Prognostic Significance of Molecular Upstaging of Paraffin-Embedded Sentinel Lymph Nodes in Melanoma Patients

Abstract

Purpose-Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome.Patients and Methods-qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (β1→4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3).Results-Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P < .0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of ≥ one marker in histopathologynegative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P = .0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1).Conclusion-Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

“…Sentinel lymph node tissue that is negative by histology and immunohistology may have RT-PCR evidence of possible occult metastases. [39][40][41][42] However, because of the inevitable cell destruction during RNA extraction, solution-phase RT-PCR cannot identify the cellular source of any amplified signals for mRNA. In this study, we have shown that it is possible to detect MART-1 mRNA and TRP-2 mRNA in archival FFPE tissue sections of melanoma by a one-step RT in situ PCR technique.…”

Molecular markers in malignant cutaneous melanoma: Gift horse or one‐trick pony?

“…Patients with positive SLNs by conventional and qRT assessment experience the worst outcomes, whereas those with negative SLNs using these combined techniques have the best prognosis. Interestingly, the risk associated with qRT-positive SLNs is not uniform; it seems to vary directly with the number of molecular markers detected [Takeuchi et al, 2004].…”

“…Investigators have improved the sensitivity and specificity of the RT-PCR assay by employing multi-marker panels in peripheral blood analysis [Hoon et al, 1995;Curry et al, 1998;Bostick et al, 1999;Hoon et al, 2000;Boi et al, 2002;Kuo et al, 2003;Takeuchi et al, 2004]. Hoon et al [1995] used a multi-marker panel of tyrosinase and tumor-associated antigens melanotransferrin (p97), melanoma antigen gene A3 (MAGE-3) and an adhesion-related glycoprotein, melanoma cell adhesion molecule (MUC18) to assay the blood of 119 stage I-IV melanoma patients by RT-PCR.…”

“…We recently developed a blood-based qRT assay incorporating markers initially used to upstage SLN [Takeuchi et al, 2004]. Serial blood draws assessed by multi-marker qRT successfully detected tumor cells in the blood of stage III melanoma patients receiving neoadjuvant biochemotherapy (BC) [Koyanagi et al, 2005].…”

“…qRT is particularly valuable when detecting occult tumor cells, as this assay better distinguishes differential biomarker expression among tumors, even at low copy numbers. Takeuchi et al [2004] performed a qRT assay on 53 histologically-confirmed positive and 162 negative PE SLN using a multi-marker panel of MART-1, MAGE-3, b1 > 4-N-acetylgalactosaminyl-transferase (GalNac-T), and paired-box homeotic gene transcription factor 3 (Pax3). GalNac-T is a key enzyme in the synthesis of cell-surface gangliosides GM2 and GD2 that are abundant in melanoma cells.…”