The New Identified Biomarkers Determine Sensitivity To Immune Check-point Blockade Therapies In Melanoma

ObjectiveImmune checkpoint blockade(ICB)therapy,such as anti-CTLA-4 and anti-PD-1,have achieved remarkable clinical benefit in advanced melanoma.However,effective clinical use of ICB agents is encumbered by the high rate of innate resistance(60%–70%).Here,we sought to assess genomic features related to clinical response and survival patterns in order to gain insights into potential mechanistic basis for immune response and identification of ICB responsders.MethodsWe aggregated and uniformly analyzed the somatic mutation data from six studies of 336 melanoma patients treated by immune checkpoint blockade(CTLA-4/PD-1).Clinicopathological information including age,gender,stage,immune blockades type,response status,and survival were curated from these studies.Gene expression profile(GEP)of 72 pre-treatment samples were avaiable for analysis,the HLA types,predicted MHC binding affinity and peptide sequence of neoantigen were curated from 224 samples.We identified significantly mutated genes(SMGs)in melanoma by using Mut Sig CV algorithm,and explored its association with immunotherapy survival by log-rank test.The CIBERSORT algothrim was used to estimate the relative abundance of tumor infiltrating lymphocyte cells with ICB response status.The differentiall expressed genes sets and signaling pathways were estimated by GSEA.Besisdes,we also extracted the mutational signatures based on Bayesian variant nonnegative matrix factorization.The neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to IEDB peptides was applied to identify immune responders.ResultsA median of 331 mutations per sample(range from 1 to 9210)in a total of 222,580 coding somatic mutations were collected from six previously published research.Overall,we found that higher tumor mutation load was significantly associated with superior immune response(median TML,390 vs.270,P = 0.0345).Mut Sig CV algorithm identified 60 significantly mutated genes(SMGs).Apart from well-known driver oncogenes in melanoma(e.g.BRAF,NRAS,NF1,PREX2,ARID2,PTEN et.al),we identified eight new SMGs(i.e.COL5A1,COL28A1,SEMA3 E,DGKG,RAPGEF5,GLDN,NCF2 and RCAN2),whose mutations were associated with improved overall survival(log-rank test,P < 0.05).Mutations in COL5A1 was associated with better immune response(log-rank test,P = 0.031;Fisher's excat exact,P = 0.045).The association between COL5A1 mutations and immunotherapy survival remained statistically significant after taking into account age,gender and tumor stage(HR,0.57 [95%CI,0.35 to 0.94],P = 0.028).The TML in samples of COL5A1 mutations are significantly higher than those without mutations.In the subgroup analysis,we found that patients with COL5A1 mutation in anti-PD-1 cohort and anti-CTLA-4 cohort exhibited a trend with better survival outcome.Furthermore,the primary melanoma GEPs analysis(CIBERSORT algorithm)indicated that CD8+ T cells,activated NK cells were enriched in COL5A1 mutant group,nevertheless,resting memory T cells and Neutrophils were enriched in COL5A1 wild-type group.Gene set enrichment analysis revealed that signaling pathways involved in cytokine mediated immune system,antigen processing presentation,cell cycle and IFN stimulation were significantly upregulated in samples with COL5A1 mutations compared with those without COL5A1 mutation.The overall mutational pattern in melanoma was dominated by C>T mutations.We extracted six mutational signatures from melanoma somatic mutation data with varying mutational activities and annotated against the COSMIC signature nomenclature(ie.signature 7,75.9%;signature 11,14.8 %;signature 3,4.2%;signature 1,2.5%;signature 26,1.5% and signature 18,1.1%),We observed that signature 3 was significantly associated with immune resistence in multivariable regression analysis with age,sex and stage(OR,2.79[95%CI,1.18-7.40],P = 0.026).Cox regression analysis also indicated that patients with signature 3 exhibited a worse survival outcome(HR,1.53[95%CI,1.00 to 2.34],P = 0.049).Besides,patients with signature 3 in anti-PD-1 cohort were also significantly associated with immune resistance,and a similar tendency in anti-CTLA-4 cohort but not statistically significant.High neoantigen quality was significantly associated with favorable overall survival in the neoantigen quality model(log-rank test,P = 0.009).Association of high neoantigen quality remained statistically significant in multivariate Cox model with age,gender,stage and hypermutation status(HR,0.56 [95%CI,0.38 to 0.82],P = 0.003).ConclusionOur study used by far the largest number of samples to identify new molecular markers(e.g.mutant COL5A1,mutational signature 3 and neoantigen quality)for the patients who may benefit from ICB treatment.We found that patients with COL5A1 mutation and high neoantigen quality were associated with better survival,whereas presence of mutational signature 3 was associated with worse survival.Therefore,we combined COL5A1 mutation,high neoantigen quality and lack of mutational signature 3 as a single marker.Samples with the combined marker exhibited better association with ICB response than either of each individual marker.