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-155- Melanoma 9/5 Room-C
CHROMOSOME 9p21 ALTERATIONS IN PRIMARY AND METASTATIC MELANOMAS.
Naohito Hatta, Reiji Morita, Akihide Fujimoto, Minoru Takata, Kazuhiko Takehara.
Department of Dermatology, Kanazawa University School of Medicine, Kanazawa, Japan.
In cutaneous melanomas, frequent loss of heterozygosity (LOH) at chromosome 9p21 has been reported. To examine the participation of tumor suppressor genes (TSG) on 9p21 in melanoma progression,we analyzed LOH at 9 microsatellite loci on chromosome 9p21-22 and p16 gene status in 12 paired primary and metastatic melanoma tumors. Six pairs of tumors (50%) showed LOH at 9p21-22. The deleted areas in all metastatic tumors were equal to the corresponding primary tumors. However, in one patient, deleted allele was inverted at every loci in primary and metastasis, respectively. p16 protein expression status in metastasis was equal to that in primary in 10 patients, but was decreased in 2 patients. p16 gene mutations has not been identified so far. These results suggest that the inactivation of TSG on 9p21-22 region occurs early before metastasis during melanoma progression. The inversion of the deleted alleles observed in one patient raises a possibility that a neoplastic clone in metastasis might not be always originate from a predominant clonal population in primary lesion
-156- Melanoma 9/5 Room-C
HYPERPLOIDY IN MALIGNANT MELANOMA CELLS DETECTED BY FISH ANALYSIS.
Seiki Satoh1, Yukio Kitano1, Tomoko Hashimoto2,Junichi Furuyama2.
1Department of Dermatology, 2Department of Genetics, Hyogo College of Medicine, Nisinomiya, Japan.
It has been well established that malignant melanoma cells showed hyperploidy,that is usually¡¡detected by flow-cytometry. However,this method issometimes difficult to apply to clinical diagnosisbecause it requires a certain amount of cells. It is also difficult to perform flow-cytometry along with morphological studies. Recently,fluorescence in situ hybridization(FISH) has been applied to detect the number of chromosomes in both metaphaseand interphase cells. Using this technique, morphlogical changes can be also detected and lessnumber of cells are required.In this study,we usedFISH analysis to detect ploidy of malignant melanoma cells including two cell lines,primary and metastatic tumors.Number of chromosomes was detected by ¦Á-satellite probes of X,Y and No.18 chromosomes. All malignant melanoma cells showed hyperploidy;one cell line tetraploidy,the other tetraploidy to octaploidy,and primary and meta- stasic tumors tetraploidy.Therefore,detection of hyperploidy using FISH may be effective to differ-ential diagnosis of malignant melanomas.
-157- Melanoma 9/5 Room-C
THE HIGH FREQUENCY OF LOSS OF HLA CLASS I IN MELANOMA LESIONS.
Toshiro Kageshita, Shunji Hirai, Tomomichi Ono.
Department of Dermatology, Kumamoto University School of Medicine, Kumamoto, Japan.
The aim of this study was to investigate the expression of HLA class I antigens in surgically removed melanoma lesions. To this end 32 primary and 11 metastatic lesions were stained in the immunoperoxidase reaction with monoclonal antibodies (mAb) to monomorphic, locus specific and polymorphic antigenic determinants. About 20% of primary and about 50% of metastatic lesions were not stained or were stained with reduced intensity by mAb to monomorphic and locus specific antigenic determinants. Moreover about 40% of primary and about 60% of metastatic lesions were not stained or were stained with low intensity, by mAb to HLA class I allospecificities. These results indicate that the frequency of abnormalities in HLA class I antigen expression is high in melanoma lesions. These abnormalities are likely to have a negative impact on T cell based immunotherapy, since they provide melanoma cells with a mechanism to escape from destruction by cytotoxic T cells.
-158- Melanoma 9/5 Room-C
PCR DETECTION OF CIRCULATING MELANOMA CELLS BY AMPLIFYING THREE DIFFERENT MELANOCYTE-SPECIFIC GENES.
Katsuhiko Tsukamoto, Atsushi Osada, Shinji Shimada.
Department of Dermatology, Yamanashi Medical University, Yamanashi, Japan.
Three different melanocyte-specific genes mRNAs are studied as the marker of circulating melanoma cells. All three genes encode proteins of tyrosinase, TRP-2 and Pmel17 which are essential for the synthesis of melanin and expressed specifically in melanocyte. Using mouse B16-F10 melanoma cells as an experimental model, we used reverse transcriptase to prepare cDNA from peripheral blood mRNA and targeted these three different melanocyte-specific gene transcription to identify melanoma cells. In vitro study showed that at least 1 melanoma cells in 0.125ml normal blood could be detected. In vivo, blood samples from the melanoma bearing mice gave positive and different sensitive results. In these three genes, Pmel17 mRNA is most sensitive to detect circulating melanoma cells compared with tyrosinase mRNA and TRP-2 mRNA. The PCR detection of all three genes mRNAs appears to make the result more sensitive and specific than the PCR detection of only tyrosinase mRNA reported before.
-159- Melanoma 9/5 Room-C
IMMUNOHISTOCHEMICAL ASSAY OF PROLIFERATION AND APOPTOSIS IN MALIGNANT MELANOMA.
Tetsuo Shukuwa, Ichiro Katayama.
Department of Dermatology, Nagasaki University School of Medicine, Nagasaki, Japan.
To elucidate the kinetics of development of malignant melanoma, we investigated both rates of proliferation and apoptosis of melanoma cells with immunohistochemical technique. The specimens of melanoma and uninvolved site were obtained by surgical resection or excisional biopsy from fifteen patients, ages 44 to 84, with malignant melanoma. The formalin-fixed and paraffin-embedded sections were examined with proliferating cell nuclear antigen (PCNA) monoclonal antibody for proliferation and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) system for apoptosis. The rate of PCNA positive melanoma cells was 25 to 30%, while the rate in uninvolved sites was 2%. On the other hand, there were a few positive melanoma cells (less than 1%) by TUNEL. Interestingly, TUNEL-positive lymphocytes infiltrating around malignant melanoma were abundant in several specimens. These results suggested that the apoptosis of malignant melanoma cells and lymphocytes may affect the development of malignant melanoma.
-160- Melanoma 9/5 Room-C
BORON NEUTRON CAPTURE THERAPY OF CANCER (BNCT) WITH MELANOGENESIS-SEEKING 10B-BPA FOLLOWING MELANOGENIC GENE TRANSFER.
Hirofumi Kondoh, Tatsuya Tsuboi, Yutaka Mishima.
Mishima Institute for Dermatological Research, Kobe, Japan.
In current BNCT using 10B-boronophenylalanine (10B-BPA), the high melanoma (Mm) selectivity of BPA limits the application of this therapy to Mm and anti-tumor effect on amelanotic melanoma (AMm) is insufficient. We performed experiments based on evidence that the chemical complex formation of BPA and melanin monomers in Mm cells is a major mechanism of BPA affinity to Mm, and obtained following results. 1)Tyrosinase cDNA was transfected into AMm cells and the cells recovered melanogenesis. 2)Incorporation of BPA into transfected-cells was increased by approxymately 200%. 3) Marked regression was observed in the tumor of transfected-cells after after BNCT. Further, we will report on increased BPA accumulation in gene-transfected fibrosarcoma cells.