Faculty Bibliography

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host's response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual's recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019.

Background/UNASSIGNED:Two primary histologic subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM), comprise the majority of all cutaneous melanomas. NM is associated with worse outcomes, which have been attributed to increased thickness at presentation, and it is widely expected that NM and SSM would exhibit similar behavior once metastasized. Herein, we tested the hypothesis that primary histologic subtype is an independent predictor of survival and may impact response to treatment in the metastatic setting. Methods/UNASSIGNED:We examined the most recent Surveillance, Epidemiology, and End Results (SEER) cohort (n = 118 508) and the New York University (NYU) cohort (n = 1621) with available protocol-driven follow-up. Outcomes specified by primary histology were studied in both the primary and metastatic settings with respect to BRAF-targeted therapy and immunotherapy. We characterized known driver mutations and examined a 140-gene panel in a subset of NM and SSM cases using next-generation sequencing. All statistical tests were two-sided. Results/UNASSIGNED:NM was an independent risk factor for death in both the SEER (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.41 to 1.70, P < .001) and NYU (HR = 1.47, 95% CI = 1.05, 2.07, P = .03) cohorts, controlling for thickness, ulceration, stage, and other variables. In the metastatic setting, NM remained an independent risk factor for death upon treatment with BRAF-targeted therapy (HR = 3.33, 95% CI = 1.06 to 10.47, P = .04) but showed no statistically significant difference with immune checkpoint inhibition. NM was associated with a higher rate of NRAS mutation (P < .001), and high-throughput sequencing revealed NM-specific genomic alterations in NOTCH4, ANK3, and ZNF560, which were independently validated. Conclusions/UNASSIGNED:Our data reveal distinct clinical and biological differences between NM and SSM that support revisiting the prognostic and predictive impact of primary histology subtype in the management of cutaneous melanoma.

Background Somatic genetic alterations have been associated with differential disposition of the intratumoral immune milieu. In contrast, the role of germline genetics remains largely unknown. The Cancer Genome Atlas (TCGA) Pan-Cancer Immune Response Working Group recently analyzed associations between immunological features of tumor microenvironment, prognosis, and tumor-intrinsic properties (including somatic mutations and copy number aberrations). The study generated a comprehensive set of per-sample immune response signatures, and identified distinct tumor immunological subtypes shared across multiple cancer types [1]. Here, we examined the germline genetic contribution to >100 such immune response signatures, considered as potential traits, in >9,000 study participants across 30 different cancer types in the TCGA. Methods We used SNP data from Affymetrix 6.0 arrays typed on normal tissue and blood. After stringent quality control, we imputed missing SNPs using the Haplotype Reference Consortium dataset and included SNPs with minor allele frequency >0.005 and imputation quality R2>0.5. We inferred genetic ancestry using principal components analysis. We estimated genome-wide heritability of the immune signatures using the genomic-relatedness-based restricted maximumlikelihood (GREML) approach implemented in genome-wide complex trait analysis (GCTA). We performed genome-wide association studies (GWAS) using linear regression models. All analyses were adjusted for cancer type, age at diagnosis, gender, and genetic ancestry using principal components 1-7. Linear regression was also applied to germline exome sequence data [2] to evaluate rare variant associations. Results Across different tumor types, we found significant heritability (FDR< 0.05) for estimates of innate and adaptive immune cell enrichment including natural killer cells, activated dendritic cells, eosinophils and neutrophils, and T-cell subsets (CD8 Cytotoxic, T-helper, T-follicular helper, T-effector memory, T-central memory, Th1 and Th17 cells) respectively, as well as for antigen-presenting machinery and interferon-related signatures. Furthermore, we found significant interactions between germline modifiers and distinct immune subtypes (FDR<0.05). Through GWAS analysis, we identified several polymorphisms associated with both immune cells and immunomodulatory signaling passing the genome-wide significance threshold (p<5E-8). Two SNPs previously associated with the risk of several auto-immune diseases [3], rs2111485 and rs1990760 mapping to Interferon Induced Helicase C Domain 1 (IFIH1) locus, were significantly correlated with interferon signaling in tumors (Figure 1). Moreover, suggestive associations between rare variants and immune response traits were found. Conclusions We demonstrated that intratumoral immune disposition is partially under germline control through systematic pan-cancer analysis. Germline variants associated with differential immune response might be used to stratify patients based on likelihood of treatment response and to prioritize targets for development of novel therapies. (Figure Preseted)

Background Autoantibody landscapes are very specific to the individual, can remain stable for many years, and contain unique features reported in association with cancer, autoimmunity, infection, neurologic conditions, CD8+ T cell behavior, and checkpoint blockade adverse events [1-11]. The goal of this work was to determine whether pre-existing antigenspecific features in melanoma patient autoantibody landscapes would associate with clinical outcomes following checkpoint blockade. Methods Pre-treatment serum samples were collected from 117 melanoma patients prior to checkpoint blockade with anti-CTLA4 (N=60), anti-PD1 (N=38), or both in combination (N=16). All data was collected with approval of the NYU Institutional Review Board at the NYU Perlmutter Cancer Center with informed consent [11]. Serum samples were run on HuProt Human Proteome Microarrays containing >19,000 human proteins by CDI Laboratories. Raw serum IgG signal intensities were processed across staining cohorts via interquartile range normalization. Pre-existing antibody responses were defined as patient-specific IgG signals >3.5 median absolute deviations above cohort median IgG background (modified Z-score). Group statistics were computed (GraphPad Prism), and gene ontology enrichment analysis was performed (Enrichr) [12]. Results Several pre-existing antigen-specific IgG autoantibody targets were observed to have associations with good outcomes (SD/PR) or objective clinical responses (PR/CR) versus patients with progressive disease (POD). While final determination of the most predictive subsets is ongoing, many targets represent genes in an axis surrounding immune signaling pathways, hereditary neurodegenerative disease, and the ubiquitin proteasome pathway (ie, UBQLN1, UBQLN2). An exemplary example was observed in the autoantibody responses shared by >10% of all patients regardless of clinical outcome. Gene ontology enrichment analysis of these shared melanoma-patient autoantibodies versus KEGG 2019 [12] demonstrates this set of proteins is strongly enriched for neurotrophin signaling-associated proteins after multi-sample correction (P=0.004) (Table 1). Several other associations were observed cohort-wide for ontologies with tissuespecific enrichment in the brain, neurons, and neuronal processes. Conclusions In this pilot study, we found strong associations across the cohort for autoantibodies against nerve-growth-inducing neurotrophins and genes like UBQLN1 and UBQLN2 which have strong associations with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's, and Alzheimer's - neurodegenerative diseases that are known to have incidences which correlate with melanoma [14-16]; this hints at a potential immunologic connection between the conditions, perhaps related to an antitumor / autoimmune axis involving the targets reported here. (Table Presented)

Background: During their lifetime about 8% of patients with single primary cutaneous melanoma (SPM) will develop multiple primary melanomas (MPM), which are associated with significantly higher mortality compared to patients with SPM. Based on the evidence that the immune system plays a role in regulating melanoma progression we explored whether germline genetic variants controlling the expression of immunomodulatory genes (immunomodulatory quantitative trait loci, eQTLs) discern risk of MPMcompared to patients with SPM or healthy controls.
Method(s): Previously, we identified 50 eQTLs significantly associated with the expression of 265 immunomodulatory genes using the MuTHer twin cohort. These 50 SNPs were genotyped in 837 SPM and 104MPM individuals using MassARRAY system. 1047 healthy controls were obtained from a publically available GWAS on CMascertained at MDAnderson (phs000187.v1.p1). We employed multivariate logistic regression to test the association of SNPs withMPM vs cancer-free controls andMPMvs SPM.
Result(s): When comparing MPMvs SPM, rs2071304, previously linked to expression of SPI1 in MuTHer data, showed a strong association with reduction ofMPM risk (OR=0.60; 95% CI=0.45-0.81; p=0.0007). Intriguingly, this variant also trended toward significance when comparing MPM vs controls (OR=0.61; 95% CI: 0.44-0.85; p=0.003). Finally, our most significant association when comparingMPM to controls was for rs2276645 (OR=0.60; 95% CI=0.45-0.81; p=0.0008), an eQTL associated with Zap-70 expression.
Conclusion(s): Our data, for the first time, indicate that the inherited host immunity impacts risk ofMPMin individuals with SPM, highlighting an importance of immune involvement in melanoma progression. The MPMrisk-predicting genetic variants identified here or in expanded efforts, currently underway, may eventually lead to a diagnostic tool allowing for enhanced screening and clinical management of patients at risk of MPM, hence reducing elevated MPM-associated mortality. Additionally, our results further support thatMPM and SPM may have different genetics underpinnings and should be treated as separate clinical entities

Cigarette smoking alters the oral microbiome; however, the effect of alternative tobacco products remains unclear. Middle Eastern tobacco products like dokha and shisha, are becoming globally widespread. We tested for the first time in a Middle Eastern population the hypothesis that different tobacco products impact the oral microbiome. The oral microbiome of 330 subjects from the United Arab Emirates Healthy Future Study was assessed by amplifying the bacterial 16S rRNA gene from mouthwash samples. Tobacco consumption was assessed using a structured questionnaire and further validated by urine cotinine levels. Oral microbiome overall structure and specific taxon abundances were compared, using PERMANOVA and DESeq analyses respectively. Our results show that overall microbial composition differs between smokers and nonsmokers (p = 0.0001). Use of cigarettes (p = 0.001) and dokha (p = 0.042) were associated with overall microbiome structure, while shisha use was not (p = 0.62). The abundance of multiple genera were significantly altered (enriched/depleted) in cigarette smokers; however, only Actinobacillus, Porphyromonas, Lautropia and Bifidobacterium abundances were significantly changed in dokha users whereas no genera were significantly altered in shisha smokers. For the first time, we show that smoking dokha is associated to oral microbiome dysbiosis, suggesting that it could have similar effects as smoking cigarettes on oral health.

BACKGROUND:Immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or the combination) enhance anti-tumor immune responses, yielding durable clinical benefit in several cancer types, including melanoma. However, a subset of patients experience immune-related adverse events (irAEs), which can be severe and result in treatment termination. To date, no biomarker exists that can predict development of irAEs. METHODS:We hypothesized that pre-treatment antibody profiles identify a subset of patients who possess a sub-clinical autoimmune phenotype that predisposes them to develop severe irAEs following immune system disinhibition. Using a HuProt human proteome array, we profiled baseline antibody levels in sera from melanoma patients treated with anti-CTLA-4, anti-PD-1, or the combination, and used support vector machine models to identify pre-treatment antibody signatures that predict irAE development. RESULTS:We identified distinct pre-treatment serum antibody profiles associated with severe irAEs for each therapy group. Support vector machine classifier models identified antibody signatures that could effectively discriminate between toxicity groups with > 90% accuracy, sensitivity, and specificity. Pathway analyses revealed significant enrichment of antibody targets associated with immunity/autoimmunity, including TNFα signaling, toll-like receptor signaling and microRNA biogenesis. CONCLUSIONS:Our results provide the first evidence supporting a predisposition to develop severe irAEs upon immune system disinhibition, which requires further independent validation in a clinical trial setting.

BACKGROUND:The United Arab Emirates (UAE) is faced with a rapidly increasing burden of non-communicable diseases including obesity, diabetes, and cardiovascular disease. The UAE Healthy Future study is a prospective cohort designed to identify associations between risk factors and these diseases amongst Emiratis. The study will enroll 20,000 UAE nationals aged ≥18 years. Environmental and genetic risk factors will be characterized and participants will be followed for future disease events. As this was the first time a prospective cohort study was being planned in the UAE, a pilot study was conducted in 2015 with the primary aim of establishing the feasibility of conducting the study. Other objectives were to evaluate the implementation of the main study protocols, and to build adequate capacity to conduct advanced clinical laboratory analyses. METHODS:Seven hundred sixty nine UAE nationals aged ≥18 years were invited to participate voluntarily in the pilot study. Participants signed an informed consent, completed a detailed questionnaire, provided random blood, urine, and mouthwash samples and were assessed for a series of clinical measures. All specimens were transported to the New York University Abu Dhabi laboratories where samples were processed and analyzed for routine chemistry and hematology. Plasma, serum, and a small whole blood sample for DNA extraction were aliquoted and stored at -80 °C for future analyses. RESULTS:Overall, 517 Emirati men and women agreed to participate (68% response rate). Of the total participants, 495 (95.0%), 430 (82.2%), and 492 (94.4%), completed the questionnaire, physical measurements, and provided biological samples, respectively. CONCLUSIONS:The pilot study demonstrated the feasibility of recruitment and completion of the study protocols for the first large-scale cohort study designed to identify emerging risk factors for the major non-communicable diseases in the region.

Background: Approximately 40% of metastatic cutaneous melanoma (CM) patients do not respond to the current immunotherapy (IT) regimens, pointing to other, yet unknown factors conferring IT resistance. In addition, > 60% of patients from single-line or combined treatment (COMBO) regimens present severe immune related adverse events (irAEs). In this study we have developed a novel genomic approach interrogating expression quantitative trait loci (eQTLs) to explore weather germline genetic variation can serve as novel personalized determinant of immunotherapy response and toxicity.
Method(s): By interrogating the genome wide expression data and SNP array datasets of healthy twin cohort (MuTHER), we have identified 85 eQTLs most significantly associated with the expression of 265 immune genes. Using the MassARRAY system, the 85 SNPs were genotyped in 138 anti-CTLA-4 treated patients, 87 PD-1 treated patients, and 69 patients from combined (COMBO) treatments, collected from multi-institutional collaborations. To test the association of SNPs with IT response and irAEs, logistic regression analyses were performed for each treatment group adjusting by demographic and clinical covariates.
Result(s): We found significant associations with COMBO IT resistance for and eQTL in IL10/IL19 (OR = 4.249, p = 0.0167), which we have recently identified for association with melanoma survival and which, interestingly, is an established locus associated with the risk of several autoimmune diseases. Additionally, we also identified eQTLs that are associated with IT sensitivity; IL1-beta with resistance to anti-CTLA-4 and SPI1 with resistance to anti-PD-1. Interestingly, genomic scan of 85 eQTLs has identified novel loci predictive of severe autoimmunity and site specific irAEs in patients treated with COMBO or single-line anti- CTLA4 IT.
Conclusion(s): In this study, we report that eQTLs from IL19/IL10 locus, previously shown to predict autoimmunity risk and CM survival, is also a surrogate marker of response to COMBO IT, indicating a strong relationship between interleukin pathways and tumor immunogenicity. Novel loci have been found as predictive markers for autoimmune toxicity, in patients treated with COMBO and anti-CTLA4 IT. This is a first evidence that immunomodulatory pathways modulated by germline genetic variation can impact susceptibility to irAEs as well as IT efficacy. Currently, a large scan is underway using genome-wide genetic screens to further test the functional validity of these findings in a large collaborative setting