The first study demonstrating the utility of multiple molecular MAA CTC markers in cutaneous melanoma patients using a large cohort of multiple clinical stages was reported in 1995 ^[23][50]. HereIn this study, four MAA gene markers: TYR, melanotransferrin (MELTF), melanoma cell adhesion molecule (MCAM), and MAGEA3 were assessed in PBMCs directly from a 10 mL blood sample, without mRNA isolation. The rationale of the multi-marker RT-PCR has been that melanoma cells are heterogeneous and could vary in gene expression during tumor progression or treatment. Within the 119 patients with AJCC stage I–IV disease, CTC detection rates were significantly higher when using four MAA markers rather than a single marker. There was a significant correlation between RT-PCR marker positivity to AJCC stage and disease progression. This initiall study demonstrated the potential of direct multi-marker MAA assessment for CTC detection in PBMCs, and from then, multi-marker RT-PCR became one of the most widely used CTC assays in cutaneous melanoma patients.

Most of the RT-PCR studies reported a significant correlation between positive MAA markers and disease status and/or survival, except for the study by Piotr et al. in 2008 ^[24][34]. HereIn this study, lymph-fluid and peripheral blood were collected from 107 AJCC stage III cutaneous melanoma patients after radical lymph node dissection. Three MAA gene markers TYR, MLANA, and universal MAGE (uMAGE) were detected by multi-marker RT-PCR. Lower estimated twenty-four-month DFS rates were observed for patients with at least one marker (18.9%; 95% CI, 1.4–37.5%; median, 9.9 months) compared to those without any marker (42.1%; 95% CI, 29.7–54.5%; median, 15.3 months) (p = 0.04) in lymph-fluid. Although, analysis of the PBMCs did not have additional prognostic value. Reasons for this phenomenon may be the time of blood collection and the volume of the samples collected. Blood samples were collected from the patients between 24 and 48 h after surgery in thereis study, in which surgical intervention may have affected the CTC analysis. Further, the collected sample volume herein this study was 5 mL per patient. Sample volumes ranged from 9–10 mL in the three other studies on CTCs with AJCC stage III melanoma patients included ^[25][26][27]in this review [35,41,48], and all three studies reported a significant correlation between the presence of CTCs and prognosis. Standardization of blood collection and downstream assays are critical factors in CTC analysis, and automated CTC enrichment and detection methodology may provide reliable and reproducible outcomes. This will be further discussed in the “CTC Enrichment and Detection using CELLSEARCH^® System” section. Unique features of thereis study are that they focused on lymph fluid as another source of CTCs, and that they utilized uMAGE for CTC detection.

Within the human MAGE family, MAGEA is the most characterized and identified gene in many cancers including melanoma ^[28][51]. MAGEA family consists of ≥12 major members that are exclusively expressed in human cancers as well as male germline cells (testis and placenta) ^[29][30][52,53]. Among them, MAGEA1, A2, and A3 were originally well-described antigens in melanoma and cancer originating from the testis ^[31][54]. MAGEA1 and 3 have high specificity and expression in various malignancies; however, individual family members are expressed at different frequencies ^[31][54]. Therefore, uMAGEA primer and probe covering MAGEA1, -A3, -A5, -A6, and -A12 was developed by Miyashiro et al. in 2001 ^[32][55]. This was to improve the logistics of assay setup and to obtain a comprehensive multi-marker MAGEA MAA family profile in a single assay. HereIn this study, the uMAGEA assay increased melanoma detection by 13% compared with the MAGEA1 assay alone, and by 17% compared with the MAGEA3 assay alone in melanoma tumors. Further, the uMAGEA assay detected CTCs in 24% of melanoma patients’ PBMCs. Although not frequently used in recent studies, uMAGEA detection may provide a more practical and sensitive approach for CTC detection compared to a single MAGEA family member assay, also requiring less mRNA compared to individual MAGEA marker assays.

All five multicenter studies included in theris article utilized multi-marker RT-PCR for CTC detection. The prognostic impact of CTCs in advanced-stage melanoma was analyzed in all studies, except for the report by Scoggins et al. in 2006 ^[33][27], which enrolled AJCC stage I/II patients. This was the largest researchtudy included herein this review, which applied multi-marker RT-PCR using primers for MLANA, pre-melanosome protein (PMEL), and TYR for CTC detection in PBMCs from serial bleeds collected from 820 patients. One hundred and fifteen (14%) of the patients had evidence of at least one RT-PCR marker present at one of their follow-ups (median follow-up, 37 months). Analysis of individual markers did not differentiate survival outcomes. Although the analysis of baseline blood samples did not show clinical significance in herethis study, the number of MAA CTC markers expressed revealed that DFS and DDFS were significantly worse for patients with ≥1 marker detected at any point during follow-up compared to patients with negative results (p = 0.006 and 0.03, respectively).

In another study by Fusi et al. ^[25][35], serial testing for TYR and MLANA by multi-marker RT-PCR was performed for a subset of patients enrolled in EORTC 18991 phase III trial ^[34][56], which evaluated the efficacy and toxicity of pegylated interferon (IFN) versus observation in resected AJCC stage III cutaneous melanoma patients. It This study aimed to evaluate the prognostic importance of CTC detection in AJCC stage III melanoma patients after sentinel lymph node (SLN) or regional lymph node dissection. Among 299 patients who underwent final analysis, positive versus negative CTCs at a given time point had no prognostic impact on DDFS. However, Cox time-dependent analysis indicated a significantly higher risk of developing distant metastasis for patients with positive CTCs compared to negative CTCs (HR, 2.23; 95% CI, 1.40–3.55; p < 0.001). These two studies indicate the importance of multi-marker RT-PCR analysis in serial bleeds and the value of CTC analysis even in early-stage melanoma.

Two recent multicenter trials included in thereis review were both reported in 2012, using multi-marker RT-PCR for CTC detection against stage III/IV diseases ^[35][26][40,41]. The study by Hoshimoto et al. ^[35][40] analyzed CTCs from patients in a prospective-multicenter international phase III clinical trial to evaluate the efficacy of irradiated whole-cell melanoma vaccine (Canvaxin) (ClinicalTrials.gov identifier: NCT00052156) ^[36][57]. After complete metastasectomy, patients rendered disease-free were prospectively randomized to adjuvant therapy with Canvaxin plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. The blood specimens were collected pre-treatment (n = 244) and during treatment (n = 214) and were evaluated by multi-marker MAA RT-PCR for MLANA, MAGEA3, and paired box 3 (PAX3) CTC mRNA biomarkers. ≥1 CTC MAA biomarkers were detected in 54% of the blood samples from pre-treated patients and in 86% of patients over the first three months of treatment. Median follow-up for the patients was 21.9 months, and during that follow-up, 71% of patients recurred and 48% expired. CTC MAA mRNA levels were not associated with known prognostic factors or treatment arm. In multivariable analysis, CTC status (>0 biomarker versus 0 biomarker) in pre-treatment blood samples were significantly associated with DFS (HR, 1.64; p = 0.002) and OS (HR, 1.53; p = 0.028). Serial bleeds CTC status was also significantly associated with DFS (HR, 1.91; p = 0.02) and OS (HR, 2.57; p = 0.012).

In another study by Hoshimoto et al. ^[26][41], CTCs were assessed in melanoma patients with SLN metastases in a phase III international multicenter clinical trial ^[36][57]. AJCC stage IIIB-D melanoma patients after metastatic lymph node resection are at high risk of developing distant organ metastasis ^[37][58]. However, there were no specific blood biomarkers to determine which patients will develop metastasis and should be triaged onto adjuvant therapy. Blood specimens were collected from patients with melanoma (n = 331) who were clinically disease-free after having a complete lymph node dissection (CLND) before entering onto a randomized adjuvant melanoma cell vaccine plus BCG versus placebo plus BCG trial from 30 centers. Blood was assessed by a multi-marker RT-PCR assay using MLANA, MAGEA3, and beta-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) as MAA markers. Individual CTC biomarker detection rates ranged from 13.4–17.5%. Cox regression analyses were used to evaluate the prognostic significance of CTC status for disease recurrence and disease-specific survival (DSS). There was no association of CTC status (≤1 biomarker versus ≥2 biomarkers) with known clinical or pathologic prognostic variables. However, ≥2 positive CTC MAA biomarkers were significantly associated with reduced DDFS (HR, 2.13; p = 0.009), RFS (HR, 1.70; p = 0.046), and DSS (HR, 1.88; p = 0.043) in multivariable analyses. The CTC MAA marker B4GALNT1 used in thereis study was originally described by Kuo et al. in 1998 ^[38][59]. B4GALNT1 is involved in the synthesis of GM2 and GD2, key melanoma cell surface glycosphingolipids ^[39][40][60,61], which is frequently found in advanced-stage melanomas and in patients showing more aggressive melanoma tumors, whereas normal donor PBMCs were negative for B4GALNT1 mRNA expression. B4GALNT1 was used in four multi-marker RT-PCR studies included in herethis review ^{[41][42][26][43]}[28,37,41,49], and all four studies reported a significant correlation between CTC detection and survival outcomes for metastatic melanoma patients.

The above multicenter studies demonstrate the utility of CTC MAA multi-marker RT-PCR assays in patients who rendered disease-free for the monitoring of recurrent distant metastatic diseases, reducing the bias observed in single-center trials.