Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation

Abstract

Background: 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 (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.

Methods: CTCs 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.

Results: We 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 BRAF^A598V.

Conclusions: CTCs 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.

Keywords: BRAF; BRAF/MEK inhibitor; Circulating tumor cell; Heterogenous mutation; Melanoma.

Fig. 1

Number of circulating tumor cells (CTCs) at stages 0–I, II, and III. a Number of CTCs in healthy individuals and melanoma patients at stages 0–I, II, and III. CTCs were counted using blood samples collected before surgical resection of the primary tumor and sentinel node biopsy. b, c Pathologic features of stage I primary melanoma. Atypical melanocytes invaded the dermis in a nodular and diffuse manner. Tumor thicknesses were 0.6 (b) and 0.8 mm (c). Clark levels were III (b) and IV (c) (× 100, hematoxylin/eosin staining)

Fig. 2

Number of CTCs during treatment with BRAF/MEK inhibitors. MMbraf1, MMbraf2, MMbraf3, and MMbraf4 were diagnosed with metastatic BRAF^v600E/K melanoma. a Monitoring the number of CTCs during the clinical course in MMbraf1. The graph shows the number of CTCs and the LDH level (upper limit of normal, 230 IU/L). Arrows indicate lung metastases in computed tomography (CT) imaging. b Monitoring the number of CTCs during the clinical course in MMbraf2. Circles indicate a right axillary lymph node (LN) metastasis in CT imaging. c Monitoring the number of CTCs during the clinical course in MMbraf3. Arrowheads indicate a liver metastasis in CT imaging. d Monitoring the number of CTCs during the clinical course in MMbraf4. Arrows indicate a right inguinal LN metastasis in CT imaging

Fig. 3

BRAF genotype and the number of CTCs in MMbraf5. a BRAF sequence chromatograms of the primary tumor and a metastatic lymph node. b BRAF sequence chromatograms of CTCs during treatment with BRAF/MEK inhibitors. c Diversity of BRAF mutations surrounding codon 600 in MMbraf5. d Number of CTCs during treatment and clinical outcome response in patient MMbraf5. Arrowheads indicate lung metastasis in CT imaging on Days − 40, 70, and 148. Far-right CT imaging shows the appearance of interstitial pneumonia on Day 148. In the upper graph, solid and dotted lines indicate the number of total CTCs and the LDH level, respectively, during the clinical course in MMbraf5. In the lower graph, pink and purple lines indicate the numbers of BRAF^V600E CTCs and total CTCs with BRAF mutations, respectively