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Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma

ABSTRACT: Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p=0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

SUBMITTER: Mr. Mark Sementsov

PROVIDER: S-SCDT-10_1038-S44321-024-00082-6 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundHuman malignant melanoma is a highly aggressive, heterogeneous and drug-resistant cancer. Due to a high number of clones, harboring various mutations that affect key pathways, there is an exceptional level of phenotypic variation and intratumor heterogeneity (ITH) in melanoma. This poses a significant challenge to personalized cancer medicine. Hitherto, it remains unclear to what extent the heterogeneity of melanoma affects the immune microenvironment. Herein, we explore the interaction between the tumor heterogeneity and the host immune response in a melanoma cohort utilizing The Cancer Genome Atlas (TCGA).MethodsClonal Heterogeneity Analysis Tool (CHAT) was used to estimate intratumor heterogeneity, and immune cell composition was estimated using CIBERSORT. The Overall Survival (OS) among groups was analyzed using Kaplan-Meier curves with the log-rank test and multivariate cox regression. RNA-seq data were evaluated to identify differentially expressed immunomodulatory genes. The reverse phase protein array (RPPA) data platform was used to validate immune responses at protein level.ResultsTumors with high heterogeneity were associated with decreased overall survival (p = 0.027). High CHAT tumors were correlated with less infiltration by anti-tumor CD8 T cells (p = 0.0049), T follicular cells (p = 0.00091), and M1 macrophages (p = 0.0028), whereas tumor-promoting M2 macrophages were increased (p = 0.02). High CHAT tumors correlated with a reduced expression of immunomodulatory genes, particularly Programmed Cell Death 1 (PD1) and its ligand PD-L1. In addition, high CHAT tumors exhibited lower immune Cytotoxic T lymphocytes (CTLs)-mediated toxicity pathway score (p = 2.9E-07) and cytotoxic pathway score (p = 2.9E-08). High CHAT tumors were also associated with a lower protein level of immune-regulatory kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) (p = 3.4e-5) and spleen tyrosine kinase (SYK) (p = 0.0011).ConclusionsHighly heterogeneous melanoma tumors are associated with reduced immune cell infiltration and immune response activation as well as decreased survival. Our results reveal that intratumor heterogeneity is an indicative factor for patient survival due to its impact on anti-tumor immune response.

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Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma

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