The clonal evolution of metastases from primary serous epithelial ovarian cancers

Several models of evolution from primary cancers to metastases have been proposed; but the most widely accepted is the clonal evolution model proposed for colorectal cancer in which tumors develop by a process of linear clonal evolution driven by the accumulation of somatic genetic alterations. Various other models of cancer progression and metastasis have been proposed, including parallel evolution and the same gene model. The aim of this study was to investigate the evolution of metastases from primary cancer in 22 patients diagnosed with high‐grade serous epithelial ovarian cancer. We established somatic genetic profiles based on the pattern of loss of heterozygosity, in several different regions of tumor tissue within the primary tumor and metastatic deposits from each case. Maximum parsimony tree analysis was used to examine the evolutionary relationship between the primary and metastatic samples for each patient. In addition, we investigated the extent of genetic heterogeneity within and between metastatic tumors compared with primary ovarian tumors. Our data suggest that most, if not all, metastases are clonally related to the primary tumors. However, the data oppose a single model of linear‐clonal evolution whereby a late stage clone within the primary tumor acquires additional genetic changes that enable metastatic progression. Instead, the data support a model in which primary ovarian cancers have a common clonal origin, but become polyclonal, with different clones at both early and late stages of genetic divergence acquiring the ability to progress to metastasis. © 2009 Wiley‐Liss, Inc.     

7th Jan Waldenstr?m Lecture. The biology of human cancer metastasis.

The process of cancer metastasis is dynamic and consists of sequential, interrelated steps. Malignant cells that produce metastases have survived a series of potentially lethal interactions that are regulated by both the intrinsic properties of the tumor cells and host factors. Although some of the steps in this process contain stochastic elements, metastases develop from the nonrandom survival of a few subpopulations of cells that preexist within the parent neoplasm. Metastases can have a clonal origin, and different metastases can originate from the proliferation of different cells. The orthotopic implantation of human cancer cells derived from surgical specimens into nude mice provides a biological model of metastasis. Using this model, clonal analysis of a human renal carcinoma, colon carcinoma, and melanoma has revealed that these tumors are heterogeneous for metastatic properties. Damage to an organ's environment is followed by inflammation and repair, and these homeostatic processes facilitate the proliferation of normal (physiology), and in some cases, tumor cells (pathology). Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be usurped by neoplastic cells. Collectively, the factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis.     

Rationale and methods for the use of nude mice to study the biology and therapy of human cancer metastasis

Summary Human neoplasms are biologically heterogeneous. The extensive cellular diversity found in malignant neoplasms is generated by the rapid emergence of clonal subpopulations of tumor cells with different properties that include invasion, metastasis and responsiveness to treatment. Studies in rodent systems have indicated that cancer metastases can be clonal in their origin and that different metastases can originate from different progenitor cells from the primary tumor. This metastatic heterogeneity of tumor cells has many ramifications for studies of tumor biology, in general, and studies of therapy, in particular.The heterogeneous nature of metastatic human neoplasms can now be studied under defined conditions in healthy athymic nude mice. The neoplasms must be free of mouse pathogens and the mice must be kept in specific-pathogen-free conditions. Careful consideration must be given to the intimate tumor-host relationship for each tumor system studied, because the metastatic potential of human neoplasms can vary with the site of implantation into nude mice.Several methods for studying the biology of human neoplasms in the nude mouse are described as well as techniques to assure the success of these studies. The data show that the healthy young nude mouse can be a useful in vivo model for ascertaining the metastatic potential of human neoplasms, for selecting and maintaining cell variants of high metastatic potential from heterogeneous human tumors, and for studying therapeutic agents directed against metastatic cells proliferating in visceral organs.     

Critical determinants of cancer metastasis: rationale for therapy

The major cause of death from cancer is metastases that are resistant to conventional therapies. Several reasons account for treatment failure in patients with metastases. First, neoplasms are biologically heterogeneous and contain subpopulations of cells with different angiogenic, invasive, and metastatic properties. Second, the process of metastasis selects a small subpopulation of cells that preexist within a parental neoplasm. Although metastases can have a clonal origin, genetic instability results in rapid biological diversification and the regeneration of heterogeneous subpopulations of cells. Third, and perhaps the most important principle for the design of new cancer therapies, is that the outcome of metastasis depends on multiple interactions ("cross-talk") of metastatic cells with homeostatic mechanisms which the tumor cells usurp. The organ microenvironment can influence the biology of cancer growth, angiogenesis, and metastasis in several different ways. For example, the survival and growth of tumor cells are dependent on angiogenesis, which is mediated by an imbalance between positive and negative regulating molecules released by tumor cells, normal cells surrounding a tumor, and infiltrating lymphoid cells. Many cytokines that stimulate or inhibit angiogenesis are present in different tissues, and thus the organ environment profoundly influences this process. Moreover, the organ microenvironment can also influence the response of metastases to chemotherapy by regulating the expression of different drug resistance genes, such as mdr-1. The finding that the resistance of metastases to some chemotherapeutic agents can be mediated by epigenetic mechanisms has obvious implications for therapy. The identification of organ-specific cytokines that can upregulate expression of mdr-1 (or other resistant mechanisms) may suggest an approach to overcome the resistance of some metastases to particular chemotherapeutic agents. Therefore therapy of metastasis should be targeted not only against metastatic tumor cells, but also the homeostatic factors that are favorable to metastasis, growth, and survival of the metastatic cells.     

Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture.

The process of metastasis is not random. Rather, it consists of a series of linked, sequential steps that must be completed by tumor cells if a metastasis is to develop. Although some of the steps in this process contain stochastic elements, as a whole, metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Moreover, metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ-specific metastases have been demonstrated in a variety of experimental tumor systems. Moreover, we have found tumor growth that is specific to a particular site within one organ. Whether the same conclusions can be reached for human cancers remained unanswered until very recently. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomical sites of nude mice can provide a suitable model of metastasis of human tumors. Clonal analysis of a human renal carcinoma, colon carcinomas, and melanomas has revealed that these tumors are indeed heterogeneous for metastatic properties, an observation made only after orthotopic implantation. Thus, growth in the environment of specific organs can be selective and the environment per se influences this process. While it is clear that vascularity and local immunity can facilitate or retard tumor growth, we have concentrated on understanding how damage to an organ and the subsequent repair process can facilitate tumor cell proliferation. Accelerated growth of human colon cancer cells was found in hepatectomized nude mice, whereas accelerated growth of human renal cancer cells was found in nephrectomized nude mice. These data suggest that systemic physiological signals can be recognized by neoplastic cells presumably by mechanisms similar to those shared by their normal cell counterparts. In summary, the critical factors that regulate metastasis are the intrinsic properties of metastatic cells and host factors involved in homeostasis. The recent increase in our understanding of metastasis should provide important leads for developing more effective approaches to the treatment of disseminated cancer.     

Comparison of 'spontaneous' and 'experimental' metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones

Rat 13762NF mammary adenocarcinoma cloned cell lines were assayed at different in vitro passage numbers and compared for their abilities to form 'spontaneous' metastases by subcutaneous injection of cells and 'experimental' metastases by intravenous injection of cells. Tumor cell clones were established from locally growing tumor and spontaneous lung metastases, and these clones were found to possess heterogeneous metastatic potentials in both metastasis assays. The rank order of clonal metastatic potentials based on either the average number of lung tumor colonies or the average total lung tumor volume was generally equivalent for 'spontaneous' and 'experimental' metastases, but some differences were noted. Ranking of 'spontaneous' metastasis by average total lung tumor volumes more closely resembled the rank order of 'experimental' metastasis than by the average number of spontaneous metastases. The results demonstrated that in the 13762NF mammary adenocarcinoma system (i) there is heterogeneity in tumor cell clonal metastatic potential using either 'spontaneous' or 'experimental' assays; (ii) these two assay methods yield generally the same rank order of metastatic potential; (iii) the metastatic potential of each of the tumor cell clones drifts with time (passage number) in cell culture, and (iv) ranking by average tumor burden calculated from total lung tumor volumes may yield a better estimate of metastatic potential than ranking by the average number of lung tumor colonies.     

Macrophages and cancer

The uncontrolled growth of metastases resistant to conventional therapeutic modalities is a major cause of death from cancer. Data from our laboratory and others indicate that metastases arise from the nonrandom spread of specialized malignant cells that preexist within a primary neoplasm. These metastases can be clonal in their origin, and different metastases can originate from different progenitor cells. In addition, metastatic cells can exhibit an increased rate of spontaneous mutation compared with benign nonmetastatic cells. These data provide an explanation for the clinical observation that multiple metastases can exhibit different sensitivities to the same therapeutic modalities. These findings suggest that the successful therapy of disseminated metastases will have to circumvent the problems of neoplastic heterogeneity and the development of resistance. Appropriately activated macrophages can fulfill these demanding criteria. Macrophages can be activated to become tumoricidal by interaction with phospholipid vesicles (liposomes) containing immunomodulators. Tumoricidal macrophages can recognize and destroy neoplastic cells in vitro and in vivo, leaving nonneoplastic cells uninjured. Although the exact mechanism(s) by which macrophages discriminate between tumorigenic and normal cells is unknown, it is independent of tumor cell characteristics such as immunogenicity, metastatic potential, and sensitivity to cytotoxic drugs. Moreover, macrophage destruction of tumor cells apparently is not associated with the development of tumor cell resistance. Macrophages are found in association with malignant tumors in a definable pattern, suggesting that the most direct way to achieve macrophage-mediated tumor regression is in situ macrophage activation. Intravenously administered liposomes are cleared from the circulation by phagocytic cells, including macrophages, so when liposomes containing immunomodulators are endocytosed, cytotoxic macrophages are generated in situ. The administration of such liposomes in certain protocols has been shown to bring about eradication of cancer metastases. Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at the start of treatment is minimal. For this reason, we have been investigating various methods to achieve maximal cytoreduction in metastases by modalities such as chemotherapy or radiotherapy prior to macrophage-directed therapy. It is important to note that even the destruction of 99.9% of cells in a metastasis measuring 1 cm2 would leave 10(6) cells to proliferate and kill the host. The ability of tumoricidal macrophages to distinguish neoplastic from bystander nonneoplastic cells presents an attractive possibility for treatment of the few tumor cells which escape destruction by conventional treatments. Macrophage-directed therapy has been studied in several human protocols, yielding important biological information about the use of liposome-encapsulated macrophage activators in cancer patients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging

It has been thought that metastases are clonal and originate from rare cells in primary tumors that are heterogeneous in genotype and phenotype. Recent studies using DNA array analysis challenge this hypothesis and suggest the genetic background of the host is the important determinant of metastatic potential implying that metastases are not necessarily clonal. Previous methods to determine clonality of metastasis used karyotype or molecular analysis that were complicated, thereby limiting the number of metastatic colonies analyzed and the conclusions that could be drawn. We describe here the use of green fluorescent protein-labeled or red fluorescent protein-labeled HT-1080 human fibrosarcoma cells to determine clonality by simple fluorescence visualization of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. In a spontaneous metastasis model originating from footpad injection in severe combined immunodeficient mice, 95% of the resulting lung colonies were either pure green or pure red, indicating monoclonal origin, whereas 5% were of mixed color, indicating polyclonal origin. In an experimental lung metastasis model established by tail vein injection in severe combined immunodeficient mice, clonality of lung metastasis was dependent on cell number. With a minimum cell number injected, almost all (96%) colonies were pure red or green and therefore monoclonal. When a large number of cells were injected, almost all (87%) colonies were mixed color and therefore heteroclonal. We conclude that spontaneous metastasis may be clonal because they are rare events, thereby supporting the rare-cell clonal origin of metastasis hypothesis. The clonality of the experimental metastasis model depended on the number of input cells. The simple fluorescence method of determining clonality of metastases described here can allow large-scale clonal analysis in numerous types of metastatic models.     

Clonal origin of metastasis in B16 murine melanoma: a cytogenetic study

A cell line isolated from the B16 melanoma and carried in continuous culture for 8 years (the parent line) exhibited great heterogeneity in terms of marker chromosome content. A lung metastasis from a C57BL/6 mouse inoculated im with cells of this line showed karyotypic homogeneity. Inoculation iv of cells from the parent line produced numerous tumor foci in various organs. Cytogenetic analyses of 18 such lesions led to the following conclusions: Cells from each metastatic colony exhibited relatively homogeneous karyotypic characteristics, indicating that metastases are of clonal origin; many parental cells with different marker chromosomes had metastatic potential; and some genomes maintained homogeneity longer than others.     

Tumor heterogeneity and drug resistance

Drug resistance has long been identified as a major reason for therapy failure in cancer patients. Concurrently, work from many laboratories in the past 10 years has established tumor heterogeneity as a phenomenon of critical importance in the natural history of individual neoplasms. The two most sinister aspects of intraneoplastic diversity in human solid tumors are the genesis of clones with metastatic potential, and the existence of drug-resistant variants in primary cancers and their metastases. Thus, recent investigations on drug resistance and on tumor heterogeneity have converged to focus attention on the clonal organization of primary tumors and their metastases as the underlying basis for anticancer drug resistance. This review examines the degree of heterogeneity observed within tumors and the relationship of this diversity to resistance that might be anticipated for any given agent. A question critical to our discussion is "How many subpopulations are there?" The impact of multiple tumor clones on therapy is next discussed in relationship to normal tissue tolerance, the barrier clinicians face regardless of the specific agent used in treatment. Finally, laboratory and clinical approaches are presented for addressing a drug resistance problem that is seemingly overwhelming because of its complex biological roots.     

Characterization of variants of a human breast-cancer cell-line isolated from metastases in different organs of nude-mice

The MDA-MB-435 human breast cancer cell line metastasizes from tumors growing in the mammary fatpad of nude mice; metastases are found in the lymph nodes and lungs of 75 to 100% of tumor-bearing mice, and at a lower incidence in other organs such as the heart, skeletal muscle, adrenal gland and brain. Variants of this breast cancer cell line were established from metastases in different organs, including the lungs and brain of nude mice; these lines were designated 435-Lung2 and 435-Br1, respectively. Karyotypic analysis of the new variants suggested a clonal origin for the different organ metastases. The lung-derived cells were as metastatic as the original cell line when re-injected in nude mice, yet the cells from the brain metastasis produced few metastases. Analyses of the invasive potential of the cell lines showed that they did not differ in expression of mRNA for 72-kDa type IV-collagenase, or in gelatinase activity (by zymography), or in ability to invade through a Matrigel-coated filter. The poorly metastatic 435-Br1 cells showed reduced binding to cultured monolayers of mouse lung-endothelial cells, compared with the metastatic 435-Lung2 and MDA-MB-435 cells. When the breast cancer cells were artificially arrested in the lungs of nude mice, by injecting Cytodex beads coated with cells, all three cell lines grew equally well, suggesting that the deficiency found in the metastatic potential of the 435-Br1 cells may be associated with interactions with endothelial cells, rather than growth potential in the lungs. Further comparisons of these two metastasis-derived variants of a heterogeneous cell line will lead to greater understanding of the metastatic phenotype of human breast cancer.     

Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis

Summary Human colon carcinomas (HCC) are heterogeneous for a varriety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop animal models for the selection and isolation of metastatic variants from human colon concers and for testing the metastatic potential of these cells. We have implanted cells from more than 100 HCC (obtained from surgical specimens) into different organs of nude mice. Regardless of their malignant potential in the patient, the HCC did not metastasize unless they were implanted orthotopically. Only when they were injected into the cecum or spleen of nude mice did they yield hepatic metastases. These metastases consisted of highly metastatic cells. The invasive phenotype was influenced by the organ environment. HCC cells in the subcutis did not produce degradative enzymes and the cells did not metastasize. In contrast, HCC cells in the cecum did both. Collectively, the results demonstrate that the orthotopic implantation of HCC cells can yield metastatic subpopulations of cells suitable for the study of metastasis.     

A study of tumor heterogeneity in a case with breast cancer

Tumor heterogeneity has been suggested based on clinical and pathological findings. Several clinical findings can be explained by tumor evolution during progression and metastasis. We herein report a case of metastatic breast cancer indicated tumor heterogeneity by clinical findings and a genomic analysis. A 64-year-old woman with advanced breast cancer was treated with primary chemotherapy, to which primary tumor responded. After a 6 month treatment pause, lung, liver, and skin metastases developed and her serum tumor markers were elevated. None of those serum markers had been elevated before the treatment, despite the large tumor burden. Notably, there was discordance in the expression of human epidermal growth factor receptor 2 (HER2) between the primary tumor and metastatic skin lesions, with the former being negative and the latter positive. A genomic analysis was performed by in-house Breast Cancer Panel, which consisted of 53 pre-selected genes. Twenty-three somatic mutations were found in primary breast tumor and 7 in the skin metastasis. None of these 30 genes matched. However, the cell-free (cf) DNA in the plasma taken at the time of skin metastasis contained 10 mutations, 7 from the primary lesion and 3 from the metastasis. These data indicate that the clonal changes or tumor heterogeneity was shown in two solid tumors by clinical and the result of a genomic analysis. Of particular interest was that cell-free DNA could be a powerful tool to look into these dynamic changes.     

Seasonal modulation of post-resection breast cancer metastasis

Background Human breast cancer incidence, histopathologic grade, invasiveness, and mortality risk vary significantly throughout each year. In order to better understand this seasonal cancer biology, we investigated the circannual pattern of post-resection breast cancer metastasis, under genetically and environmentally controlled conditions. Methods Over a span of 14 consecutive years, we conducted 22 similar experiments to investigate metastatic biology of breast cancer among 1,214 C3HeB/FeJ female mice. All mice were kept in temperature-controlled environment with 12 h light:12 h dark photoperiod, with food and water freely available, from birth until death. At 10-13 weeks of age, each mouse received 20,000 viable syngeneic mammary cancer cells subcutaneously and the tumor bearing leg was resected 10-12 days after tumor inoculation for potential cure. Once 10% of resected mice were found moribund, due to autopsy proven pulmonary metastases, all remaining mice were sacrificed and metastatic lung nodules were counted. Results The incidence of post-resection pulmonary metastasis was not randomly distributed throughout the year, but peaked prominently in Summer and Winter. Although tumor volume at resection was strongly associated with metastatic potential, a significantly higher probability of pulmonary metastasis was observed if surgery was performed in Summer and Winter, regardless of tumor volume at resection, compared to Spring and Fall. Conclusion These results support the likelihood that human breast cancer seasonality is real and of biological origin. There are implications of this cancer chronobiology for breast cancer prevention, screening, diagnosis, and treatment.     

Clonal drift and role of chromosome dosage in human melanoma metastatic cell lines: a statistical analysis.

Karyotypic analyses were performed on twenty human melanoma clones and variants all deriving, following in vivo selections and/or in vitro cloning, from the parental M4Be cell line, but expressing different tumorigenicity (expressed as mean tumor weight) and metastatic ability (expressed as pulmonary metastasis frequency) after s.c. injection in antithymocyte immunosuppressed newborn rats. These cells were hypertriploid, showing quite comparable modal numbers (around 70). They all expressed a wide range of chromosome number per mitosis (28 to 198), as well as a large extent of karyotypic heterogeneity, showed by extensive clonal drifts within the different cell lines. Their common origin was ascertained by five clonal abnormal marker chromosomes deriving from chromosomes 6, 7, 8, 9, 11 and 14. Twenty-one additional marker chromosomes, most of them non clonal, were observed in the different cell lines. We developed a statistical analysis to search for putative relationship between the expression of tumorigenicity and metastatic ability and the evolution of specific subclones within the different cell lines. We showed that the expression of a high mean tumor weight and/or a high metastasis incidence was related to the modification of the ratio of different subclones within each cell line, and more especially to the emergence of subclones presenting partial losses of individual chromosomes or chromosomal fragments, which were encountered with a lower frequency in the low metastatic cell lines. The biological relevance of these findings in terms of clonal evolution and role of chromosome dosage in tumor progression is discussed.     

Metastatic heterogeneity of cells from Lewis lung carcinoma

To allow investigations of the role of tumor cell proteases in invasion and metastasis, an attempt was made to obtain well-defined homogeneous populations of Lewis Lung carcinoma cells differing widely in their metastatic potential. From a single Lewis lung carcinoma, a parental line of cells was established and subsequently cloned so as to provide 18 clonal tumor cell lines. These clones differed in their ability to produce spontaneous, macroscopically visible metastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less metastatic than the parental line. The parental line expressed a metastatic behavior close to that of the high-metastatic cell subpopulations that it contained. There was, within certain limits, a good correlation between the potential for spontaneous lung metastases arising from a primary tumor and that for "artificial" lung colonies obtained after i.v. injection of the Lewis lung carcinoma cells. Although positively correlated with the growth rate of the tumor cells, the metastatic ability of the clones could not be considered as a mere reflection of the proliferation rates of the cells constituting the primary tumors. Differences in metastatic behavior observed among clones persisted in several cases after the cells had been maintained in culture for prolonged periods. However, this stability of the clones in vitro was not absolute. Indeed, some subclones isolated from the low-metastatic clone H122 displayed metastatic abilities which were lower than that of the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged culture period of that same clone, H122. Thus, new metastatic phenotypes can emerged under in vitro culture conditions. However, the relative rarity of this event suggests that some metastatic heterogeneity already preexisted in vivo among the tumor cells.     

Bisphosphonate treatment and radiotherapy in metastatic breast cancer

Patients with advanced breast cancer frequently develop metastasis to bone. Bone metastasis results in intractable pain and high risk of pathologic fractures due to osteolysis. The treatment of breast cancer patients with bone metastases requires a multidisciplinary approach. Radiotherapy is an established treatment for metastatic bone pain. It may be delivered either as a localized low dose treatment for localized bone pain or systemically for more widespread symptoms. Bisphosphonates have been shown to reduce morbidity and bone pain from bone metastases when given to patients with metastatic bone disease. In vivo studies indicate that early bisphosphonates administration in combination with radiotherapy improves remineralization and restabilization of osteolytic bone metastases in animal tumor models. This review focused on a brief discussion about biology of bone metastases, the effects of radiotherapy and bisphosphonate therapy, and possible mechanisms of combination therapy in metastatic breast cancer patients.     

Clonal dominance of primary tumours by metastatic cells: genetic analysis and biological implications

A new method is described for analysing the clonal evolutionary dynamics of tumour growth and the lineage relationship of primary tumours to their metastases. It exploits random integrations of transfected plasmid or retroviral infected (proviral) DNA as a means of generating very large numbers of uniquely marked cell clones in a single-step selection whose fates can then be tracked during progressive tumour growth. Using a mouse breast adenocarcinoma we undertook experiments in which syngeneic mice were injected with a mixture of very large numbers of uniquely marked cell clones, only one or a few of which were metastatic, or with reconstituted mixtures containing a genetically tagged metastatic clone with an excess of non-marked non-metastatic tumour cells. Among the results we summarize is the finding that spontaneous metastases developed in a non-random fashion from genotypically distinct cell clones. They were clonal or biclonal at the time of analysis. We also found that the progeny of a single metastatic clone could eventually overgrow the primary tumour. Thus malignant (metastatic) cells may manifest a striking growth advantage within the primary tumour site as well as for dissemination and growth at distant, secondary sites. As a result, late-stage advanced primary tumours, if left intact, may evolve to become biologically similar or equivalent to distant metastases. This 'clonal dominance' phenomenon can reconcile many of the discrepant experimental findings with respect to the putative selective nature of metastatic phenotype. Furthermore, it has important consequences for understanding one source of biological variability in experiments in which different primary tumours are compared to each other or to metastases; it also has implications for theories regarding the clonal origin of neoplasms, and for the physiological and biochemical changes that cause malignant disease.     

Placental metastasis (author's transl)]

Rarity of placental metastasis is only apparent, for only few placentas of cancerous mothers have been examined histologically. However, it may show biological and immunological conditions which are characteristics of foeto-placental unit. During metastatic spread of solid tumors or hematologic malignancies in the mother, tumor emboli may be localized in intervillous spaces, without being real placental metastasis. Rarely tumor emboli are able to invade the struma of chorionic villi and produce true placental metastases: twelve such observations have been published, seven of which were malignant melanomas. It is even more exceptional that metastatic spread reaches the foetus. In most of the cases, it is thus protected against maternal cancer. This historical observation holds true. The fear of transplacental graft to the foetus is not an argument favorable of terminating a cancer associated pregnancy and foetal metastasis of maternal origin are not among the causes of congenital cancers in children.     

Clonal heterogeneity in sporadic melanomas as revealed by loss-of-heterozygosity analysis

The major obstacle preventing effective treatment of melanoma is the biological heterogeneity of tumor cells. This study was performed to determine clonal genetic heterogeneity within primary melanoma and the evolution of these heterogeneous sub-clones during disease progression. DNA samples were obtained from 44 morphologically distinct areas identified within 10 primary tumors and from 15 metastases in the same patients. Loss of heterozygosity (LOH) analyses were performed using 17 microsatellite markers that mapped to chromosomes 6q, 9p, 10q and 18q, the most frequently deleted in melanoma. Of 10 primary tumors, 8 were revealed to have intratumoral genetic heterogeneity in terms of LOH of the 4 chromosome arms examined, 7 containing at least 2 different sub-clones harboring LOH of different chromosome areas, while the remaining one tumor showed prominent intratumoral genetic heterogeneity consisting of at least 6 genetically distinct sub-clones. LOH of 6q was detected only in a sub-set of multiple microdissected samples in most of the primary tumors, but was most frequently detected in metastases, suggesting that loss of this chromosome arm occurred late and played an important part in metastatic progression. Comparison of LOH between sub-clones within primary tumors and within metastases showed the divergence of metastatic clones from dominant populations within the primary tumor in 5 patients, whereas in the remaining three patients parent sub-clones were not identified, or constituted only a minor sub-population within the primary tumors. These results, showing considerable genetic heterogeneity in sporadic melanoma, have profound implications for the choice of future therapeutic strategies.