The value of reverse transcription-polymerase chain reaction in malignant melanoma
Authors: R. Russell-Jones
Journal: British Journal of Dermatology (2001)

There are two main theories as to how cutaneous melanoma metastasizes. One proposes an orderly spread from the primary lesion to the draining lymph node. The other proposes that micrometastatic disease occurs at multiple sites de novo and that a positive sentinel node is merely an index of metastatic spread. On this basis, removal of the sentinel node would not be expected to influence survival. Under both scenarios, sentinel node status is the best prognostic indicator available, and has been included in the new American Joint Committee on Cancer staging system for malignant melanoma.1 However, sentinel node status is not an absolute guide to outcome, as 10% of patients who are sentinel node negative will relapse within 3 years.2 There are three reasons why this might occur: haematogenous spread that bypasses the local lymph nodes, in-transit disease which has not yet reached the sentinel node and failure to identify micrometastatic disease within the sentinel node. Reverse transcription–polymerase chain reaction (RT–PCR) is therefore a potentially useful technique. In sentinel nodes it is an order of magnitude more sensitive than conventional histology with immunostaining, and it allows blood to be examined for the presence of circulating melanoma cells. RT–PCR relies upon the presence of mRNA belonging to a gene that is highly expressed in malignant melanoma. However, several different techniques can be used to detect mRNA, and assays of different genes will vary in their sensitivity and specificity. Thus, the identification of tyrosinase mRNA in a lymph node could indicate the presence of melanoma, benign naevus cells or even neural tissue. mRNA for MelanA is specific for melanocytes, but this antigen is not expressed by all melanomas and there is still the concern that macrophages might have ingested mRNA from the primary tumour, and transported this to the local nodes. In order to be certain that a signal is melanoma-derived, it is necessary to use in situ PCR, but if one has to resort to tissue sections, then one might as well rely on conventional histology and immunostaining. It is therefore crucial, when establishing the value of such techniques, to compare RT–PCR results with a well-established prognostic indicator. In a paper published in this issue,3 Blaheta and coworkers report their findings in small numbers of patients who underwent RT–PCR for both tyrosinase and MelanA in lymph nodes, blood and bone marrow. The key finding is that RT–PCR status of lymph nodes correlates well with Breslow thickness, whereas RT–PCR results in blood do not. The lymph node findings are very much in line with what one might expect from data already available in the literature. Thus, it is known that sentinel node positivity defined histologically increases with Breslow thickness, and one would anticipate that RT–PCR analysis would yield similar findings.2, 4 The failure to correlate Breslow thickness with RT–PCR results in blood is also of interest and reinforces the data published earlier this year by Rona MacKie's group, who showed that blood RT–PCR could not be used to monitor disease progression in melanoma patients.5 There are several possible explanations for this: in particular, that melanoma cells are present only intermittently in the peripheral circulation, or that haematogenous spread occurs in only a minority of melanoma patients. In either event, it is clear that RT–PCR in peripheral blood is of limited clinical value, and cannot be used as a substitute for sentinel node status, as was suggested in an editorial last year in the British Medical Journal.6 By contrast, RT–PCR of lymph nodes does yield information additional to that provided by histological examination alone. Thus, Shivers et al. employed tyrosinase RT–PCR to detect metastatic melanoma in the sentinel nodes of 114 patients, and found that all subjects who were histologically positive were also RT–PCR positive, and had a relapse rate of 67% over a median follow-up of 28 months. Patients who were negative by both techniques had relapse rates of only 2%, while patients who were histologically negative, but RT–PCR positive, had a relapse rate of 13%.7 In a separate study to the one published in this issue, Blaheta and coworkers reported recurrence rates of 67%, 6% and 20% in 116 patients at 19 months of follow-up.8 These findings are remarkably consistent and demonstrate that tyrosinase positivity in lymph nodes is of prognostic value and identifies a group of patients with relapse rates intermediate between those who are PCR negative and those who are histologically positive. In addition, RT–PCR in lymph nodes is being used in the Sunbelt study as a criterion for randomizing patients to different treatment arms. Even so, the technique cannot be recommended in routine clinical practice until better data are available as to the sensitivity and specificity of the different genes, and this can only be obtained through follow-up. Preliminary data presented at the 5th World Melanoma Conference in Venice9 showed that tyrosinase RT–PCR is superior to the histology of sentinel nodes in predicting recurrence (88% vs. 47% sensitivity), whereas histology is superior to RT–PCR in patients whose melanoma had not recurred (87% vs. 38% specificity). These data underline the need for a more specific melanoma marker before RT–PCR results can be used to guide management in individual patients.