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From the Department of Molecular Oncology, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, California.
Correspondence: Address correspondence and reprint requests to: Dave S. B. Hoon, MSc, PhD, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404; Fax: 310-449-5282; E-mail: Hoon{at}jwci.org
ABSTRACT
Lymphatic mapping and sentinel lymph node (SLN) biopsy have been validated in malignant melanoma. We hypothesized that histopathologically negative SLNs may contain occult metastases that can be identified by sensitive molecular approaches such as quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. We previously demonstrated that RT-PCR-based detection of occult metastasis in frozen SLNs upstaged early-stage cutaneous melanomas and that a multiple specific mRNA marker (MM) RT-PCR assay was an independent predictor of disease outcome. We recently developed an MM RT-PCR using electrochemiluminescence (ECL) and quantitative real-time detection assays. These assays can detect occult metastatic melanoma cells in paraffin-embedded (PE) SLNs and are of clinical utility for retrospective analysis of specimens with long-term follow-up. The molecular detection of occult metastasis in PE SLNs has significant clinicopathologic and logistic advantages over molecular analysis of frozen SLNs.
Key Words: Melanoma • RT-PCR • Sentinel lymph node
The sentinel lymph node (SLN) concept developed by Morton et al.1 is well established in the treatment of patients with malignant melanoma. This concept allows a more focused and efficient pathologic analysis of micrometastatic disease in the regional lymph nodes. The addition of serial sectioning and immunohistochemical (IHC) analysis of SLNs (e.g., with HMB-45 and melanoma-associated antigen antibodies) has further improved occult tumor cell detection in comparison with hematoxylin and eosin (H&E) staining alone.1–3 Nevertheless, 20% to 30% of melanoma patients with histopathologically negative SLNs will develop recurrent disease within 10 years.
We hypothesized that histopathologically negative SLNs may contain occult metastases that can be identified by a sensitive molecular approach. Moreover, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLNs may provide additional prognostic information. To date we have demonstrated that RT-PCR-based detection of occult metastasis in frozen SLNs upstaged melanoma.3 Furthermore molecular detection of micrometastasis in the SLN has been shown to be an independent predictor of disease outcome for patients with early-stage cutaneous melanoma.3,4
RT-PCR AND SOUTHERN BLOT ASSAY
In a previous study using frozen sections of SLNs, we demonstrated that the tumor status of histopathologically negative SLNs could be upstaged by RT-PCR and Southern blot analysis using multiple specific mRNA markers (MM RT-PCR) that consisted of tyrosinase, MART-1, and MAGE-A3.3 This was the first major report to demonstrate the clinical utility of MM RT-PCR for analysis of SLNs. Tyrosinase, MART-1, and MAGE-A3 are known antigens that are well-expressed in melanomas.5–7 In this study, 20 (36%) of 55 patients had histopathologically negative SLNs that expressed two or more mRNA markers.3 These patients were at significantly increased risk of recurrent disease by multivariate analysis. However, molecular analysis of frozen SLNs is limited by sampling error, logistic difficulties in multicenter studies, and quality control. Sampling error occurs when metastatic tumor cells are missed because of the bias of tissue sampling. Contamination in the PCR reactions at early stages of tissue preparation can cause false-positive results (erroneous upstaging). We therefore developed a more sensitive PCR-based assay that utilizes paraffin-embedded (PE) SLN specimens (Fig. 1).
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Here we describe two approaches for assessing PE SLNs by RT-PCR. The concept of using PE SLNs is highly important because the approach can be applied easily at most university and community hospitals, as well as multicenter international sites. Traditional RT-PCR assay for SLNs has been based primarily on assessment of frozen lymph node tissues and sections with use of gel-based systems, which are often subjective in interpretation and limited in sensitivity. In developing an RT-PCR assay for PE SLNs, we modified and redesigned primers and probes to address RNA degradation, limited marker mRNA copy numbers, specificity, and sensitivity. Significant time was required to design primers and probes to sequences of gene mRNA that are relatively stable. Although mRNA degradation occurs during the interval between tissue resection and fixation, the degree of degradation depends in part on the mRNA’s half-life. Housekeeping gene analysis can be used for overall analysis of mRNA degradation, but degradation time can vary among individual cells and gene transcripts. The level of a specific marker mRNA in any given tumor cell will fluctuate according to its "state." This is a significant factor often ignored in RT-PCR analysis of a specific marker mRNA.
A multiple marker approach can circumvent these problems. We have developed a solution-phase capture MM RT-PCR using an electrochemiluminescence (ECL) detection system (Fig. 2). This sensitive semiquantitative assay can detect melanoma occult metastasis in the PE SLN.4 We initially utilized tyrosinase, MART-1, and tyrosinase-related protein (TRP)-1 and TRP-2 as specific mRNA markers for analysis of PE SLNs. At a median follow-up of 55 months, patients with histopathologically negative but 0 or 1 mRNA marker–positive SLN had a significantly improved disease-free and overall survival versus those expressing 
2 markers.4 This study demonstrated the clinicopathological utility of molecular diagnosis based on PE SLNs.
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ACKNOWLEDGMENTS
This study was supported by National Institutes of Health grants (NCI PO1 CA 20925 Project II and PO1 CA 12528 Project II) and the Roy E. Coates Foundation.
The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.
FOOTNOTES
The molecular detection of occult metastasis in paraffin-embedded sentinel lymph nodes has significant clinicopathologic and logistic advantages over molecular analysis of frozen sentinal lymph nodes.
Received for publication September 23, 2003. Accepted for publication November 20, 2003.
REFERENCES
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