Molecular staging by multimarker reverse transcriptase-polymerase chain reaction assay of lymphatic drainage and blood from melanoma patients after lymph node dissection

Abstract

Reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated detection of melanoma cells may be a prognostic factor for disease outcome. We investigated the presence of melanoma cells in lymphatic drainage and blood in melanoma patients after lymph node dissection (LND) via the highly sensitive multimarker (MM) RT-PCR assay. We collected 24-h lymph fluid (LY) and peripheral blood (BL) from 107 stage III melanoma patients after radical LND (59 axillary and 48 ilioinguinal LND). Tyrosinase, MART1 and uMAGE mRNA levels were determined by RT-PCR to detect melanoma cells, and the presence of at least one marker signified a positive result. All patients underwent follow-up (median for survivors, 21 months, range: 4-37 months). Forty patients (37.4%) were positive for LY MM RT-PCR and 28 (26.2%) were positive based on BL MM RT-PCR. No differences for disease-free survival (DFS) curves according to BL MM RT-PCR were observed, but we found significant differences in the estimated 24-month DFS rate for patients with at least one marker and those without any marker in lymph fluid [18.9% (95% confidence interval: 1.4-37.5%) and 42.1% (95% confidence interval: 29.7-54.5%), median: 9.9 and 15.3 months, respectively] (P=0.04). Detection of multiple markers in lymph fluid correlated with shorter DFS. Approximately 37% of lymph fluid after radical LND were positive by MM RT-PCR, which correlated significantly with early melanoma recurrences and shorter survival. The LY MM RT-PCR seems to be an effective prognostic tool for stage III melanoma patients. The MM RT-PCR analysis of single peripheral blood sample in these patients did not have additional prognostic value.

Prognostic value of microphthalmia-associated transcription factor and tyrosinase as markers for circulating tumor cells detection in patients with melanoma

“…Indeed, several studies have shown that multiple-marker analysis allows for the detection of circulating melanoma cells in a higher percentage of melanoma patients than single-marker analysis [9][10][11][24][25][26][27][28]30,36]. Among the markers analyzed in these studies, some have shown high specificity for melanoma cells (MART-1, TRP-1, TRP-2, MAGE-3, paired box homeotic gene transcription factor 3, b1-4-N-acetylgalactosaminyltransferase), whereas others were found to be positive also in healthy volunteer samples (p97, MUC18, gp100) [9][10][11][24][25][26][27]30,36]. Our study has confirmed the advantage of multi-marker analysis for the detection of circulating melanoma cells that had been shown in earlier studies.…”

“…Several studies have shown that the presence of circulating melanoma cells detected by RT-PCR is associated with shorter overall and progression-free survival [9][10][11][12][13][14][15][16][17][18][19][20][21][28][29][30][31][32]. But other studies did not confirm the prognostic value of RT-PCR detection of circulating melanoma cells [22,23,[33][34][35][36]. metastases and negative values of investigated markers.…”

“…In 23 different studies analyzed by Tsao et al [6], 45% of melanoma patients with distant metastases were positive for tyrosinase on average. It has been shown that multiple-marker analysis enables detection of circulating melanoma cells in a higher percentage of melanoma patients than single-marker (usually tyrosinase) analysis [9][10][11][24][25][26][27][28]30,36].…”

Multimarker Reverse Transcriptase-Polymerase Chain Reaction Assay in Lymphatic Drainage and Sentinel Node Tumor Burden

“…The samples were centrifuged again, and the pellets were collected. Total cellular RNA was prepared from fresh or frozen pellets as described previously 12. RNA was further purified from trace amounts of DNA according to the method recommended by the DNase I (RNase-free) producer (Gibco-BRL), except that one-fourth of the recommended amount of DNase I was used.…”

“…We have demonstrated previously with using two-marker or multimarker reverse transcriptase-polymerase chain reaction (MM RT-PCR) technique in the group of cutaneous melanomas with regional node metastases (microscopic and macroscopic), that positive result of this assay correlated significantly with early melanoma recurrence and shorter survival 10–12…”