Tyrosine-protein kinase SYK-related gene signature in baseline immune cells associated with adjuvant immunotherapy-induced immune-related adverse events in melanoma.

PURPOSE: Immune checkpoint inhibition (ICI) shows benefits in adjuvant (AT) and neoadjuvant (NAT) melanoma treatments. However, ICI frequently induces severe immune-related adverse events (irAEs). Unlike metastatic disease, where irAEs are a clinical trade-off for treatment that improves survival, the toxicity burden from ICI in AT is a substantial clinical problem urging for irAE-predictive biomarkers. EXPERIMENTAL DESIGN: We assessed post-surgical, pre-ICI treatment peripheral CD4+ and CD8+ T cells from clinical trial patients (CheckMate-915) treated with AT nivolumab (NIVO, n=130) or ipilimumab/nivolumab (COMBO, n=82). Performing RNA-seq differential gene expression analysis we tested baseline differences associated with severe (grade 3-5) irAEs and constructed an irAE-predictive model using LASSO-regularized logistic regression. RESULTS: The analysis of predicted protein-protein interactions among differentially expressed genes (DEGs) in peripheral CD4+ cells revealed significant enrichment of the spleen tyrosine kinase (SYK) pathway, associated with severe irAEs in COMBO-treated patients. This gene-expression signature predicted severe-irAE COMBO patients (chi-square p-value=0.001) with 73% accuracy and was independent of disease recurrence (p=0.79). The irAE-predictive model incorporating this gene-expression signature demonstrated 82% accuracy (chi-square p-value=8.91E-06). CONCLUSIONS: We identified baseline gene-expression differences in key immune pathways of peripheral blood T cells from COMBO-treated patients with grade 3-5 irAEs, and defined a SYK-related gene signature correctly identifying ~60% of COMBO-treated patients with grade 3-5 irAE. This finding aligns with our previous work linking anti-CTLA-4 irAEs with a germline variant associated with high SYK expression. This gene signature may serve as a baseline biomarker of severe grade 3-5 irAE risk, which is especially important in AT.

Germline immunomodulatory expression quantitative trait loci (ieQTLs) associated with immune-related toxicity from checkpoint inhibition.

BACKGROUND: Immune checkpoint inhibition (ICI) has improved clinical outcomes for metastatic melanoma patients; however, 65-80% of patients treated with ICI experience immune-related adverse events (irAEs). Given the plausible link of irAEs with underlying host immunity, we explored whether germline genetic variants controlling the expression of 42 immunomodulatory genes were associated with the risk of irAEs in melanoma patients treated with the single-agent anti-CTLA-4 antibody ipilimumab (IPI). METHODS: We identified 42 immunomodulatory expression quantitative trait loci (ieQTLs) most significantly associated with the expression of 382 immune-related genes. These germline variants were genotyped in IPI-treated melanoma patients, collected as part of a multi-institutional collaboration. We tested the association of ieQTLs with irAEs in a discovery cohort of 95 patients, followed by validation in an additional 97 patients. RESULTS: We found that the alternate allele of rs7036417, a variant linked to increased expression of SYK, was strongly associated with an increased risk of grade 3-4 toxicity [odds ratio (OR) = 7.46; 95% confidence interval (CI) = 2.65-21.03; p = 1.43E-04]. This variant was not associated with response (OR = 0.90; 95% CI = 0.37-2.21; p = 0.82). CONCLUSION: We report that rs7036417 is associated with increased risk of severe irAEs, independent of IPI efficacy. SYK plays an important role in B-cell/T-cell expansion, and increased pSYK has been reported in patients with autoimmune disease. The association between rs7036417 and IPI irAEs in our data suggests a role of SYK overexpression in irAE development. These findings support the hypothesis that inherited variation in immune-related pathways modulates ICI toxicity and suggests SYK as a possible future target for therapies to reduce irAEs.

A genome-wide association study of germline variation and melanoma prognosis.

Background: The high mortality of cutaneous melanoma (CM) is partly due to unpredictable patterns of disease progression in patients with early-stage lesions. The reliable prediction of advanced disease risk from early-stage CM, is an urgent clinical need, especially given the recent expansion of immune checkpoint inhibitor therapy to the adjuvant setting. In our study, we comprehensively investigated the role of germline variants as CM prognostic markers. Methods: We performed a genome-wide association analysis in two independent cohorts of N=551 (discovery), and N=550 (validation) early-stage immunotherapy-naïve melanoma patients. A multivariable Cox proportional hazard regression model was used to identify associations with overall survival in the discovery group, followed by a validation analysis. Transcriptomic profiling and survival analysis were used to elucidate the biological relevance of candidate genes associated with CM progression. Results: We found two independent associations of germline variants with melanoma prognosis. The alternate alleles of these two SNPs were both associated with an increased risk of death [rs60970102 in MELK: HR=3.14 (2.05-4.81), p=1.48×10-7; and rs77480547 in SH3BP4: HR=3.02 (2.02-4.52), p=7.58×10-8, both in the pooled cohort]. The addition of the combined risk alleles (CRA) of the identified variants into the prognostic model improved the predictive power, as opposed to a model of clinical covariates alone. Conclusions: Our study provides suggestive evidence of novel melanoma germline prognostic markers, implicating two candidate genes: an oncogene MELK and a tumor suppressor SH3BP4, both previously suggested to affect CM progression. Pending further validation, these findings suggest that the genetic factors may improve the prognostic stratification of high-risk early-stage CM patients, and propose putative biological insights for potential therapeutic investigation of these targets to prevent aggressive outcome from early-stage melanoma.

Tumor immunogenomic signatures improve a prognostic model of melanoma survival.

BACKGROUND: Tumor mutation burden (TMB) has been associated with melanoma immunotherapy (IT) outcomes, including survival. We explored whether combining TMB with immunogenomic signatures recently identified by The Cancer Genome Atlas (TCGA) can refine melanoma prognostic models of overall survival (OS) in patients not treated by IT. METHODS: Cox proportional-hazards (Cox PH) analysis was performed on 278 metastatic melanomas from TCGA not treated by IT. In a discovery and two validation cohorts Cox PH models assessed the interaction between TMB and 53 melanoma immunogenomic features to refine prediction of melanoma OS. RESULTS: Interferon-γ response (IFNγRes) and macrophage regulation gene signatures (MacReg) combined with TMB significantly associated with OS (p = 8.80E-14). We observed that patients with high TMB, high IFNγRes and high MacReg had significantly better OS compared to high TMB, low IFNγRes and low MacReg (HR = 2.8, p = 3.55E-08). This association was not observed in low TMB patients. CONCLUSIONS: We report a model combining TMB and tumor immune features that significantly improves prediction of melanoma OS, independent of IT. Our analysis revealed that patients with high TMB, high levels of IFNγRes and MacReg had significantly more favorable OS compared to high TMB patients with low IFNγRes and low MacReg. These findings may substantially improve current melanoma prognostic models.

Ultra Low-Coverage Whole-Genome Sequencing as an Alternative to Genotyping Arrays in Genome-Wide Association Studies.

An array-based genotyping approach has been the standard practice for genome-wide association studies (GWASs); however, as sequencing costs plummet over the past years, ultra low-coverage whole-genome sequencing (ulcWGS <0.5× coverage) has emerged as a promising alternative that provides superior genomic coverage with substantial reduction of genotyping cost. To evaluate the potential utility of ulcWGS, we performed a whole-genome sequencing (WGS) of 72 European individuals to a target coverage of 0.4× and compared its performance with the widely used Infinium Global Screening Multi-Disease Array (GSA-MD). We showed that the number of variants captured by ulcWGS is comparable with imputed GSA-MD platform, particularly for low-frequency (95.5%) and common variants (99.9%), with high imputation R2 accuracy (mean 0.93 for SNPs and 0.86 for indels). Using deep-coverage 30× WGS as the "truth" genotypes, we found that ulcWGS has higher overall nonreference genotype concordance compared with imputed GSA-MD for both SNPs (0.90 vs. 0.88) and indels (0.86 vs. 0.83). In addition, ulcWGS proved to be as sensitive as the genotyping-based method in sex imputation and ancestry prediction producing similar principal component (PC) scores. Our findings provide important evidence that the cost efficient ulcWGS of <0.5× generates high genotype accuracy, outperforming the standard genotyping arrays, making it an attractive alternative to the array-based method in next-generation GWAS design.

Immunomodulatory germline variation associated with the development of multiple primary melanoma (MPM).

Multiple primary melanoma (MPM) has been associated with a higher 10-year mortality risk compared to patients with single primary melanoma (SPM). Given that 3-8% of patients with SPM develop additional primary melanomas, new markers predictive of MPM risk are needed. Based on the evidence that the immune system may regulate melanoma progression, we explored whether germline genetic variants controlling the expression of 41 immunomodulatory genes modulate the risk of MPM compared to patients with SPM or healthy controls. By genotyping these 41 variants in 977 melanoma patients, we found that rs2071304, linked to the expression of SPI1, was strongly associated with MPM risk reduction (OR = 0.60; 95% CI = 0.45-0.81; p = 0.0007) when compared to patients with SPM. Furthermore, we showed that rs6695772, a variant affecting expression of BATF3, is also associated with MPM-specific survival (HR = 3.42; 95% CI = 1.57-7.42; p = 0.0019). These findings provide evidence that the genetic variation in immunomodulatory pathways may contribute to the development of secondary primary melanomas and also associates with MPM survival. The study suggests that inherited host immunity may play an important role in MPM development.

Autoimmune genetic risk variants as germline biomarkers of response to melanoma immune-checkpoint inhibition.

Immune-checkpoint inhibition (ICI) treatments improve outcomes for metastatic melanoma; however, > 60% of treated patients do not respond to ICI. Current biomarkers do not reliably explain ICI resistance. Given the link between ICI and autoimmunity, we investigated if genetic susceptibility to autoimmunity modulates ICI efficacy. In 436 patients with metastatic melanoma receiving single line ICI or combination treatment, we tested 25 SNPs, associated with > 2 autoimmune diseases in recent genome-wide association studies, for modulation of ICI efficacy. We found that rs17388568-a risk variant for allergy, colitis and type 1 diabetes-was associated with increased anti-PD-1 response, with significance surpassing multiple testing adjustments (OR 0.26; 95% CI 0.12-0.53; p = 0.0002). This variant maps to a locus of established immune-related genes: IL2 and IL21. Our study provides first evidence that autoimmune genetic susceptibility may modulate ICI efficacy, suggesting that systematic testing of autoimmune risk loci could reveal personalized biomarkers of ICI response.

Germline genetic host factors as predictive biomarkers in immuno-oncology.

In immuno-oncology (IO), the baseline host factors attract significant clinical interest as promising predictive biomarker candidates. Growing evidence from experimental or population-based studies suggests that the host genetic factors contribute to the immunological status of a patient as it plays out at the multiple rate-limiting steps of the cancer immunity cycle. Recent observations suggest that germline genetics may be associated with tumor microenvironment phenotypes, autoimmune toxicities and/or efficacy of immunotherapy regimens and overall cancer survival. Despite these highly intriguing indications, the potential of germline genetic factors as personalized biomarkers of immune-checkpoint inhibition (ICI) remains vastly unexplored. Here, we review the rationale for exploring the germline genetic factors as novel biomarkers predictive of IO outcomes, including ICI efficacy, toxicity and survival, and discuss the comprehensive approaches for the identification of such germline genetic indicators. In addressing the current limitations, we highlight a need for large collaborative consortia in these efforts. We also outline possible avenues for incorporating germline genetic factors into emerging multifactorial tools for a more personalized prediction of ICI outcomes.

Association between number of children and carotid intima-media thickness in Bangladesh.

Previous studies on the association between number of children and carotid intima-media thickness (cIMT) were limited to Western populations. Pregnancy in women is associated with physiologic changes that may influence the risk of cardiovascular disease. Comparing the association between number of children and cIMT in men and women can provide insights on whether the association may be due to pregnancy. We investigated the association between number of children and cIMT among 718 female (mean age 37.5 years) and 417 male participants (mean age 41.3 years), randomly selected from the Health Effect of Arsenic Longitudinal Study (HEALS), a population-based cohort study in Bangladesh. Multivariate linear regression was used to assess the association and to control for education attainment, history of diabetes, age, smoking, betel use, BMI, systolic blood pressure, and diastolic blood pressure. The average number of children was 4.43 for women and 3.74 for men. There were no nulliparous women. We observed a positive association between number of children and cIMT in women. Mean cIMT increased by 4.5 µm (95% CI, 0.8-8.1) per increment of one birth (P = 0.02). Compared to women with two children, cIMT in women with 4 children and ≥5 children was 23.6µm (95%CI, 2.6-44.7; P = 0.03) and 25.1 µm (95%CI, 3.5-46.6; P = 0.02) greater, respectively. The association was not modified by BMI, SBP, betel use or age. Data in men showed no evidence of association (P = 0.4). The finding suggests a role of high parity in atherosclerosis in women of a low-income, high parity population.