PCR Assay Finds Occult Melanoma Metastases in Sentinel Nodes, Promises More Accurate Staging

BUENOS AIRES--The techniques of lymphatic mapping, sentinel lymphnode biopsy, and polymerase chain reaction (PCR) determinationof occult metastases promise to provide a more accurate stagingof the melanoma patient with more conservative surgery. This couldsave the health-care industry dollars and save patients the morbidityand expense of complete node dissection, Douglas Reintgen, MD,said at the Sixth World Congress on Cancers of the Skin.

In melanoma, routine histologic examination of the lymph nodescommonly misses micrometastatic disease. Although serial sectioningand immunohistochemical staining can double the rate of positivenodal dissections, these techniques have not been incorporatedinto standard pathologic practice because of the time and expenseinvolved.

Lymphatic mapping and sentinel lymph node biopsy (see "SentinelNode ID Allows Selective Lymphadenectomy"), however, allowfull nodal staging from a detailed examination of only one ortwo nodes from the lymphatic basin. With this technique, use ofserial sectioning and immunohistochemical staining becomes morepractical. However, the rate-limiting step continues to be thenumber of sections of the node examined. Thus, an assay was neededfor the accurate identification of all patients with occult or"submicroscopic" metastases.

Dr. Reintgen, associate professor of surgery, Moffitt Cancer Centerand the University of South Florida, Tampa, said that initiallyhis group tested cell culture techniques. Although accurate, theseassays take up to 4 weeks to obtain results and are not widelyapplicable, since many community hospitals do not have tissueculture laboratories.

The Moffitt Cancer Center group then focused its research on developmentof an assay to analyze the sentinel lymph node preparation forthe presence of tyrosinase messenger RNA (mRNA). The theory wasthat all cells of the body would have the gene for tyrosinase,but only those cells that are actively producing pigment wouldexpress the message for the gene.

A PCR assay was developed to analyze for the mRNA for the tyrosinasegene, with the hypothesis that mRNA found in the sentinel nodeis good evidence of the presence of metastatic melanoma cells,Dr. Reintgen said.

With PCR technology, one melanoma cell can be found in a backgroundof 106 normal cells, orders of magnitude greater in sensitivitythan routine histologic examination, a process that can identifyone abnormal melanoma cell in a background of 104 normal lymphocytes.

In tests of the new assay, 44% of the histologic node-negativepopulation was upstaged using the PCR technique, and the patientswhose sentinel nodes were histologic negative but PCR positivehad an increased recurrence rate.

If confirmed in other studies with good clinical correlation,the finding of "submicroscopic" disease with molecularbiology techniques promises to provide a more accurate stagingfor the melanoma patient, Dr. Reintgen said.

With the use of lymphatic mapping, it becomes realistic to examinethe sentinel lymph node with the PCR assay, whereas if all thenodes of the basin had to be submitted for PCR, the expense wouldmake the technique impractical.

Dr. Reintgen pointed out that a recent report in the literaturefrom ECOG has suggested that adjuvant interferon-alfa has a rolein decreasing recurrence and increasing survival in patients withnodal metastases.

Thus, it becomes even more important to identify node-positivepatients so that they can receive this adjuvant treatment, andto identify node-negative patients who can thus be excluded fromthis expensive therapy.

Currently, a National Cancer Institute-sponsored trial is investigatingthe role of sentinel lymph node mapping in the surgical care ofmelanoma patients.

Patients are being randomized to receive either wide local excisionand observation of the primary melanoma or wide local excisionand sentinel node biopsy. Only those patients with a positivesentinel node will undergo a complete nodal dissection. The endpointof this trial is whether there is a survival advantage with thissurgical strategy.

However, even if this trial is negative for a survival benefit,the emerging interferon data may make accurate nodal staging anecessity, Dr. Reintgen said, adding that "lymphatic mappingtechniques are the cheapest and easiest way to obtain these data."