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Prospective Molecular Profiling of Circulating Tumor Cells from Patients with Melanoma Receiving Combinatorial Immunotherapy.

ABSTRACT: BACKGROUND:Blood molecular profiling of circulating tumor cells (CTCs) can enable monitoring of patients with metastatic melanoma during checkpoint inhibitor immunotherapy (CII) and in combination with targeted therapies. We developed a microfluidics-based CTC platform to explore CTC profiling utility in CII-treated patients with melanoma using a melanoma messenger RNA (mRNA)/DNA biomarker panel. METHODS:Blood samples (n = 213) were collected prospectively from 75 American Joint Committee on Cancer-staged III/IV melanoma patients during CII treatment and those enriched for CTCs. CTC profiling was performed using 5 known melanoma mRNA biomarkers and BRAF V600E DNA mutation. CTC biomarker status associations with clinical outcomes were assessed. RESULTS:CTCs were detected in 88% of blood samples from patients with melanoma. CTC-derived biomarkers and clinical variables analyzed using classification and regression tree analysis revealed that a combination of lactate dehydrogenase, CTC-mRNA biomarkers, and tumor BRAF-mutation status was indicative of clinical outcomes for patients with stage IV melanoma (n = 52). The panel stratified low-risk and high-risk patients, whereby the latter had poor disease-free (P = 0.03) and overall survival (P = 0.02). Incorporation of a DNA biomarker with mRNA profiling increased overall CTC-detection capability by 57% compared to mRNA profiling only. RNA sequencing of isolated CTCs identified significant catenin beta 1 (CTNNB1) overexpression (P <0.01) compared to nondisease donor blood. CTC-CTNNB1 was associated with progressive disease/stable disease compared to complete-responder patient status (P = 0.02). Serial CTC profiling identified subclinical disease in patients who developed progressive disease during treatment/follow-up. CONCLUSIONS:CTC-derived mRNA/DNA biomarkers have utility for monitoring CII, targeted, and combinatorial therapies in metastatic melanoma patients.

SUBMITTER: Lin SY

PROVIDER: S-EPMC7193771 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Prospective Molecular Profiling of Circulating Tumor Cells from Patients with Melanoma Receiving Combinatorial Immunotherapy.

Lin Selena Y SY Chang Shu-Ching SC Lam Stella S Irene Ramos Romela R Tran Kevin K Ohe Shuichi S Salomon Matthew P MP Bhagat Ali Asgar S AAS Teck Lim Chwee C Fischer Trevan D TD Foshag Leland J LJ Boley Christine L CL O'Day Steven J SJ Hoon Dave S B DSB

Clinical chemistry 20200101 1

<h4>Background</h4>Blood molecular profiling of circulating tumor cells (CTCs) can enable monitoring of patients with metastatic melanoma during checkpoint inhibitor immunotherapy (CII) and in combination with targeted therapies. We developed a microfluidics-based CTC platform to explore CTC profiling utility in CII-treated patients with melanoma using a melanoma messenger RNA (mRNA)/DNA biomarker panel.<h4>Methods</h4>Blood samples (n = 213) were collected prospectively from 75 American Joint C ...[more]

PMID: 31672856

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Project description:BackgroundAbout 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients.MethodsCTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn's post-test was used for comparisons between groups. Statistical significance was set at p?<?0.05.ResultsEpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis.ConclusionsOur results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.

Project description:Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.

Dataset Information

Prospective Molecular Profiling of Circulating Tumor Cells from Patients with Melanoma Receiving Combinatorial Immunotherapy.

Publications

Prospective Molecular Profiling of Circulating Tumor Cells from Patients with Melanoma Receiving Combinatorial Immunotherapy.

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