Although a number of studies of the possible clonal origin of double malignancies have been conducted to date, none to our knowledge have involved double primary melanomas. This absence may be due to the fact that most dermato-pathologists do not perceive the misdiagnosis of a metastasis as a second primary as a likely occurrence or as a diagnostic problem. However, the high incidence of reported multiple primaries in this disease could be due in part to the misdiagnosis of metastases as independent primaries. Our study was constructed to provide preliminary evidence on this issue. The results would appear to support the conclusion that most second primary melanomas diagnosed on the basis of their clinical and pathologic characteristics are indeed independent occurrences of the disease.
It is of interest to examine more closely the two cases that showed patterns suggestive of clonal relatedness. Case #30 exhibited concordant LOH at 6 separate genetic loci, yet the tumors have different cell types, occurred 2.4 years apart, and were located in distant anatomic sites. A re-inspection of the pathological characteristics indicated that both tumors had significant epidermal components extending beyond bulky dermal components. Case #34 had only two concordant mutations, but the overall patterns were very similar, that is most of the loci exhibited no mutations on either tumor. The two tumors were synchronous, with the same cell type in the same general anatomic site, the chest, although the tumors were in the left and right portions of the chest, and well apart. Re-examination of the pathology in recut sections showed that the two tumors were mostly epidermal, and thus appeared pathologically to be independent primaries. Since we expect one false positive finding for every 20 statistical tests performed at the 5% significance level, the observation of only 2 significant results in this set of 19 is broadly consistent with the conclusion that few, if any, of these melanoma pairs, and very few in general, are of clonal origin.
Our study has technical, epidemiological and statistical limitations. We obtained specimens from both primaries for 19 cases, but these cases were selected based on the availability of sufficient tissue samples. This opportunistic selection of cases, and the small sample size, limits our ability to estimate accurately the proportion of cases that may be mis-diagnosed. Furthermore the cases were obtained from a specialized melanoma treatment center where the pathologic reviews were accomplished by dermato-pathologists specializing in melanoma, and where full clinical histories were also available. All such information is rarely available to either clinicians or pathologists at the time of initial diagnosis in routine clinical practice and hence misdiagnosis of a metastasis as a primary may be somewhat more common in everyday clinical practice, particularly outside of specialist centers. Nonetheless, most patients with a second skin melanoma designated as a second primary have clinical courses consistent with a new primary and more favorable than would be expected for stage IV melanomas. For a subset of markers we encountered amplification failures. This could be a result of primers being unable to anneal to their specific sequences due to homozygous deletions or duplications, but more likely the high rate of failures encountered for D2S131, D2S2291, D6S275, D6S457, D10S185 and D13S153 was due to the suboptimal quality of the DNA.
Melanoma is a disease that is frequently characterized by small tumors. We had hoped as part of this study to conduct array comparative genomic hybridization on all pairs of samples as an alternative genomic approach to profiling the tumors. However, sufficient DNA of high molecular weight suitable for array CGH for both tumors in the pair was available only for 4 cases (data not shown). In general, for a technology of this nature to be applicable in a clinical diagnostic setting, we would need a minimum of 0.6μg of high molecular weight DNA from each tumor and counterpart normal sample if extracted from fresh frozen tissue, or 1.5μg of DNA extracted from formalin-fixed, paraffin-embedded tissue. Such quantities will typically not be available from both tumors. With a polymerase chain reaction (PCR)-based method such as the one presented here, one would require no more than 0.5μg of DNA if testing a relatively high number of microsatellite repeats, and approximately 0.2μg when working with fresh-frozen tissue.
We employed a statistical test that was designed specifically for the purpose of detecting clonal relatedness (Begg et al, 2007
). This test is based on the simplifying assumptions that the mutations at different loci are independent, that the probabilities of mutations are similar for each locus, and that each allele is equally likely to experience a mutation. Each of these assumptions is clearly approximate. Validation studies show that the test is robust to modest departures from the latter two assumptions (Begg et al, 2007
). In fact, in our presumptively independent cases 66% of the losses that occurred in both members of the tumor pair occurred on the same allele. This modest preponderance of concordances could be the result of clonality in some of the pairs (such as cases 30 and 34), but it may also be explained by the possibility that allelic changes do not occur with equal probability for the alleles at specific genetic loci, especially if located within or nearby a gene involved in the development of the tumor. If this is true then we expect to see a modest correlation in mutational profiles even for independent tumors. The statistical power of the test is, of course, dependent on the number of independent genetic markers evaluated. In practice one could increase power by examining more loci for allelic gains and losses, and by testing for presence of common point mutations such as the V600E variant on the BRAF gene.
In summary, our study provides evidence that most melanomas that are classified as independent second primaries on the basis of comprehensive clinicopathologic analysis in a specialist melanoma treatment center are indeed independent occurrences of melanoma. In clinical use, this technology could, on present evidence, be a supplement to but not a replacement for detailed clinical and pathologic evaluation of the lesions.