Abstract
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.
Highlights
While molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses
Circulating tumor cells (CTC) detection in patients with stage 0–III melanoma Peripheral blood was collected from melanoma patients with stage 0–III disease before surgical resection of the primary tumor and sentinel node biopsy (Table S2)
Monitoring CTCs in BRAF-mutated advanced melanoma To evaluate the usefulness of CTCs as biomarkers of responsiveness to treatment, we analyzed blood from five patients with BRAF-mutated melanoma (MMbraf1–5), who were treated with BRAF/MEK inhibitors (Table S1)
Summary
While molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. 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. Targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma. Prediction of drug response and optimization of treatment order are required. Pretreatment tumor biopsies provide useful information, including driver mutations, expression levels of programmed death-ligand 1, infiltration of CD8-positive T-cells within tumors, and microsatellite instability. Those markers are insufficient for predicting response to treatment because they reflect tumor status at a single time point. Additional biopsies may be desired during treatment, tissue biopsies may not accurately reflect systemic tumor status due to intertumoral and intratumoral heterogeneity [3]
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