Detection of circulating tumor cells (CTC) in peripheral blood has been investigated for its prognostic ability, and its potential to measure the effectiveness of treatment(s) in patients with melanoma. However, a highly sensitive and specific assay is required to detect CTC in patients’ blood. We have developed a multimarker quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assay for detecting CTC directly from peripheral blood specimens without the need of separating CTC from leukocytes (PBL). We selected and optimized four mRNA biomarkers (MART-1/Melan-A, MAGE-A3, PAX3, and GalNAc-T) for detection and prediction of clinical outcome in melanoma patients. Our protocol has both high sensitivity and specificity for CTC in blood specimens—detecting approximately one to five melanoma cells in 107 PBL. We have demonstrated the significance of this assay for serial bleed assessment of CTC in clinical trials and for daily clinical usage.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This study was supported by Dr. Miriam & Sheldon G. Adelson Medical Research Foundation, Leslie and Susan Gonda (Goldshmied) Foundation (Los Angeles, CA), Ruth and Martin H. Weil Fund (Los Angeles, CA), and Grant No. P01 CA012582 Project II and Core from the National Institutes of Health/National Cancer Institute, USA. This work was supported in part by the fund from Japan Society for the Promotion of Science (JSPS) for the “Institutional Program for Young Researcher Overseas Visits.”
Balch CM, Gershenwald JE, Soong SJ et al (2009) Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 27(36):6199–6206CrossRefPubMedGoogle Scholar
AJCC Cancer Staging Manual (2010) In: Edge S, Byrd D, Compton C et al. (eds) 7th edn. Springer, NewYork, NYGoogle Scholar
Hoshimoto S, Faries MB, Morton DL et al (2011) Assessment of prognostic circulating tumor cells in a phase III trial of adjuvant immunotherapy after complete resection of stage IV melanoma. Ann Surg 255(2):357–362CrossRefGoogle Scholar
Koyanagi K, O'Day SJ, Gonzalez R et al (2005) Serial monitoring of circulating melanoma cells during neoadjuvant biochemotherapy for stage III melanoma: outcome prediction in a multicenter trial. J Clin Oncol 23(31):8057–8064CrossRefPubMedGoogle Scholar
Hoon DS, Bostick P, Kuo C et al (2000) Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence. Cancer Res 60(8):2253–2257PubMedGoogle Scholar
Nicholl MB, Elashoff D, Takeuchi H et al (2010) Molecular upstaging based on paraffin-embedded sentinel lymph nodes: ten-year follow-up confirms prognostic utility in melanoma patients. Ann Surg 253(1):116–122CrossRefGoogle Scholar
Takeuchi H, Morton DL, Kuo C et al (2004) Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients. J Clin Oncol 22(13):2671–2680CrossRefPubMedGoogle Scholar
Koyanagi K, O'Day SJ, Boasberg P et al (2010) Serial monitoring of circulating tumor cells predicts outcome of induction biochemotherapy plus maintenance biotherapy for metastatic melanoma. Clin Cancer Res 16(8):2402–2408CrossRefPubMedGoogle Scholar
Mocellin S, Hoon D, Ambrosi A et al (2006) The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis. Clin Cancer Res 12(15):4605–4613CrossRefPubMedGoogle Scholar
Cristofanilli M, Budd GT, Ellis MJ et al (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351(8):781–791CrossRefPubMedGoogle Scholar
Kitago M, Koyanagi K, Nakamura T et al (2009) mRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with high molecular weight melanoma-associated antigen-specific monoclonal antibody beads. Clin Chem 55(4):757–764CrossRefPubMedGoogle Scholar
Koyanagi K, Kuo C, Nakagawa T et al (2005) Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: relation to disease stage in melanoma patients. Clin Chem 51(6):981–988CrossRefPubMedGoogle Scholar