Bao, Riyue

Abstract Purpose: Germline pathogenic variants (PV) in ATM increase the risk of pancreatic ductal adenocarcinoma (PDAC), but the underlying tumor biology of PDAC associated with germline PV in ATM has not been adequately explored. Experimental Design: Whole-genome, whole-exome, and RNA sequencing were performed on PDAC tumors from 25 germline ATM PV carriers diagnosed at Mayo Clinic between 2007 and 2017. Somatic and copy-number alterations, mutational signatures, transcriptomic subtypes, and the immune landscape were evaluated. Results: High-quality whole-exome and whole-genome sequencing were obtained from 21 and 15 tumors, respectively. Biallelic inactivation of ATM was observed in 87%, KRAS PV in 90%, CDKN2A homozygous loss in 60%, and TP53 alterations in <10% of these tumors. A predominant clock-like mutational signature was present in all samples. Whole-transcriptome analysis identified that the aberrantly differentiated endocrine exocrine subtype accounted for 18% of PDAC and was consistently associated with >5-year overall survival. In addition, a 28-gene expression–based signature associated with overall survival was identified and further validated in The Cancer Genome Atlas cohort. Immune landscape analysis through CODEX identified enriched CD4 T-helper cell/tumor interactions and reduced B7H3-high cell/tumor interactions in ATM PV carriers compared with noncarriers. Conclusions: The observed absence of TP53 PV and enrichment for CDKN2A alterations in ATM tumors, along with differences in the mutational signatures, transcriptomic subtypes and immune landscape, improve our understanding of the mechanistic pathways involved in PDAC development in germline ATM PV carriers and help identify potential targeted therapeutic strategies.

Abstract Purpose: DROSHA, DGCR8, and DICER1 regulate miRNA biogenesis and are commonly mutated in cancer. Although DGCR8 and DICER1 germline pathogenic variants (GPV) cause autosomal dominant tumor predisposition, no association between DROSHA GPVs and clinical phenotypes has been reported. Experimental Design: After obtaining informed consent, sequencing was performed on germline and tumor samples from all patients. The occurrence of germline DROSHA GPVs was investigated in large pediatric and adult cancer datasets. The population prevalence of DROSHA GPVs was investigated in the UK Biobank and Geisinger DiscovEHR cohorts. Results: We describe nine children from eight families with heterozygous DROSHA GPVs and a diagnosis of pineoblastoma (n = 8) or Wilms tumor (n = 1). A somatic second hit in DROSHA was detected in all eight tumors analyzed. All pineoblastoma tumors analyzed were classified as miRNA processing–altered 1 subtype. We estimate the population prevalence of germline DROSHA loss-of-function variants to be 1:3,875 to 1:4,843 but find no evidence for increased adult cancer risk. Conclusions: This is the first report of DROSHA-related tumor predisposition. As pineoblastoma and Wilms tumor are also associated with DICER1 GPVs, our results suggest that the tissues of origin for these tumors are uniquely tolerant of general miRNA loss. The miRNA processing–altered 1 pineoblastoma subtype is associated with older age of diagnosis and better outcomes than other subtypes, suggesting DROSHA GPV status may have important clinical and prognostic significance. We suggest that genetic testing for DROSHA GPVs be considered for patients with pineoblastoma, Wilms tumor, or other DICER1-/DGCR8-related conditions and propose surveillance recommendations through research studies for individuals with DROSHA GPVs.

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy globally, and the vast majority of cases are treated with primary surgical resection and adjuvant therapy. Our group has pioneered the use of HNSCC circulating tumor DNA (ctDNA) in surgical effluent collected postoperatively from surgical drains (surgical drain fluid, SDF) as a novel proximal bioanalyte for measuring minimal residual disease after surgery. We now profile the immune cell composition of SDF compared to paired peripheral blood mononuclear cells (PBMCs), and correlate SDF immune cell profiles with clinical variables and oncologic outcomes in HNSCC. Method: SDF and peripheral venous blood were collected 24 hours postoperatively from 58 HNSCC patients, including 11 HPV (+) and 47 HPV (-) patients undergoing standard of care surgical resection, lymph node dissection, and adjuvant therapy. Freshly collected SDF cells and PBMCs were cryopreserved until profiling by multicolor flow cytometry for respective leukocyte compositions. Analyses were used to define the following viable CD45+ leukocyte subsets: total T cells, CD4 T-cells CD8 T-cells, Treg cells, B-cells, non- classical monocytes, classical monocytes, natural killer (NK) cells, classical dendritic cells (cDC), plasmacytoid DC (pDC), macrophages, and monocytic myeloid-derived suppressive cells (MDSCs). Immune cell percentages were correlated with clinical characteristics and cancer recurrence. Results: CD45+ leukocytes comprised >99.5% of SDF cells, with very few EPCAM+ tumor cells (< 10^-4). HPV status did not significantly affect SDF immune cell composition, and no significant differences were noted by tumor site (larynx, oral cavity, oropharynx), or extracapsular lymph node extension. Among 13 HPV (-) HNSCC patients with paired peripheral blood drawn concurrently, SDF had a higher proportion of B-cells(16.8% vs. 9%, p=0.044), NK cells(56% vs. 12%, P<0.001), monocytes(14% vs. 1%, P<0.001), and macrophages(3.1% vs. 0.46%, P<0.001) compared to PBMCs, and a lower proportion of CD4+ T cells(1% vs. 24%, P<0.001), CD8+ T cells(0.6% vs. 18%, P<0.001), MDSCs(2.1% vs. 3.4%, P=0.029), and pDCs(0.1% vs. 0.2%, P=0.014). HPV (-) HNSCC patients that went on to recur (n=9) had a significantly higher proportions of SDF monocytes (total, classical, and non- classical) and CD56dimCD16+ NK cells (all P<0.001) compared to non-recurrent patients (n=38). Patients who went on to recur also had a significantly lower proportion of SDF B-cells (P<0.001), CD56bright NK cells(P=0.01), and macrophages (P=0.018) compared to non- recurrent cases. No significant differences were noted with respect to proportions of CD3+, CD4+, CD8+ T-cells, DC, and MDSC cells. Conclusion: SDF from HNSCC patients contains unique immune cell populations and may provide new insights into postoperative tumor immunity. Differences in SDF immune cell composition by oncologic outcome in patients that go on to recur, highlighting potential prognostic and predictive applications of SDF immune cell profiling for adjuvant therapy decision-making. Citation Format: Zhongping Xu, Noah Earland, Lucien Khalil, Lazar Vujanovic, Danny Azmi Elias, Riyue Bao, Jason J. Luke, Aadel A. Chaudhuri, Jose P. Zevallos. Immune cell composition in surgical drain fluid as a novel prognostic marker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B031.

Abstract BACKGROUND Glioblastoma (GBM), one of the deadliest primary tumors of the central nervous system, is characterized by a highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAMs) are a dominant population of immune cells in the GBM TME and substantially contribute to immunosuppression, tumor progression, and treatment resistance. Elucidating the molecular mechanisms underlying TAM behavior is crucial to the development of effective immuno-therapeutic strategies for treating GBM. The adaptor protein, Caspase Recruitment Domain-containing protein 9 (CARD9) is primarily expressed in myeloid cells and its activation triggers assembly of the CARD9-BCL10-MALT1 (CBM) complex, which then promotes activation of NF-κB and/or MAPK pathways. The aim of this study was to investigate the role of the CBM complex in the GBM TME and assess its potential as a therapeutic target. METHODS/RESULTS In silico analyses revealed that GBM tumors demonstrate increased CARD9 and MALT1 expression in comparison to non-tumor brain tissue, with CARD9 expressed only within the myeloid compartment. Using co-culture, scratch wound, and transwell assays, we found that GBM tumor cells induce macrophage M2 polarization and migration via a mechanism that is dependent on CARD9/MALT1 signaling within macrophages. scRNA-Seq and flow-cytometric analysis of orthotopic syngeneic immunocompetent GBM tumor models revealed that the GBM TME is dominated by macrophages. Disruption of MALT1 protease activity within the TME leads to reduced tumor infiltration by immunosuppressive TAMs, remodeling of TAMs toward an immunoreactive anti-tumor phenotype, and improved survival of GBM-bearing mice. Additionally, we found that tumor growth was abrogated when GBM-bearing mice were treated with either a MALT1-protease inhibitor or a CARD9 inhibitor. CONCLUSIONS Our study suggests that blockade of the CARD9-MALT1 signaling axis impairs GBM-induced macrophage recruitment and M2 polarization and inhibits tumor progression. These findings nominate the CARD9-MALT1 signaling axis as a new target for TAM-directed immunotherapy for the treatment of GBM.

Abstract The field of oncology has been transformed by immune checkpoint inhibitors (ICI) and other immune-based agents; however, many patients do not receive a durable benefit. While biomarker assessments from pivotal ICI trials have uncovered certain mechanisms of resistance, results thus far have only scraped the surface. Mechanisms of resistance are as complex as the tumor microenvironment (TME) itself, and the development of effective therapeutic strategies will only be possible by building accurate models of the tumor-immune interface. With advancement of multi-omic technologies, high-resolution characterization of the TME is now possible. In addition to sequencing of bulk tumor, single-cell transcriptomic, proteomic, and epigenomic data as well as T-cell receptor profiling can now be simultaneously measured and compared between responders and nonresponders to ICI. Spatial sequencing and imaging platforms have further expanded the dimensionality of existing technologies. Rapid advancements in computation and data sharing strategies enable development of biologically interpretable machine learning models to integrate data from high-resolution, multi-omic platforms. These models catalyze the identification of resistance mechanisms and predictors of benefit in ICI-treated patients, providing scientific foundation for novel clinical trials. Moving forward, we propose a framework by which in silico screening, functional validation, and clinical trial biomarker assessment can be used for the advancement of combined immunotherapy strategies.

Acral melanoma (AM) has distinct characteristics as compared with cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICIs). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3 + CD8 + PD1 + intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low versus high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared with responders across cancers, including AM, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation.

Abstract Purpose: To assess whether MUC1 peptide vaccine produces an immune response and prevents subsequent colon adenoma formation. Patients and Methods: Multicenter, double-blind, placebo-controlled randomized trial in individuals age 40 to 70 with diagnosis of an advanced adenoma ≤1 year from randomization. Vaccine was administered at 0, 2, and 10 weeks with a booster injection at week 53. Adenoma recurrence was assessed ≥1 year from randomization. The primary endpoint was vaccine immunogenicity at 12 weeks defined by anti-MUC1 ratio ≥2.0. Results: Fifty-three participants received the MUC1 vaccine and 50 placebo. Thirteen of 52 (25%) MUC1 vaccine recipients had a ≥2-fold increase in MUC1 IgG (range, 2.9–17.3) at week 12 versus 0/50 placebo recipients (one-sided Fisher exact P < 0.0001). Of 13 responders at week 12, 11 (84.6%) responded to a booster injection at week 52 with a ≥2-fold increase in MUC1 IgG measured at week 55. Recurrent adenoma was observed in 31 of 47 (66.0%) in the placebo group versus 27 of 48 (56.3%) in the MUC1 group [adjusted relative risk (aRR), 0.83; 95% confidence interval (CI), 0.60–1.14; P = 0.25]. Adenoma recurrence occurred in 3/11 (27.3%) immune responders at week 12 and week 55 (aRR, 0.41; 95% CI, 0.15–1.11; P = 0.08 compared with placebo). There was no difference in serious adverse events. Conclusions: An immune response was observed only in vaccine recipients. Adenoma recurrence was not different than placebo, but a 38% absolute reduction in adenoma recurrence compared with placebo was observed in participants who had an immune response at week 12 and with the booster injection.

Uveal melanoma is a rare form of melanoma with particularly poor outcomes in the metastatic setting. In contrast with cutaneous melanoma, uveal melanoma lacks BRAF mutations and demonstrates very low response rates to immune-checkpoint blockade. Our objectives were to study the transcriptomics of metastatic uveal melanoma with the intent of assessing gene pathways and potential molecular characteristics that might be nominated for further exploration as therapeutic targets. We initially analyzed transcriptional data from The Cancer Genome Atlas suggesting PI3K/mTOR and glycolysis as well as IL6 associating with poor survival. From tumor samples collected in a prospective phase II trial (A091201), we performed a transcriptional analysis of human metastatic uveal melanoma observing a novel role for epithelial-mesenchymal transition associating with survival. Specifically, we nominate and describe initial functional validation of neuropillin-1 from uveal melanoma cells as associated with poor survival and as a mediator of proliferation and migration for uveal melanoma in vitro. These results immediately nominate potential next steps in clinical research for patients with metastatic uveal melanoma.

Abstract Purpose: The surface receptor MET is highly expressed on primary uveal melanoma; MET inhibitors demonstrated early clinical signals of efficacy in slowing uveal melanoma growth. The primary objective of our study was to compare the progression-free survival rate at 4 months (PFS4) of patients with uveal melanoma treated with cabozantinib or chemotherapy. Patients and Methods: Patients with metastatic uveal melanoma and RECIST measurable disease were randomized 2:1 to receive either cabozantinib (arm 1) versus temozolomide or dacarbazine (arm 2) with restaging imaging every two cycles. Cross-over from arm 2 to cabozantinib after progression was allowed (arm 2X). Available tumor specimens were analyzed by whole-exome sequencing (WES) and results were correlated with outcome. Results: Forty-six eligible patients were accrued with 31, 15, and 9 in arms 1, 2, and 2X, respectively. Median lines of prior therapy, including hepatic embolization, were two. Rates of PFS4 in arm 1 and arm 2 were 32.3% and 26.7% (P = 0.35), respectively, with median PFS time of 60 and 59 days (P = 0.964; HR = 0.99). Median overall survival (OS) was 6.4 months and 7.3 months (P = 0.580; HR = 1.21), respectively. Grade 3–4 Common Terminology Criteria for Adverse Events were present in 61.3%, 46.7%, and 37.5% in arms 1, 2, and 2X, respectively. WES demonstrated a mean tumor mutational burden of 1.53 mutations/Mb and did not separate OS ≤ or >1 year (P = 0.14). Known mutations were identified by WES and novel mutations were nominated. Conclusions: MET/VEGFR blockade with cabozantinib demonstrated no improvement in PFS but an increase in toxicity relative to temozolomide/dacarbazine in metastatic uveal melanoma.

9506 Background: Survival is poor in mUM and treatment options are limited. MET kinase is over-expressed on UM and the MET inhibitor cabo showed activity in an early trial. Methods: A091201 was a 2:1 randomized phase II study testing progression-free survival (PFS) of cabo (60 mg daily) vs chemo (DTIC/TMZ). We studied baseline metastatic tumor samples (n = 19; 1 lung, 18 liver) by whole exome sequencing (WES) and RNAseq. We correlated data with OS and made comparisons for mUM vs. primary tumors from TCGA (n = 80). Results: 46 patients were accrued with 96% and 63% with liver metastases and elevated LDH, respectively. Toxicities were similar to prior reports of cabo and chemo. The trial stopped at interim analysis due to no difference in PFS (p = 0.964; HR = 0.99) or OS (p = 0.580; HR = 1.21). WES showed tumor mutational burden of 46±4 (mean±SEM) and did not separate OS at 1 year (p = 0.14, two-sided Wilcoxon rank sum test) in A091201. Recurrent known mutations included GNAQ/11, SF3B1, BAP1; novel mutations included GOLGA6L10, PKD1L3, and FAM228B. Gene expression signatures differed significantly between A091201 and TCGA cohorts including MET signaling (p = 7.87e-22), T cell-inflamed (p = 0.004), homologous recombination deficiency (p = 0.004), proliferation (p = 0.009) and hypoxia (p = 5.2e-10) (two-sided Student’s t-test). Tumor immune cell enrichment analysis revealed significant differences with lower M1:M2 macrophage (p = 1.2e-10) and higher Tregs (p = 6.0E-21) in mUM relative to TCGA (two-sided Wilcoxon rank sum test). Epithelial-mesenchymal transition gene expression signature was significantly associated with worse OS in A091201 (p = 0.02) with angiogenesis signature trending toward significance (p = 0.21) (log-rank test). OS separated by differentially expressed genes with OS ≤ 1 year associating with increased expression of the angiogenesis/immune-associated molecule neuropilin 1. Conclusions: These results provide insights between primary and mUM indicating potential novel therapeutic approaches. Support: U10CA180821, U10CA180882, T32GM007019, Exelixis. https://acknowledgments.alliancefound.org ; Clinical trial information: NCT01835145.