Detection of chromosome aberrations in paraffin sections of seven gonadal yolk sac tumors of childhood

Yolk sac tumors are the most frequent kind of malignant pediatric germ cell tumor and may have a fundamentally different pathogenesis than adult germ cell tumors. Since few cytogenetic studies have been performed so far, in situ hybridization was applied to interphase cell nuclei of seven gonadal yolk sac tumors of childhood in routine paraffin-embedded tissue sections. The panel of chromosome-specific DNA probes was selected on the basis of their relevance in adult germ cell tumors and consisted of five DNA probes specific for the (peri)centromeric regions of chromosomes 1, 8, 12, 17 and/or X and/ or one DNA probe specific for the subtelomeric region of chromosome 1 (p36.3). As in adult germ cell tumors, all pediatric gonadal yolk sac tumors had an increased incidence of numerical chromosome aberrations. All tumors showed an overrepresentation of at least three chromosomes. Gains of chromosome 12, which is highly specific in adult germ cell tumors, were diagnosed in six pediatric gonadal yolk sac tumors. The DNA indices determined in the paraffin-embedded tumor material correlated well with the in situ hybridization findings. A chromosome was either over- or underrepresented, compared with the corresponding DNA indices, in only a few cases. The short arm of chromosome 1 in adult germ cell tumors is often involved in structural aberrations. In pediatric germ cell tumors, the short arm of chromosome 1 is also a nonrandom site of structural aberrations. Moreover, the presence of a deletion at 1p36.3 in four out of five tumors suggests that the loss of gene(s) in this region is an important event in the pathogenesis of gonadal yolk sac tumors of childhood.     

Primary mediastinal malignancies: findings in 219 patients

The purpose of this study was to determine the demographics, histology, methods of treatment, and survival in primary mediastinal malignancies. We did a retrospective review of the statewide New Mexico Tumor Registry for all malignant tumors treated between January 1, 1973 and December 31, 1995. Benign tumors and cysts of the mediastinum were excluded. Two hundred nineteen patients were identified from a total of 110,284 patients with primary malignancies: 55% of tumors were lymphomas, 16% malignant germ cell tumors, 14% malignant thymomas, 5% sarcomas, 3% malignant neurogenic tumors, and 7% other tumors. There were significant differences in gender between histologies (P < 0.001). Ninety-four percent of germ cell tumors occurred in males, 66% of neurogenic tumors were in females; other tumors occurred in males in 58% of cases. There were also significant differences in ages by histology (P < 0.001). Neurogenic tumors were most common in the first decade, lymphomas and germ cell tumors in the second to fourth decades, and lymphomas and thymomas in patients in their fifth decades and beyond. Stage at presentation (P = 0.001) and treatment (P < 0.001) also differed significantly between histologic groups. Five-year survival was 54% for lymphomas, 51% for malignant germ cell tumors, 49% for malignant thymomas, 33% for sarcomas, 56% for neurogenic tumors, and 51% overall. These survival rates were not statistically different (P > 0.50). Lymphomas, malignant germ cell tumors, and thymomas were the most frequently encountered malignant primary mediastinal neoplasms in this contemporary series of patients. Demographics, stage at presentation, and treatment modality varied significantly by histology. Despite these differences, overall five-year survival was not statistically different.     

Chromosome X modulates incidence of testicular germ cell tumors in <Emphasis Type="Italic">Ter</Emphasis> mice

Germ cell tumor development in humans has been proposed to be part of testicular dysgenesis syndrome (TDS), which manifests as undescended testes, sterility, hypospadias, and, in extreme cases, as germ cell tumors. Males of the Ter mouse strain show interesting parallels to TDS because they either lack germ cells and are sterile or develop testicular germ cell tumors. We found that these defects in Ter mice are due to mutational inactivation of the Dead-end (Dnd1) gene. Here we report that chromosome X modulates germ cell tumor development in Ter mice. We tested whether the X or the Y chromosome influences tumor incidence. We used chromosome substitution strains to generate two new mouse strains: 129-Ter/Ter that carry either a C57BL/6J (B6)-derived chromosome (Chr) X or Y. We found that Ter/Ter males with B6-Chr X, but not B6-Chr Y, showed a significant shift in propensity from testicular tumor development to sterile testes phenotype. Thus, our studies provide unambiguous evidence that genetic factors from Chr X modulate the incidence of germ cell tumors in mice with inactivated Dnd1. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Telomerase activity in Kaposi's sarcoma, squamous cell carcinoma, and basal cell carcinoma

Patients with acquired immune deficiency syndrome (AIDS) often develop Kaposi's sarcoma (KS), an unusual skin tumor. The malignant nature of KS has long been disputed. Telomerase activity that maintains telomere length and ensures chromosomal stability, a frequently appearing marker in human malignancies, has been proposed to play a critical role in supporting continued cell growth, hence formation of tumors. We examined telomerase activity in tissue extracts from 22 KS, 10 squamous cell carcinoma (SCC), and 22 basal cell carcinoma (BCC) using the telomeric repeat amplification protocol (TRAP). All of the tumor tissues were previously cryopreserved at -80 degrees C. In this study, all tumor samples tested were positive for telomerase activity. Consistent with the presence of the enzyme activity, the skin tumors had relatively long telomeres. Inhibitors in the tissue extracts of some samples needed to be diluted or extracted by phenol before the enzyme activity was detected in the TRAP assay. All KS as well as two other skin carcinoma samples revealed positive telomerase activity. Our finding supports telomerase's role in tumor cell immortality and suggests the true neoplastic nature of KS.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Serum lactate dehydrogenase isoenzyme 1 activity in patients with testicular germ cell tumors correlates with the total number of copies of the short arm of chromosome 12 in the tumor

Summary The aim of our study was to assess the relationship between the serum lactate dehydrogenase isoenzyme 1 (S-LDH-1) activity in patients with testicular germ cell tumors and the number of copies of the short arm of chromosome 12 (12p) present in tumor. Twenty-seven adult patients with measurable tumor lesions were studied. Twenty-five had three or more copies of chromosome 12 per cell in the tumors. Nineteen had one or more copies of a specific chromosomal abnormality, an isochromosome of the short arm of chromosome 12, i(12p). Fourteen had increased S-LDH-1 levels. S-LDH-1 activity correlated significantly with the product of total tumor volume and the total number of copies of the short arm of chromosome 12 present per cell (total tumor 12p). We conclude that the total number of copies of the short arm of chromosome 12 in the tumors is most probably a factor contributing to the LDH-1 activity released from the tumors.     

Meiotic studies of a 38,XY/39,XXY mosaic boar

Klinefelter's syndrome (KS) is the most common sex chromosome abnormality identified in human males. This syndrome is generally associated with infertility. Men with KS may have a 47,XXY or a 46,XY/47,XXY karyotype. Studies carried out in humans and mice suggest that only XY cells are able to enter and complete meiosis. These cells could originate from the XY cells present in mosaic patients or from XXY cells that have lost one X chromosome. In pig, only 3 cases of pure 39,XXY have been reported until now, and no meiotic analysis was carried out. For the first time in pig species we report the analysis of a 38,XY/39,XXY boar and describe the origin of the supplementary X chromosome and the chromosomal constitutions of the germ and Sertoli cells.     

The incidence of male genital tumors: a cellular model for the age dependence.

Most human tumors, including most male genital tumors, exhibit an exponential increase in incidence with advancing age of the host. This exponential age-incidence pattern can be explained by the accumulation of mutations in the stem cells of the tissues of tumor origin. The age-incidence pattern for testicular tumors, however, is unique with a large linear increase in incidence from age 14 to 30 and a linear decline in incidence from age 30 to 60. After age 60, the incidence of testicular tumors remains low and constant. The probability of testicular tumorigenesis is determined by the susceptibility of male germ cells to neoplastic mutation and/or the neoplastic mutagenicity of the male germ cell environment. Since there is no evidence for an environmental mutagen which is specific for male germ cells, and since male germ cells are unusually susceptible to mutation, we interpret the variation in testicular tumor incidence with age as a reflection of the susceptibility of male germ cells to neoplastic mutation. Cell are most susceptible to mutation during genome replication and we propose a model for testicular tumorigenesis which is consistent with the available data on male germ cell proliferation and with the data on testicular tumor incidence.     

Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

The dynamics of imprinted X inactivation during preimplantation development in mice

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.     

Testicular germ cell tumor susceptibility genes from the consomic 129.MOLF-Chr19 mouse strain

Chromosome substitution strains (CSS or consomic strains) are useful for mapping phenotypes to chromosomes. However, huge efforts are needed to identify the gene(s) responsible for the phenotype in the complex context of the chromosome. Here we report the identification of candidate disease genes from a CSS by using a combination of genetic and genomic approaches and by using knowledge about the germ cell tumor disease etiology. We used the CSS 129.MOLF-Chr19 chromosome substitution strain, in which males develop germ cell tumors of the testes at an extremely high rate. We were able to identify three protein-coding genes and one microRNA on chromosome 19 that have previously not been implicated to be testicular tumor susceptibility genes. Our findings suggest that changes in gene expression levels in the gonadal tissues of multiple genes from Chr 19 likely contribute to the high testicular germ cell tumor (TGCT) incidence of the 129.MOLF-Chr19 strain. Our data advance the use of CSS to identify disease susceptibility genes and demonstrate that the 129.MOLF-Chr19 strain serves as a useful model to elucidate the genetics and biology of germ cell transformation and tumor development. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Age-specific incidence data indicate four mutations are required for human testicular cancers

Normal human cells require a series of genetic alterations to undergo malignant transformation. Direct sequencing of human tumors has identified hundreds of mutations in tumors, but many of these are thought to be unnecessary and a result of, rather than a cause of, the tumor. The exact number of mutations to transform a normal human cell into a tumor cell is unknown. Here I show that male gonadal germ cell tumors, the most common form of testicular cancers, occur after four mutations. I infer this by constructing a mathematical model based upon the multi-hit hypothesis and comparing it to the age-specific incidence data. This result is consistent with the multi-hit hypothesis, and implies that these cancers are genetically or epigenetically predetermined at birth or an early age.     

Many different tumor types have polyclonal tumor origin: evidence and implications

Few ideas have gained such strong acceptance in the scientific community as the monoclonal origin of tumors; the idea that tumors start with a single mutated cell (or a single clone of cells) that go on to accumulate additional mutations as a tumor develops. The certainty with which this concept is held by the scientific community reflects the length of time it has been unchallenged and the experimental difficulty in obtaining direct evidence to the contrary. Yet, recent findings regarding X chromosome inactivation patch size indicate that the X-linked marker data previously interpreted as evidence of monoclonal tumor origin is actually more consistent with polyclonal tumor origin, a situation where two or more cells or clones of cells interact to initiate a tumor. Although most tumors show homotypy for X-linked markers (as expected given the bias conferred by X chromosome inactivation patch size), the literature contains numerous examples of tumors with X-linked marker heterotypy, examples of which encompass 24 different tumor types. Chimeric models have yielded direct unequivocal demonstrations of polyclonality in rodent and human tumors. Also, mutational data are consistent with polyclonal tumor origin. Methods that analyze levels of tumor-associated oncogene and tumor suppressor gene mutations demonstrate that initiated cells are much more common in normal tissues than previously realized. Also, while tumors have higher levels of mutation than normal tissues, oncogenic mutations frequently are present as subpopulations within tumors, rather than as the pure mutant populations expected to develop from a single initiated cell. Understanding the mutational basis of tumor etiology has important practical significance for assessing cancer risk, as well as in modeling and treating cancer. Therefore, the scientific community needs to re-examine this issue and consider the implications of polyclonal origin for, perhaps, a majority of tumors, encompassing many different tumor types.     

Expression of intermediate filaments and other special markers by testicular germ cell tumors. With reference to embryogenesis.

Distribution of intermediate filament proteins (IFs) and several special markers was studied in 39 testicular germ cell tumors and 8 embryos and foetuses. The similarity and difference between development of germ cell tumor and embryogenesis were immunohistochemically investigated. Seminoma and embryonal carcinoma, as tumoral counterparts of undifferentiated germ cells, were characterized by little IF expression. This study revealed that the maturing and differentiating process in germ cell tumor is different from normal embryonal development and the tumor cells showed leaping maturing steps in tumorigenesis. Immunostaining for IFs helped to discover the further differentiation occurring in embryonal carcinoma and to demonstrate heterogeneous elements in non-seminoma germ cell tumors, which sometimes might not be apparent by light microscopical observation of H&E staining section. According to the findings, two patterns in mixed germ cell tumors are suggested; i.e., combined and diffuse types. The mechanism of tumorigenesis of the two types is supposed to be different. Clinically, the prognosis of most patients with testicular germ cell tumor is fairly good because of the improved chemotherapies that are dependent on histological diagnosis.     

Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation

The patterns of cytoskeletal differentiation were studied in 20 testicular non-seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin-fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)