Antigenic profiles of individual-matched pairs of primary and melanoma metastases.

A unique collection of individual-matched pairs of primary and melanoma metastases were studied immunohistochemically with a panel of 6 monoclonal antibodies directed to gp-100, pigmentation-associated antigen, tyrosinase-related protein, human leukocyte antigen DR, MAA-1, and MAA-2 (high molecular weight melanoma-associated antigens). The antigenic profile of immunoreactive pigment cells was compared with the stage of tumor progression. Our data show consistent antigenic profiles of primary melanomas and their metastases within the same patient. Expression of tyrosinase-related protein and pigmentation-associated antigen was observed in the radial growth phase of primary melanomas but showed diminished or complete loss of expression in the vertical growth phase and in metastatic melanomas. HLA-DR was negative in the most primary lesions, but melanoma cells and a larger proportion of immunoreactive cells were observed at the metastatic site. The melanoma-associated antigens MAA-1 and MAA-2 were expressed throughout tumor progression. Although no clear distinction could be made between primary and secondary melanoma lesions for both melanoma-associated antigens, there was a profound variability in the topographical antigen distribution when compared with HLA-DR. The loss of expression of pigmentation-associated antigen and tyrosinase-related protein in the vertical growth phase of the primary lesions and metastatic melanomas did not reach statistical significance but still may be related to tumor progression. This indicates that primary melanomas can be distinguished from their metastases by evaluation of the antigenic profile and in this respect facilitate the recognition of tumor progression stages.

Multiple complementary transcripts of pCMa1, a novel gene located at chromosome 11p15.1-2, and melanocytic cell transformation.

The cDNA clone pCMa1 (0.45 kb) is one of the 12 novel cDNAs, previously identified when comparing RNA expression profiles of melanocytes, naevus cells, and non-metastatic melanoma cells. This clone did not reveal a unique long open reading frame. The pCMa1 gene localized to the distal, telomere proximal region on the short arm of chromosome 11.p15.1-2. Northern blot analyses with single-stranded cRNA probes revealed the presence of various complementary pCMa1 transcripts of different lengths, which are not enriched in the poly(A)(+) RNA fraction. The arbitrarily defined plus strand (used as a probe) mainly hybridized to 0.45 kb and 4.0 kb minus transcripts in total RNA samples, and the minus strand (used as a probe) hybridized to a major plus transcript of 4.0 kb. By RNA in situ hybridization, the highest levels of the plus transcripts were observed in melanocytic naevi (12/12), particularly in congenital naevi, whereas normal skin melanocytes (12/12) were negative. pCMa1 plus transcripts were detected in naevus cell nests (100%) near the dermo-epidermal junction. Expression, however, diminished to some extent in the deeper parts of the melanocytic naevi. Although most of the cutaneous primary melanoma lesions (11/15) showed detectable, but variable levels of plus transcripts of pCMa1 in the papillary to reticular dermis, not more than 10% of the melanoma cells were positive. The majority of melanoma metastases (6/7) were negative, while the positive lesion originated from a patient with a positive primary melanoma. Furthermore, plus transcripts were present in the nuclei of non-metastatic melanoma cells in culture, whereas metastatic cells showed elevated expression both in the nucleus and in the cytoplasm. Briefly, the data show transient up-regulation of pCMa1 in neoplastic progression of melanocytic cells, with peak levels occurring during naevus stages, and suggest that pCMa1 is a molecular marker in melanocytes for the early changes from the proliferating phenotype to malignant transformation.

Gene expression patterns in melanocytic cells: candidate markers for early stage and malignant transformation.

Different stages of differentiation of human melanocytic cells, such as normal melanocytes, naevus and melanoma cells, reflect distinct gene expression patterns. A PCR-based subtractive hybridization and display method was applied to identify genes that are differentially expressed in melanocytic cells in relation to early stage and malignant transformation. This resulted in the identification of a number of candidate cDNAs differentially expressed among melanocytes, naevus cells, and (non)-metastatic melanoma cells. Out of this collection of cDNAs, 16 clones were screened that comprised 12 novel genes, one previously identified expressed sequence tag related to vesicular trafficking (Ras-related protein Rab5b). The other three were also known genes that were either related to cell motility (beta-tubulin), pre-mRNA splicing (small nuclear protein U1A), or of unknown function (the human TI227-H gene). The differential expression patterns of Rab5b and two novel gene fragments (pCMa1, pCMn2) were further assessed in melanocytic cells. pCMa1 was expressed more in metastatic melanoma than in primary melanoma cells. In contrast, pCMn2 was expressed in both non-metastatic and metastatic melanoma cells, but was not detectable in either normal melanocytes or naevus cells. The Ras-related protein Rab5b showed lower levels of expression in highly metastatic than in other melanoma cells. These three cDNAs may therefore be involved in the early stage and malignant transformation of melanocytes.