Comparison of nuclear DNA content in primary and metastatic malignant melanoma.

The DNA patterns obtained from 23 primary malignant melanomas and 35 corresponding metastases were compared and found to differ in many cases. In eight cases the primary tumors and their metastases had a ploidy type I ("euploid") DNA pattern. One case had a type I primary tumor and both type I and type II metastases. Five cases had type I primary tumors and ploidy type II ("aneuploid") DNA pattern metastases. In five cases the primary tumors and corresponding metastases were type II, and in another four cases the primary tumors were type II, whereas the metastases were type I. We interpret these data as indicating that malignant melanomas (more often than adenocarcinomas) are composed of genetically heterogeneous tumor sublines that frequently give rise to heterogeneously composed metastases. Since we sometimes observed a change in the DNA content in malignant melanomas, it seems to be more difficult to obtain prognostic information from DNA analysis in malignant melanoma as compared to the more stable adenocarcinomas.     

Complete spontaneous regression of cutaneous primary malignant melanoma.

Melanomas may first present as nodal metastasis. Most of these cases have a discernible primary source. A proportion of these, however, have no apparent primary. A very few patients in this latter group actually have an identifiable primary source that regressed and disappeared. There is a set of stringent clinical and histologic criteria that must be met before a melanoma can be classified as complete spontaneous regression, and only 24 cases in the literature meet all these criteria. This report reviews those cases and presents the first report to provide sequential photographic documentation of a complete spontaneous regression of a cutaneous malignant melanoma. It also gives a 10-year follow-up, the longest in the literature.     

Cellular morphology of human malignant melanoma in primary culture

Summary Early monolayer outgrowths of cells from human cutaneous malignant melanomas mostly derived from metastatic lesions were examined microscopically. Cells resembling the two dendritic types of melanoma previously described in the established lines could readily be recognized. Of 22 specimens, 14 consisted of cells with a triangular dendritic morphology, four had both triangular and elongated dendritic morphology, and one had a cuboidal morphology. The remaining three specimens showed only fibroblastic outgrowths. It is concluded that cells with a triangular dendritic morphology are either the most common type of the secondary cutaneous melanomas, or alternately the most adaptable to the present culture conditions. An association of a more favorable prognosis with the homogeneous triangular dendritic cell type is noted. This study was supported in part by grants from The Medical Research Council of Canada and The Ontario Cancer Treatment and Research Foundation.     

Second primary malignant tumors in patients with ocular melanoma

Multiple primary malignant tumors have been documented with increased frequency over the last two decades. Continuously increasing success of modern oncotherapy has led to long-term remissions in many cases, but this success rate poses a growing risk for the development of second primary malignancies. The incidence of those involving an intraocular tumor is relatively rare. In the present study we report five ocular melanoma patients with second primary malignant tumors diagnosed during a fourteen-year period in our department. We wish to emphasize that an intraocular mass lesion in a patient with a history of a previous malignancy should not be dismissed as a metastatic lesion. The diagnosis of an intraocular lesion as a separate primary tumor drastically changes the prognosis and the therapeutic approach.     

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.     

Flow cytometric DNA ploidy analysis of soft tissue sarcomas. A comparative study of preoperative fine needle aspirates and postoperative fresh tissues and archival material

Flow cytometric (FCM) DNA ploidy measurements on frozen fresh samples of soft tissue sarcomas were compared with the corresponding analyses on preoperative fine needle aspirates and postoperative formalin-fixed archival tissues from the same tumors. A concordance in ploidy status (diploid versus non-diploid) was obtained for 63% of the fresh tissue-fine needle aspiration (FNA) sample comparisons and for 85% of the fresh tissue-archival material comparisons. The majority of discordances in the fresh tissue-FNA sample comparisons could be explained by FNA sampling errors. In the remaining discordant cases (3 of 27 FNA sample comparisons and 6 of 40 archival material comparisons), sampling errors could not explain the differences in ploidy status. The discordant cases were evenly distributed among the different sampling methods. Method reproducibility was not responsible for the differences in ploidy determinations; tumor heterogeneity may be an explanation for the discrepancies. This study showed that archival soft tissue sarcoma samples are as well suited for DNA ploidy analysis as are fresh frozen tissues.     

10 kb DNA replication intermediates are formed from Okazaki-fragments in human malignant melanoma cells

Human melanoma cells were synchronized with hydroxyurea. The DNA replication intermediates formed either during the hydroxyurea-treatment or after the release of the hydroxyurea-block were investigated. The intermediates were released from the parental DNA by lysing the cells in dilute alkali and were then analyzed by agarose gel electrophoresis. After the release of the hydroxyurea block radioactivity was first detected in Okazaki-fragments and then later also in larger replication intermediates (ranging in size up to 10 kb). The results indicate that the 10 kb DNA replication intermediate is derived from the Okazaki-fragments. Thus during its maturation to high molecular weight DNA the Okazaki-fragments form a large, well defined DNA replication intermediate, which later forms the high molecular weight DNA. However, it is not possible to determine whether all or only a part of the Okazaki-fragments give rise to the 10 kb DNA.     

Application of DNA flow cytometry to paraffin-embedded archival material for the study of aneuploidy and its clinical significance

By using a recently developed flow cytometric method we have analyzed cellular DNA content of paraffin-embedded histological material from cancer patients. This method allows the retrospective study of tumors from patients whose clinical outcome is already known, and we have applied it to ovarian cancers, stage II breast cancers, and to metastatic adenocarcinoma of unknown primary site. In addition to knowledge of patient survival, comprehensive information was available about other prognostic determinants and treatment received, and we have used multivariate analysis in an attempt to determine the prognostic significance of cellular DNA content. In ovarian cancer, it is a major prognostic variable except in stage IV disease, whereas in metastatic adenocarcinoma of unknown primary site cellular DNA content has no influence on survival. For stage II breast cancer the situation is more complex and requires larger numbers to be studied. However, aneuploid tumors tend to have more extensive involvement of axillary lymph nodes and a poorer overall disease-free survival. This influence of DNA content on disease-free survival appears to be confined to premenopausal patients, and has no effect on patient survival following disease recurrence. Although we need to study more patients and more tumor types, taken together the results so far show a generally more favorable prognosis for patients with diploid tumors, except in the presence of recurrent or metastatic disease. The better prognosis associated with diploid tumors could be due to the fact that they are more commonly found in earlier clinical stages rather than to their being inherently less aggressive than aneuploid tumors.     

A distinct octamer-binding protein present in malignant melanoma cells.

The octamer-binding proteins present in HeLa cells, B-cells and malignant melanoma cells were compared by a gel-electrophoresis DNA-binding assay. Using an extract from the malignant melanoma cells a complex was formed using a variety of octamer containing probes that was distinct from those found using either a HeLa or B-cell extract. DNAase 1 footprints and methylation interference patterns of the melanoma-specific octamer-binding protein were indistinguishable from those obtained with the HeLa factor NF-A1, except for preferential binding of the melanoma-specific factor to DNA methylated at two G residues 16 base-pairs 3' to the octamer motif. Competition analyses using a variety of wild-type and mutant probes showed that mutations affecting binding of NF-A1 similarly affected binding of the melanoma octamer-binding factor. These data also revealed the extreme flexibility of the octamer-binding site, with one probe sharing only 4 bases with the 8 base consensus sequence binding efficiently.     

Nuclear deoxyribonuclease activities in human lymphoblastoid and mouse melanoma cells A comparative study

Deoxyribonuclease activities were examined in isoelectric focusing fractions of non-histone, chromatin-associated and nucleoplasmic proteins of isolated normal human lymphoblastoid and mouse melanoma cell nuclei using parallel procedures. A very similar series of eight DNA endonucleases, each active on calf thymus DNA and containing no exonuclease activity, were found in the chromatin proteins of both cell lines. Several differences were observed: an activity in human cells at pI 6.6 was absent from murine cells, and there was an increased activity in mouse cells at pI 4.4 and a decreased activity at pI 7.3, as compared with corresponding human cell activities. Assay of these fractions against supercoiled, circular phage PM2 DNA showed greater activity among the fractions with acidic pI valves and slightly lower activities in the murine cells than in the human cells. Analysis of the nucleoplasmic fractions showed a series of DNA endonuclease and exonuclease activities which were again very similar between the two cell lines, although greater endonuclease activity at pI 4.4 occurred in mouse than in human nucleoplasm. These results demonstrate an entire series of deoxyribonuclease activities in both chromatin and nucleoplasm which are nearly identical in two very different mammalian cell lines, suggesting that many of these enzymes are ubiquitous in mammalian cell nuclei.     

DNA flow cytometry in metastases and a recurrency of malignant melanomas. A comparison of results from fresh and paraffin embedded material

Single cell suspensions from 16 biopsies from 15 patients with metastatic or recurrent malignant melanoma were prepared according to the method described by Vindel?v (1977) and the nuclear DNA content was measured by a laboratory-built flow cytometer. The DNA histograms thus obtained were compared with those obtained from suspensions of single nuclei from the same biopsies after formalin fixation and paraffin embedding, according to the method of Hedley et al. (1983). Linear regression analysis of ploidy values from fresh material compared with those from paraffin blocks showed a strong correlation (R2 = 0.85), while that of the S-phase fraction was somewhat weaker (R2 = 0.66). It is concluded that archival wax preparations of malignant melanoma cell populations are suitable for FCM analysis of ploidy, and to a lesser extent for analysis of fraction of cells in various cell cycle phases.     

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [³H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.