Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma
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ABSTRACT:
SUBMITTER: Mr. Mark Sementsov
PROVIDER: S-SCDT-10_1038-S44321-024-00082-6 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:BackgroundWhile molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. Circulating tumor cells (CTCs) are released from primary and/or metastatic tumors into the peripheral blood. We examined whether CTCs have potential as biomarkers by checking the number of CTCs, as well as the BRAF genotype of individual CTCs, in melanoma patients undergoing BRAF/MEK inhibitor treatment.MethodsCTCs were isolated from peripheral blood using a high-density dielectrophoretic microwell array, followed by labeling with melanoma-specific markers (MART-1 and/or gp100) and a leukocyte marker (CD45). The numbers of CTCs were analyzed in fifteen patients with stage 0-III melanoma. Furthermore, changes in CTC numbers were assessed in five patients with stage IV melanoma at four time points during BRAF/MEK inhibitor treatment, and the BRAF genotype was analyzed in CTCs isolated from one patient.ResultsWe examined CTCs in patients with stage 0-III (five samples per stage: stage 0-I, stage II, and stage III), and detected CTCs even in patients with early disease (stage 0 and I). Interestingly, recurrence occurred in the lymph nodes of one stage I patient 2 years after the detection of a high number of CTCs in the patient's blood. The total number of CTCs in four of five patients with stage IV melanoma fluctuated in response to BRAF/MEK inhibitor treatment, suggesting that CTC number has potential for use as a drug response marker in advanced disease patients. Interestingly, one of those patients had CTCs harboring seven different BRAF genotypes, and the mutated CTCs disappeared upon BRAF/MEK inhibitor treatment, except for those harboring BRAFA598V.ConclusionsCTCs are present even in the early stage of melanoma, and the number of CTCs seems to reflect patients' responses to BRAF/MEK inhibitor treatment. Furthermore, genetic heterogeneity of BRAF may contribute to resistance to BRAF/MEK inhibitors. Our findings demonstrate the usefulness of CTC analysis for monitoring responses to targeted therapies in melanoma patients, and for understanding the mechanism of drug resistance.
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Project description:Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml(-1)). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.
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Project description:Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2'-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate ∼2-6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and ∼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design.
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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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