Gloria Balaban

Characteristics of Cultured Human Melanocytes Isolated from Different Stages of Tumor Progression

Normal melanocytes and melanocytes of normal nevi, primary melanoma in the radial (RGP) and vertical (VGP) growth phases, and metastatic melanoma exhibited and maintained phenotypic differences when grown in tissue culture or in experimental animals. Only metastatic and VGP primary melanoma cells were tumorigenic in athymic nude mice and had nonrandom chromosomal abnormalities involving chromosomes 1, 6, and 7. The colony-forming efficiency in soft agar was also highest in these two cell types. A cell line of RGP primary melanoma had characteristics of both benign and malignant cells: nevus-like morphology; nontumorigenicity in nude mice; but karyotypic abnormality of chromosome 6. It also had a ganglioside pattern similar to that of normal melanocytes but not melanomas, i.e., a high GM3 ganglioside content compared to the amounts of GM2, GD2, and GD3 gangliosides. Binding of monoclonal antibodies secreted by hybridomas generated by immunization of mice with VGP primary and metastatic melanoma was highest with cells and supernatants of cultures from advanced melanoma and least with nevus cells. There was no binding to normal melanocytes except with the monoclonal antibodies specific for nerve growth factor receptor or 9-O-acetyl-GD3 ganglioside. On the other hand, monoclonal anti-nevus antibodies bound to melanocytes, nevus cells, and RGP primary melanoma cells but not to VGP primary or metastatic melanoma cells. Cultured human melanocytic cells appear to be a unique model for the study of tumor progression.

Cytogenetics of human malignant melanoma and premalignant lesions

Chromosome studies were done on direct preparations, early passage cultures, and/or cell lines derived from melanocytic lesions of 17 patients. There were 5 nevi (3 dysplastic); 1 early primary melanoma (radial growth phase); 1 advanced primary melanoma (vertical growth phase) with multiple metastases; and 10 metastatic lesions. The 5 nevi had normal karyotypes, while each of the tumors had a predominantly abnormal karyotype. The early melanoma was pseudodiploid, including a 6p;22 translocation. Ten of the 11 advanced melanomas had one or more aberrations involving chromosome #1, with 9 having deletions or translocations of lp that involved the proximal segment 1p12----1p22 9 times in 8 lesions. Six advanced tumors had additional material involving 7q, including extra #7s (4 cases) and 7q+ (2 cases). Nine melanomas, including the early tumor, had alterations in chromosome #6. Three had additional copies of 6p (as iso6p or t6p); the others showed no consistent pattern. In one advanced tumor, the primary lesion and 5 metastases (removed seriatim over an 18-month period) had nearly identical karyotypes, indicating the clonal nature of the neoplasm. The nonrandom cytogenetic changes suggest that genes important in melanoma carcinogenesis are located on the proximal portion of 1p, on 7q, and on chromosome #6. More data on early lesions are needed to identify the relation of these various cytogenetic changes to the different stages of malignant melanoma development.

Expression of the receptor for epidermal growth factor correlates with increased dosage of chromosome 7 in malignant melanoma

Epidermal growth factor (EGF) receptor is expressed selectively by human melanoma cells which show the presence of an extra copy of chromosome 7. None of the cells of benign pigmented lesions (nevi) or radial growth phase (nonmetastatic) primary melanoma expressed EGF receptor and none of these cells showed an extra copy of chromosome 7. The results indicate that a single extra dose of a gene (for EGF receptor) may provide a selective advantage to cells in the late stages of tumorigenesis.

Karyotypic evolution in human malignant melanoma

Chromosome studies were performed on direct preparations, early passage cultures, and cell lines derived from melanocytic lesions of 37 patients. There were six congenital or common acquired nevi, six dysplastic nevi, one early primary melanoma (radial growth phase), three complex melanomas (RGP with foci of vertical growth phase), six advanced primary melanomas (VGP), and 26 metastases. The karyotype was normal in the six common nevi. A chromosomally abnormal clone with a single karyotypic alteration was found in two dysplastic nevi. All melanomas had clones with multiple cytogenetic changes. Nonrandom abnormalities involving translocations or deletions in the short arm of chromosome #1, either arm of chromosome #6, and/or extra copies of the short arm of chromosome #7 were present in all melanomas. These were not obviously associated with a particular stage of disease, except that the only nonrandom alteration in the early (RGP) melanoma involved chromosome #6. In four cases, cytogenetic data were available on both a primary melanoma and its metastases. In each instance there were common alterations (demonstrating the clonality of the disease), as well as additional changes in the metastases. Our findings indicate that demonstrable somatic genetic abnormalities increase in severity with clinical progression of melanocytic disease, but additional data are required to establish the significance of specific karyotypic changes (and the involved genes) in the clinical evolution of these disorders.

Abnormalities of chromosome #13 in retinoblastomas from individuals with normal constitutional karyotypes

Constitutional chromosome abnormalities have been associated with retinoblastoma, Wilms tumor, and a familial form of renal carcinoma. For each tumor type, the particular chromosome segment involved in the observed rearrangements is different: in retinoblastoma, that segment is band q14 on chromosome #13. We now present evidence that in retinoblastoma, structural abnormalities involving the particular chromosome segment identified in the constitutional cases can also occur in the tumors of individuals with normal constitutional karyotypes. Six cases with retinoblastoma in one or both eyes were analyzed; deletions/rearrangements involving 13q14 were found in the tumor cell karyotypes of five of the six. These observations suggest that changes in a gene or genes at a common site (13q14) play a role in tumorigenesis in all forms of retinoblastoma, sporadic as well as heritable.

Possible involvement of the chromosome region 10q24----q26 in early stages of melanocytic neoplasia.

A recent report described a translocation involving 10q24 in a compound nevus. We have examined our series of melanocytic tumors ranging from common nevi to metastatic melanomas with the following results. In 24 nevi (congenital or common acquired), no karyotypically abnormal clones were found. Two of 10 dysplastic nevi had abnormal clones: one had an unidentified marker chromosome, the other had a t(9;10)(p24;q24) translocation. Of the three complex primary melanomas studied (lesions with both radial and vertical growth phase present), one had a t(10;?)(q26;?) and one showed a loss of chromosome 10. Among 51 advanced melanomas (primary and metastatic), all but one had multiple alterations, and 18 of these had lost one or more copies of chromosome 10. The one invasive melanoma without multiple abnormalities had a complex three-way rearrangement: 46,XY,t(5;6;10) with breakpoints on chromosome 10 at both q23 and q25. These data support the view that the terminal region of 10q may harbor one or more genes involved in the early stages of melanocytic neoplasia.

Karyotypic Progression and Metastasis Formation of Human Tumors

In recent years, chromosome studies have contributed significantly to our understanding of fundamental tumor biology in a number or areas. Based on the work from many laboratories, four general statements can be made about karyotypic alterations in neoplasia: 1) most tumors have chromosome abnormalities, which are not present in other cells of the body, 2) in a given tumor, all the neoplastic cells often have the same cytogenetic change, or related changes, 3) although chromosome alterations often differ between tumors, there are nonrandom patterns, and 4) chromosome abnormalities are more extensive in advanced tumors.

The 13p HSR in a human neuroblastoma cell line does not contain rRNA genes

We have studied a human neuroblastoma cell line containing a homogeneously staining region (HSR) on the short (p) arm of chromosome #13. The HSRs are believed to represent sites of gene amplification, containing multiple copies of one or a small number of genes. The 18S and 28S ribosomal RNA genes are the only structural genes so far assigned to the p arms of the human acrocentric chromosomes, which include number #13. Using two recombinant DNA probes specific for sequences in the 18S/28S ribosomal RNA (rRNA) gene complex for in situ hybridization, we have been able to establish that the 13pHSR in this line does not contain rRNA genes.

Clonal Evolution and Childhood Tumors

Many of the clinical and biological characteristics of tumor development appear to fit with the view that most neoplasms are unicellular in origin (i.e., ‘clones’) and that tumor progression results from ‘clonal evolution’, the sequential appearance within unstable neoplastic clones of subpopulations which are more and more genetically aberrant. These concepts, and the suggested underlying mechanisms, will be reviewed briefly, along with some of the supporting evidence. Although relatively few data are available from tumors occurring in the pediatric age group, some examples from both hematopoietic and nonhematopoietic neoplasms will be summarized. The findings support the general applicability of these concepts to pediatric neoplasms, but certain special characteristics of this age group require consideration, and these will be discussed from the standpoint of both their theoretical and practical implications.