necessary and sufficient for antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated L.reuteri’s ability to restrain melanoma growth. Further, a tryptophan-enriched diet potentiated both L.reuteri- and ICI-induced antitumor immunity. Finally, we provide evidence for a potential role of I3A in promoting ICI therapy efficacy and survival in advanced melanoma patients. Citation Format: Mackenzie Bender, Alex McPherson, Catherine Phelps, Diwakar Davar, Dario A. Vignali, Hassane Zarour, Marlies Meisel. Dietary tryptophan catabolite released by intratumoralLactobacillus reuterifacilitates aPD-L1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1253.

Abstract 3275: Combination of talimogene laherparepvec (T-VEC) with pembrolizumab (pembro) in advanced melanoma patients following progression on a prior PD-1 inhibitor: SWOG S1607

Abstract We hypothesize that a significant number of patients do not respond to PD-1/L1 blockade because there are no pre-existing tumor antigen-specific T-cells, and this can be addressed by combination therapy with an oncolytic virus such as T-VEC. S1607 is a single arm Phase 2 study of T-VEC plus pembro in patients with advanced melanoma after PD-1/L1 inhibitor progression. The primary endpoint is ORR by modified RECIST (progression at the first follow-up disease assessment had to be confirmed). Secondary endpoints include durable response rate (response ≥ 6 months), ORR in injected, non-visceral non-injected, and visceral lesions, PFS, OS and toxicity. In Cohort A patients must have at least one measurable visceral lesion; in Cohort B patients must not have any visceral lesions. Each cohort had an independent accrual goal with a 2-stage design. All received intratumoral T-VEC and pembro 200mg IV every 21 days. Tumor biopsy and research blood are taken at baseline and on Day 28 (both injected and non-injected lesions). Tumor assessments are performed every 12 weeks for up to 2 years. 38 evaluable patients were enrolled. As of July 26, 2022, the median follow up was 28 months. Treatment was well tolerated, with 5/38 (13%) grade 3 AE (no grade 4/5) including injection site reactions, lymphocyte count decrease, and hypoxia. Cohort A was closed after stage I (n=11) with no confirmed responses. In Cohort B (n=27), there were 7 confirmed responses (26%; 2 CR, 5 PR; this rejected H0: ORR = 10%, p=0.01). Clinical outcomes are summarized in Table 1. Baseline tumor mutational burden from 17 patients in Cohort B were not different between responder vs non-responders (p=0.96). Translational study is ongoing for pharmacodynamic confirmation. T-VEC plus pembro in melanoma patients who have progressed on prior anti-PD1/L1 therapy has efficacy in the subset of melanoma patients who have non-visceral metastases. Table 1 Cohort A (Visceral) Cohort B(Non-Visceral) N (%; 95% CI) 11 27 Confirmed PR + CR 0 (0%; 0%-28%) 7 (26%; 11%-46%) Confirmed + Unconfirmed 1 (9%; 0%-41%) 9 (33%; 17%-54%) Durable response 0 (0%; 0%-28%) 4 (15%; 4%-34%) Median PFS in months 2.1 (0.7-5.5) 2.3 (1.9-6.2) INJECTED LESIONS 11 27 Confirmed PR + CR 0 (0%; 0%-28%) 6 (22%; 9%-42%) Confirmed + Unconfirmed, PR + CR 1 (9%; 0%-41%) 8 (30%; 14%-50%) NON-INJECTED, NON-VISCERAL LESIONS 8 19 Confirmed PR + CR 0 (0%; 0%-37%) 3 (16%; 3%-40%) Confirmed + Unconfirmed, PR + CR 0 (0%; 0%-37%) 5 (26%; 9%-51%) VISCERAL LESIONS 11 Confirmed PR + CR 0 (0%; 0%-28%) Confirmed + Unconfirmed, PR + CR 1 (9%; 0%-41%) ACQUIRED RESISTANCE 3 2 Confirmed PR + CR 0 (0%; 0%-71%) 2 (100%; 16%-100%) Confirmed + Unconfirmed, PR + CR 0 (0%; 0%-71%) 2 (100%; 16%-100%) Median PFS in months 2.1 (2.0-4.1) NR (8.0-∞) PRIMARY RESISTANCE 8 25 Confirmed PR + CR 0 (0%; 0%-37%) 5 (20%; 7%-41%) Confirmed + Unconfirmed, PR + CR 1 (13%; 0%-53%) 7 (28%; 12%-49%) Median PFS in months 1.8 (0.3-6.2) 2.1 (1.9-3.3) Citation Format: Siwen Hu-Lieskovan, James Moon, John Hyngstrom, Katie M. Campbell, Gino K. In, Theodore F. Logan, Kari L. Kendra, Ding M. Wang, Douglas B. Johnson, Gary C. Doolittle, Alan Tan, Ann W. Silk, Kenneth F. Grossmann, Christopher W. Ryan, Sapna P. Patel, Shay Bellasea, Michael C. Wu, John M. Kirkwood, Helen X. Chen, Antoni Ribas. Combination of talimogene laherparepvec (T-VEC) with pembrolizumab (pembro) in advanced melanoma patients following progression on a prior PD-1 inhibitor: SWOG S1607 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3275.

Abstract 1851: Mechanisms underlying focused ultrasound-induced immunogenicity in melanoma

Abstract Advanced melanoma is an aggressive skin cancer characterized by low survival rates. The generation of a robust anti-tumor response relies on dendritic cells (DCs) to process tumor antigens, migrate to draining lymph nodes, and activate T-cells. Within the tumor microenvironment, immunosuppressive factors impede DC differentiation and activation, and the subsequent immature DC phenotype is a major mechanism underlying the ineffective T-cell response in melanoma patients. We have shown that low-intensity focused ultrasound (LOFU), a novel therapy that delivers mechanical and thermal stresses locally at the tumor site, may enhance DC function in a B16 murine melanoma model. We hypothesize that LOFU enhances the dendritic cell-mediated anti-tumor response by inducing changes in tumor cell and dendritic cell autophagy and increasing pro-immunogenic factors in the TME. Autophagy is a lysosome-dependent process of cellular content degradation that has previously been implicated in the regulation of multivesicular body formation/degradation, and thus in the secretion of extracellular vesicles. Autophagy may subsequently modify the cargo of tumor-derived extracellular vesicles, such as exosomes, in a pro-immunogenic manner. We have found that LOFU treatment is associated with an increase in autophagic flux in melanoma cells and have identified at least 82 proteins within melanoma-derived extracellular vesicles that are significantly enriched or reduced with LOFU treatment, with enriched proteins partaking in essential processes including antigen processing, intracellular trafficking, and cellular metabolism. DCs pulsed with vesicles from LOFU-treated melanoma cells have shown enhanced activation of both CD4+ and CD8+ T-cell activation, suggesting that the LOFU-induced increase in tumor cell autophagy and modification of vesicular cargo may promote a DC-activated anti-tumor antigen activation response. Characterization of other mechanisms by which LOFU may enhance DC function, such as direct changes in DC autophagy, is critical to elucidate DC-mediated pathways involved in the anti-tumor response. Further understanding of DC involvement in the anti-tumor response will provide novel targets for the development of immunotherapies to improve survival and quality of life in advanced melanoma patients. Citation Format: Sandra Pelka, Sanmay Bandyopadhyay, Sanjay Pandey, Chandan Guha, Fernando Macian. Mechanisms underlying focused ultrasound-induced immunogenicity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1851.

Abstract 2283: An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients

Abstract The development of antibodies to block immune checkpoints like PD-1, CTLA-4, PD-L1 or LAG-3 has been a major advance in cancer therapy. This has led to the approval of multiple antibodies for metastatic melanoma and other malignancies. A large proportion of the patients do not benefit from CTLA-4 or PD-1 antagonistic antibodies. Understanding the mechanisms of resistance to immunotherapy is thus critical to improve its effectiveness. Previously we identified and validated a novel population of cells characterized by phenotype CD3+CD4+CD127-GARP-CD38+CD39+ that was associated with non-responsiveness to immune checkpoint blockade (ICB) in metastatic melanoma patients (Woods et al, 2020). CD38 and CD39 are ectonucleotidases, that convert intracellular ATP or NADPH to extracellular adenosine which has suppressive effect on T cell functions. We used high-dimension flow cytometry and bulk RNA seq to characterize the phenotype of these cells and evaluated the functions by T cell suppression assays. All characterizations and functional experiments were performed on peripheral blood samples from metastatic melanoma patients undergoing anti-PD1 therapy. The characterization of this population by flow cytomtery shows an enhanced expression of activation markers (4-1BB, HLA-DR, OX-40, GITR), inhibitory receptors (Tim3, CTLA-4, PD-1) and ligands (PD-L1, CD244) and an exhausted phenotype (TOXhiTCF1low). Further functional studies performed on these cells showed a suppressive phenotype, as observed by a significant decrease in the proliferation of target autologous CD4 (p=0.001) and CD8 (p=0.0012) T cells when in co-culture with these cells, compared to the controls. The transcriptomic features of these cells showed an upregulation of genes associated with proliferation (e.g. CDC20, TK1, DHFR, MYL6B), as well as exhaustion (e.g. upregulated TOX), consistent with the flow cytometry data. We also evaluated the presence of these cells and their correlation with the outcome in other melanoma datasets. By performing gene set variation analysis (GSVA) on existing single-cell RNA seq datasets of tumor derived immune cells in melanoma, we have found that an intra-tumoral CD3+CD4+CD127-GARP-CD38+CD39+ signature is associated with resistance to immunotherapy (p= 0.000003). We identified a novel population of ectonucleotidase expressing T cells in peripheral blood and tumor to be associated with immunotherapy resistance in metastatic melanoma patients. This population showed an exhausted phenotype and suppressed autologous T-cells in vitro. Our data identifies CD3+CD4+CD127-GARP-CD38+CD39+ cells as a potential target to enhance the efficacy of immunotherapy in advanced melanoma patients. Citation Format: Ankita Mitra, Brian Thompson, David M. Woods, Jeffrey S. Weber. An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2283.

Abstract 4444: Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy

Abstract Hyperactivation of the PI3K pathway has been reported to correlate with resistance to immune checkpoint blockade therapy (ICB) in melanoma, highlighting the therapeutic potential of combining PI3K inhibition (PI3Ki) with ICB. To maximize the clinical benefit of PI3Ki-based immune oncology (IO) combination, we characterized the role of PI3K isoforms in tumor and T cells and determined the immunological impacts of PI3Ki alone or in combination with ICB. Inhibitions of PI3K were achieved by either genetic knockdown (KD) or the bioactive compound in PTEN-present (B16/MC38), PTEN-absent (BP/D4M) tumor cell lines, and CD8+ T cells (Pmel-1). Following PI3Ki, we determined the activation status of the PI3K pathway (p-AKT level), transcriptional profile, and cellular function of these cells. We found both in vitro KD and pharmacological inhibition of either PI3Kα or PI3Kβ displayed a dramatic reduction of the PI3K pathway in tumor cells but moderate or no reduction in T cells, whereas the PI3K pathway significantly decreased in T cells with PI3Kγ or PI3Kδ inhibition. KD of PI3Kα or β isoforms drastically sensitized both D4M and MC38 tumors to αPD1 in vivo. We also observed that only PI3Kγ or PI3Kδ inhibition profoundly suppressed cytokine production and cytotoxicity of CD8+ T cell, suggesting that PI3Kα or PI3Kβ isoform inhibition can achieve tumor specific PI3Ki with limited impacts on T cell function. Furthermore, we used multiple syngeneic melanoma models to determine whether PI3K isoform inhibition can synergize the antitumor activity of ICB in vivo. In PTEN-present tumors, BYL719 (BYL, a PI3Kα inhibitor) synergized with αPD1 to delay tumor growth and extend survival (median survival of MC38-bearing mice in control (Ctrl), BYL, αPD1, and combination (Comb) groups: 30, 36, 33, and >45 respectively; p<0.05: Ctrl/BYL/αPD1 vs Comb). However, a limited combinatorial effect between GSK2636771(a PI3Kβ inhibitor) and αPD1 was observed in PTEN-present tumor models. Moreover, the combination of BYL and αPD1 exhibits superior antitumor activity in a spontaneous Braf-mutant, PTEN-loss melanoma model when compared with either reagent. Mechanistically, the combination of BYL and αPD1 improved CD8+ T cells tumor infiltration (14 days treatment, mean CD8+ number/mg of the tumor, Ctrl:1392.9, BYL:2073.9, αPD1:1545.2, Comb:4691.8; p<0.01: Ctrl/BYL/αPD1 vs Comb) and reduced MDSCs in MC38 tumors (p<0.05: Ctrl vs Comb). Multi-omics profiling of tumor cells with in vitro and in vivo PI3K isoform inhibition is ongoing. Collectively, our results demonstrate that PI3Kα inhibitor can potentiate T cell-mediated antitumor immune responses regardless of PTEN status, providing a strong rationale for the clinical development of the BYL-based IO combination. In collaboration with Novartis, MD Anderson Cancer Center will launch a Phase I/II trial of the FDA-approved BYL in combination with αPD1 in advanced melanoma and breast cancer patients. Citation Format: Ritu Bohat, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, Ashley Guerrero, Roshni Jaffery, Elizabeth M. Burton, Han Liang, Hussein Tawbi, Michael A. Davies, Weiyi Peng. Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4444.

Abstract 4997: PLK1 promotes the metastasis and drug resistance in melanoma

Abstract Melanoma is one of the most frequently diagnosed cancers in the caucasian population of both genders. The survival rate of advanced melanoma patients will be exceedingly poor, as low as 27.3%. BRAF-V600E is the most common mutation found clinically, resulting in the constitutive activation of the MAPK signaling pathway. Vemurafenib, a specific inhibitor of mutant BRAF, has shown an impressive response in phase 3 clinical trials and was approved by FDA in 2011. However, due to the rapid development of resistance, the duration of response under a single treatment is frequently short, highlighting the urgent requirement for novel therapy in melanoma treatment. Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, participates in multiple mitotic processes, including centrosome maturation, mitotic entry, spindle assembly, sister chromatid segregation, mitotic exit, and cytokinesis. Compared to normal tissues, the expression level of PLK1 significantly upregulates in multiple cancers. More importantly, the expression level of PLK1 negatively correlates with the melanoma patients’ survival rate based on the TCGA database. Besides, PLK1 is identified as an oncogene to promote proliferation, motility, and resistance to various drugs. In the present study, we found that overexpression of PLK1 promotes tumor growth, metastasis, metabolic reprogramming, and vemurafenib resistance in the BrafCA/Ptenloxp mouse melanoma model. Through phosphorylation, overexpressed PLK1 stabilizes BACH1, a critical transcriptional factor of multiple metastatic and metabolic genes, and protects it from degradation. Subsequently, a higher level of BACH1 leads to increasing metastasis, upregulation of glycolysis, and suppression of OXPHOs. Besides, we have validated the strong synergy between PLK1 inhibitor volasertib and vemurafenib in treating melanoma carrying BRAF-V600E. Our in vitro and in vivo experiments have shown an improved efficacy of this combined therapy on inhibition of cell proliferation, induction of cell death, and suppression of cell metastasis compared to mono-treatment. In short, our data has indicated PLK1 as a potent and promising target in cancer treatment. Citation Format: Fengyi Mao, Yifan Kong, Chaohao Li, Yanquan Zhang, Xiongjian Rao, Qiongsi Zhang, Xinyi Wang, Zhiguo Li, Xiaoqi Liu. PLK1 promotes the metastasis and drug resistance in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4997.

Abstract 1361: Astrocytes activate neuronal programming in melanoma brain metastases

Abstract Melanoma Brain Metastases (MBM) pose a major challenge for treating metastatic melanoma. Up to 70% of advanced melanoma patients develop MBM, resulting in significant morbidity and mortality. Melanocytes, the originating cell type of melanomas, are derived from neural crest cells in development. Melanoma’s proclivity to metastasize to the brain has been anecdotally connected to its neural crest origins. Recently, transcriptomic analyses have demonstrated that MBM express a neuronal-like phenotype, upregulating neural crest factors such as NCAM (Neural cell adhesion molecule), L1CAM (L1 cell adhesion molecule protein), while expressing melanocytic markers like MITF (melanocyte induction transcription factor). The activation of neuronal development programming may be a key mediator in brain-metastatic growth, and thus necessitates a better understanding of how melanoma cells adapt to the brain. Astrocytes have emerged as the major host cell type that cancer cells encounter and interact with during brain metastasis formation. Typically, astrocytes regulate brain homeostasis and support neuronal function. Their response to injury, termed reactive astrocytosis, is a process defined by increased secretion of growth factors and inflammatory cytokine production. Post-mortem analyses of MBM patients reveal perilesional reactive astrocytosis. Here we demonstrate that astrocyte reactivity promotes MBM growth, using in vitro and in vivo model systems. We identify that astrocytes secrete factors that increase tumor growth and expression of neural development factors. Moreover, pharmacological inhibition of neural development pathways, like Wnt signaling, attenuate the effects of astrocyte secreted factors. We developed a brain adapted melanoma line, D4M3a BrJ, and demonstrate the time course of astrocyte reactivity in vivo in its relationship with tumor growth. Our findings provide evidence that astrocyte reactivity promotes MBM growth and activation of neural development programming, unveiling a new therapeutic avenue for targeting the brain metastatic niche. Citation Format: Julia K. Farnan, Maria R. Cavallo, Edward J. Hartsough, Joshua G. Jackson. Astrocytes activate neuronal programming in melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1361.

Abstract 5701: Predictive biomarker models of immunotherapy response in patients with metastatic melanoma: genomic, transcriptomic, and immune profiles from the Personalised Immunotherapy Program (PIP)

Abstract Background: While immune checkpoint inhibitors (ICI) have become the standard-of-care for advanced melanoma patients, only half of treated patients will survive beyond 5-years and many develop significant toxicity. The PIP study is combining pre-treatment clinical, molecular, and immunological profiles of melanoma patients to provide accurate prediction of response to ICI therapies. Methods: 504 patients with advanced melanoma who received anti-PD-1±CTLA-4 ICI were studied to develop predictive models of resistance to ICI. Resistance was defined as patients with progressive disease as best response, or partial response/stable disease with <6 months progression-free survival (RECIST 1.1). Machine learning models were developed using clinicopathological characteristics, tumor mutation burden (TMB, Qiaseq TMB IO), gene expression profiling ((GEP), Nanostring Pancancer 360 IO) and spatial quantitative pathology immune profiling (multiplex immunofluorescence, (MIF)) of baseline melanoma biopsies. Models were developed in a discovery cohort (n=247), validated in an independent cohort (n=97), with an accruing prospective cohort (n=160). Model predictive performance was assessed using the area under the curve (AUC). Results: Models were developed and validated using sequential addition of omics features to relevant clinical factors. Baseline clinical data alone achieved an AUC of 68%. Clinical plus three-tier TMB (<10, 10-20, >20 mut/mb) achieved an AUC of 78%. Clinical data plus GEP achieved 79% AUC, inclusive of tumor inflammation, antigen presentation and T-cell related signatures. Clinical and MIF spatial pathology achieved an 82% AUC including the distances between T-cells, CD16+ cells and melanoma cells as features in the model. Finally, the combination of TMB and GEP achieved an 83% AUC. Prospective validation of the models is awaiting follow-up milestones. Conclusion: Multi-omic models using pre-treatment tissue and clinicopathology can significantly improve the accuracy of predicting patient outcomes to ICI treatments compared to baseline clinical data alone. Several models may be required based on different omic combinations to account for the reality of biopsy suitability and assay failures. These findings prove personalized precision treatment of patients with immunotherapies is possible in the clinical setting and such approaches should become routine care. Citation Format: Tuba N. Gide, Nurudeen A. Adegoke, Yizhe Mao, Monica Lennox, Saurab Raj, Camelia Quek, Ismael A. Vergara, Nigel Maher, Alison Potter, Robyn P. Saw, John F. Thompson, Andrew J. Spillane, Kerwin F. Shannon, Matteo S. Carlino, Maria Gonzalez, Serigne N. Lo, Alexander M. Menzies, Inês Pires da Silva, Richard A. Scolyer, Georgina V. Long, James S. Wilmott. Predictive biomarker models of immunotherapy response in patients with metastatic melanoma: genomic, transcriptomic, and immune profiles from the Personalised Immunotherapy Program (PIP). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5701.

Abstract 5188: Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models

Abstract Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5188.

Abstract 5864: An angiogenic target of immune exclusion with checkpoint blockade in melanoma

Abstract With the development of immune checkpoint blockade to treat a variety of malignancies, better predictive biomarkers as well as new targets to further improve anti-tumor immunity remains a need. Our understanding of the role of angiogenesis in immune regulation continues to improve, and combinations of anti-angiogenesis and immune checkpoint blockade demonstrate improved clinical efficacy, as well as mechanisms for improved immune cell trafficking. Tumor biopsies from a phase I study with 46 advanced melanoma patients treated with Ipilimumab and Bevacizumab combination (Ipi-Bev) therapy, revealed activated endothelium accompanied by lymphocyte infiltration. To discover possible targets involved in the observed clinical benefit, a serological screen was performed using human protein arrays with post-treatment plasma samples of long-term patients with clinical benefit to Ipi-Bev therapy (Wu et. al., 2017; Hodi et. al., 2014). EDIL3 (EGF Like Repeats and Discoidin Domains 3), a pro-angiogenic extracellular matrix protein, was identified. Humoral responses against EDIL3 were associated with favorable clinical outcomes including improved response rates (p=0.003) and overall survival (p=0.03) to Ipi-Bev therapy. The anti-EDIL3 response was also observed to be significantly (p=0.03) related to Ipi-Bev treatment when compared with patients treated with Ipilimumab alone, anti-PD1 therapy alone, or the combination of Ipilimumab and Nivolumab in a combined analysis. Transcriptome analyses of TCGA SKCM (n=469) and BMS Checkmate 064 trial (n=90) related upregulated EDIL3 expression with enrichment of EMT, TGFβ signaling in fibroblasts, and angiogenesis signatures. Interestingly, TIDE analysis demonstrated a role for EDIL3 in T cell exclusion and immunosuppressive cancer-associated fibroblasts (CAFs) as its source in the tumor microenvironment. EDIL3 was also predicted as a biomarker of non-response to immune checkpoint blockade treatment. In vitro studies confirmed the upregulation of EDIL3 expression and secretion by patient-derived CAFs along with its role in modulating TGFβ1 mediated EMT. Mechanistically, EDIL3 was found to interfere with leukocyte-endothelial interactions by disturbing LFA-1/ICAM1 mediated adhesion and inhibited T cell transmigration in 2-D and microfluidic 3-D coculture studies with patient-derived tumor endothelial cells. Our findings suggest that the humoral response against EDIL3 may serve as one of the underlying mechanisms involved in effectuating an excluded to an inflamed phenotype observed in responders of checkpoint blockade treatment. This study develops EDIL3 as a potential target to enhance cytotoxic immune responses and improve the outcomes of combination immune therapy. Studies are ongoing for targeted inhibition of EDIL3 with implications for therapeutic application to promote anti-tumor immunity. Citation Format: Saba Tabasum, Dinesh Thapa, Anita Giobbie-Hurder, Jason L. Weirather, Xinqi Wu, Marco Campisi, Xiaoyun Li, Jingjing Li, Michael P. Manos, David A. Barbie, F. Stephen Hodi. An angiogenic target of immune exclusion with checkpoint blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5864.

Development and Validation of a Prognostic Risk Model for Patients with Advanced Melanoma Treated with Immune Checkpoint Inhibitors.

Risk stratification tools for patients with advanced melanoma (AM) treated with immune checkpoint inhibitors (ICI) are lacking. We identified a new prognostic model associated with overall survival (OS). A total of 318 treatment naïve patients with AM receiving ICI were collected from a multi-centre retrospective cohort study. LASSO Cox regression identified independent prognostic factors associated with OS. Model validation was carried out on 500 iterations of bootstrapped samples. Harrel's C-index was calculated and internally validated to outline the model's discriminatory performance. External validation was carried out in 142 advanced melanoma patients receiving ICI in later lines. High white blood cell count (WBC), high lactate dehydrogenase (LDH), low albumin, Eastern Cooperative Oncology Group (ECOG) performance status ≥1, and the presence of liver metastases were included in the model. Patients were parsed into 3 risk groups: favorable (0-1 factors) OS of 52.9 months, intermediate (2-3 factors) OS 13.0 months, and poor (≥4 factors) OS 2.7 months. The C-index of the model from the discovery cohort was 0.69. External validation in later-lines (N = 142) of therapy demonstrated a c-index of 0.65. Liver metastases, low albumin, high LDH, high WBC, and ECOG≥1 can be combined into a prognostic model for AM patients treated with ICI.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 Tcells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 Tcells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 Tcells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 Tcell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Brain metastasis and survival outcomes after first-line therapy in metastatic melanoma: a multicenter DeCOG study on 1704 patients from the prospective skin cancer registry ADOREG

BackgroundDespite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis...

CLO23-038: The Impact of Dysbiosis Agents on the Effectiveness of Immune Checkpoint Inhibitors in Advanced Melanoma Patients: A Systematic Review With Meta-Analysis

"CLO23-038: The Impact of Dysbiosis Agents on the Effectiveness of Immune Checkpoint Inhibitors in Advanced Melanoma Patients: A Systematic Review With Meta-Analysis" published on 31 Mar 2023 by National Comprehensive Cancer Network.

CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.

Description of supportive care and feasibility of physical exercise program to improve quality of life in advanced melanoma patients.

Physical activity is the first-line treatment of cancer-related fatigue. It has shown benefits on patient's quality of life (QoL) when practiced during and after treatment. New treatments have drastically changed the prognosis of melanoma. Still, few data are available about research program of supportive care in advanced melanoma. The primary outcome was to assess the feasibility of setting up a prospective study evaluating the benefits of Adapted Physical Activity (APA) on the QoL of patients with advanced melanoma. Feasibility was defined with a combination of five criteria including completion of questionnaire, recruitment, participant retention, patient adhesion to supportive care, and absence of adverse event. Between September 2019 and March 2021, 271 melanoma patients were questioned. Around 60% of stage IV melanoma patients were interested in support care. Patient retention at 3 months was sufficient. Only one patient could not be evaluated after 3 months of enrolment because of deterioration of the general state. Adhesion to exercise and sessions was good. Supervised APA program appeared to be safe and well tolerated as no adverse events or discontinuations were reported. Setting up a prospective research program evaluating the benefits of physical activity in advanced melanoma patients seems feasible. With melanoma becoming a chronic disease, supportive care may reduce fatigue, improve QoL and help maintain a healthy lifestyle. Data supporting its benefits on this survivor population are needed.

Response to checkpoint inhibition and targeted therapy in melanoma patients with concurrent haematological malignancies

BackgroundPatients diagnosed with haematologic malignancies (HMs) have a higher risk of developing subsequent solid tumours, such as melanoma. Patients with HM were mostly excluded from clinical trials but potentially derive less benefit from immune checkpoint inhibitors (ICIs) due to disease- or treatment-related T- or B-cell dysfunction. MethodsAll advanced melanoma patients treated with anti-PD-1-based treatment or targeted therapy between 2015 and 2021 were included from the prospective nationwide Dutch Melanoma Treatment Registry. Progression-free survival (PFS) and melanoma-specific survival (MSS) were analysed for patients with HM (HM+) and without HM (HM−). A cox model was used to account for confounders associated with PFS and MSS. ResultsIn total, 4638 advanced melanoma patients received first-line anti-PD-1 monotherapy (n = 1763), ipilimumab-nivolumab (n = 800), or BRAF(/MEK) inhibitors (n = 2075). Concurrent HMs were present for 46 anti-PD1-treated patients, 11 ipilimumab-nivolumab-treated patients and 43 BRAF(/MEK)-inhibitor-treated patients. In anti-PD-1-treated patients, the median PFS was 2.8 months for HM+ and 9.9 months for HM− (p = 0.01). MSS was 41.2 months for HM+ and 58.1 months for HM− (p = 0.00086). In multivariable analysis, the presence of an HM was significantly associated with higher risk of melanoma progression (HRadj 1.62; 95% confidence interval [95% CI] 1.15–2.29; p = 0.006) and melanoma-related death (HRadj 1.74; 95% CI 1.09–2.78; p = 0.020). Median PFS and MSS for first-line BRAF(/MEK-) inhibitor-treated HM+ and HM− patients were not significantly different. ConclusionsPatients with HM and advanced melanoma show significantly worse melanoma-related outcomes when treated with ICI, but not targeted therapy, compared to patients without HM. Clinicians should be aware of potentially altered effectiveness of ICI in patients with active HM.

PD-L1 Tumor Expression as a Predictive Biomarker of Immune Checkpoint Inhibitors' Response and Survival in Advanced Melanoma Patients in Brazil.

Immune checkpoint blockade (ICB) agents are prominent immunotherapies for the treatment of advanced melanoma. However, they fail to promote any durable clinical benefit in a large cohort of patients. This study assessed clinical and molecular predictors of ICB response and survival in advanced melanoma. A retrospective analysis was performed on 210 patients treated with PD-1 or CTLA-4 inhibitors at Barretos Cancer Hospital, Brazil. PD-L1 expression was assessed by immunohistochemistry using formalin-fixed paraffin-embedded tumor tissues collected prior to ICB therapy. Patients were divided into responders (complete and partial response and stable disease for more than 6 months) and non-responders (stable disease for less than 6 months and progressive disease). Among them, about 82% underwent anti-PD-1 immunotherapy, and 60.5% progressed after the ICB treatment. Patients that received ICB as first-line therapy showed higher response rates than previously treated patients. Higher response rates were further associated with superficial spreading melanomas and positive PD-L1 expression (>1%). Likewise, PD-L1 positive expression and BRAF V600 mutations were associated with a higher overall survival after ICB therapy. Since ICBs are expensive therapies, evaluation of PD-L1 tumor expression in melanoma patients should be routinely assessed to select patients that are most likely to respond.

Time interval from primary melanoma to first distant recurrence in relation to patient outcomes in advanced melanoma.

Since the introduction of BRAF(/MEK) inhibition and immune checkpoint inhibition (ICI), the prognosis of advanced melanoma has greatly improved. Melanoma is known for its remarkably long time to first distant recurrence (TFDR), which can be decades in some patients and is partly attributed to immune-surveillance. We investigated the relationship between TFDR and patient outcomes after systemic treatment for advanced melanoma. We selected patients undergoing first-line systemic therapy for advanced melanoma from the nationwide Dutch Melanoma Treatment Registry. The association between TFDR and progression-free survival (PFS) and overall survival (OS) was assessed by Cox proportional hazard regression models. The TFDR was modeled categorically, linearly, and flexibly using restricted cubic splines. Patients received anti-PD-1-based treatment (n=1844) or BRAF(/MEK) inhibition (n=1618). For ICI-treated patients with a TFDR <2 years, median OS was 25.0months, compared to 37.3months for a TFDR >5 years (P= .014). Patients treated with BRAF(/MEK) inhibition with a longer TFDR also had a significantly longer median OS (8.6months for TFDR <2 years compared to 11.1months for >5 years, P= .004). The hazard of dying rapidly decreased with increasing TFDR until approximately 5 years (HR 0.87), after which the hazard of dying further decreased with increasing TFDR, but less strongly (HR 0.82 for a TFDR of 10 years and HR 0.79 for a TFDR of 15 years). Results were similar when stratifying for type of treatment. Advanced melanoma patients with longer TFDR have a prolonged PFS and OS, irrespective of being treated with first-line ICI or targeted therapy.

Circulating tumor DNA-based molecular residual disease detection for treatment monitoring in advanced melanoma patients.

Immune checkpoint inhibitors (ICIs) have substantially improved overall survival in patients with advanced melanoma; however, the lack of biomarkers to monitor treatment response and relapse remains an important clinical challenge. Thus, a reliable biomarker is needed that can risk-stratify patients for disease recurrence and predict response to treatment. A retrospective analysis using a personalized, tumor-informed circulating tumor DNA (ctDNA) assay on prospectively collected plasma samples (n=555) from 69 patients with advanced melanoma was performed. Patients were divided into three cohorts: cohort A (N=30), stage III patients receiving adjuvant ICI/observation; cohort B (N=29), unresectable stage III/IV patients receiving ICI therapy; and cohort C (N=10), stage III/IV patients on surveillance after planned completion of ICI therapy for metastatic disease. In cohort A, compared to molecular residual disease (MRD)-negative patients, MRD-positivity was associated with significantly shorter distant metastasis-free survival (DMFS; hazard ratio [HR], 10.77; p=.01). Increasing ctDNA levels from the post-surgical or pre-treatment time point to after 6weeks of ICI were predictive of shorter DMFS in cohort A (HR, 34.54; p<.0001) and shorter progression-free survival (PFS) in cohort B (HR,22; p=.006). In cohort C, all ctDNA-negative patients remained progression-free for a median follow-up of 14.67months, whereas ctDNA-positive patients experienced disease progression. Personalized and tumor-informed longitudinal ctDNA monitoring is a valuable prognostic and predictive tool that may be used throughout the clinical course of patients with advanced melanoma.