Ron F. Suijkerbuijk

Reviews of Chromosome Studies in Urological Tumors. III. Cytogenetics and Genes in Testicular Tumors

PURPOSEnWe reviewed available cytogenetic and molecular findings in testicular germ cell tumors, and their possible application to clinical, pathological and basic parameters.nnnMATERIALS AND METHODSnFindings in the literature on testicular germ cell tumors as well as those from our laboratory were summarized, including a listing of the cytogenetic findings on testicular germ cell tumors to date with some illustrations.nnnRESULTSnTesticular germ cell tumors are characterized in most cases by the presence of an i(12p) isochromosome. In tumors without such an abnormal chromosome studies using fluorescence in situ hybridization and molecular approaches have demonstrated either masking of the i(12p) or the presence of extra 12p sequences in the karyotype. Although testicular germ cell tumors are often associated with chromosome changes in addition to the i(12p), no other specifically recurrent structural chromosome changes have been found. Based on the cytogenetic and molecular findings in testicular germ cell tumors, a hypothetical scheme for the genetic events leading to these tumors is presented.nnnCONCLUSIONSnThe genetic events leading to genesis of testicular germ cell tumors in men appear to be related to aneuploidization followed by the formation of an i(12p) isochromosome, the latter characterizing the preponderant number of testicular germ cell tumors. The exact role of the i(12p) isochromosome in testicular germ cell tumor pathogenesis remains to be determined, as is true of the genes involved in or affected by these tumors. Based on presently available information, a hypothetical pathogenetic and oncogenetic model for the development of testicular germ cell tumors is presented.

Amplification of chromosome subregion 12p11.2-p12.1 in a metastasis of an I(12p)-negative seminoma: relationship to tumor progression?

Cytogenetic analysis of a metastasis of a human testicular germ cell tumor (seminoma) revealed multiple numerical and structural anomalies, including an abnormally banding region (ABR) present on the short arm of one of the chromosome 12 homologs. Fluorescence in situ- and comparative genomic hybridization experiments revealed that the ABR results from the amplification of 12p11.2-p12.1 derived sequences. We speculate that this particular region may harbor gene(s) relevant for testicular germ cell tumor progression.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Increasing Levels of MYC and MET Co-Amplification During Tumor Progression of a Case of Gastric Cancer

The cytogenetic study of a nodal metastasis from a gastric carcinoma, after two passages in nude mice, revealed a large number of double minutes. Comparative genomic in situ hybridization (CGH) analysis using DNA extracted from this xenograft revealed the existence of three clear amplification units that originated from the chromosomal subregions 6q24-25, 7q31-32, and 8q24 in the xenograft DNA. Similar, though less prominent, CGH results were found with DNAs extracted from the primary tumor and its metastasis, implying that the same amplicons were also present, albeit less abundantly, in the DNAs of these neoplastic tissues. Southern analysis of the second-passage xenograft detected 18- and 10-fold amplification of MET (located at 7q31) and MYC (located at 8q24), respectively. The retrospective study of the first passage of the xenograft, as well as of the metastatic and primary tumors before xenografting, showed amplification levels of MET of, respectively, 12-, 9-, and 5-fold and MYC of, respectively, 8-, 7-, and 5-fold. Our results suggest that increased levels of co-amplification of MYC and MET correlate with enhanced growth potential in this case of gastric carcinoma.

Fluorescence in situ hybridization analysis of chromosome 12 anomalies in semen cells from patients with carcinoma in situ of the testis

Carcinoma in situ (CIS) of the testis is the precursor of seminomas and non‐seminomatous germ cell tumours of the adult testis. A marked cytogenetic anomaly, the isochromosome of the short arm of chromosome 12 [i(12p)], has been demonstrated in over 80 per cent of all histological varieties of testicular germ cell tumours (TGCTs). In the remaining group of i(12p)‐negative TGCTs, an overrepresentation of chromosome 12p sequences has been found. The i(12p) chromosome and overrepresentation of 12p sequences in CIS cells have also been reported. In order to establish whether numerical and/or structural aberrations of chromosome 12 can be found in CIS cells exfoliated into seminal fluid, semen specimens from ten patients with CIS lesions were investigated using bicolour double fluorescence in situ hybridization (FISH). The two DNA probes used, p12H8 and YAC 5, specifically detect the centromeric region of chromosome 12 and a subregion, p11.2–p12.1, on the short arm of chromosome 12, respectively. Ejaculates of ten azoospermic or oligozoospermic infertile males, presumably CIS‐free, were used as negative controls. Nuclei exhibiting three or more chromosome 12 signals were found to be present in a significantly larger number in the patient samples than in the control samples. Nuclei with five or more chromosome 12 signals were observed in eight out of the ten patients. Morphologically similar arrangements to i(12p) were observed in some of the ejaculates. These results demonstrate the potential of FISH in the early detection of CIS and TGCTs in males at high risk.Copyright

Fluorescent in situ identification of human marker chromosomes using flow sorting and Alu element-mediated PCR

A novel approach to the identification of human chromosomes has been developed. Chromosomal in situ hybridization (or chromosome painting) has been performed using Alu element-mediated PCR products from small quantities (250-500) of flow-sorted normal and abnormal chromosomes. Chromosome paints for various normal chromosomes, including 5, 6, 7, 14, 18, 19, 21, and 22, were generated and shown to be effective in the identification of the appropriate chromosomes. In addition, certain abnormal chromosomes, including a mental retardation-associated deletion chromosome 11 (q22-q23), the products of the constitutional translocation t(11;22), and the CML-associated t(9;22), were used to generate region-specific paints. In each case, the appropriate regions of the chromosomes were highlighted and this strategy is, therefore, well suited to the identification of previously unidentified marker chromosomes. A further direct consequence of this work is that chromosome paints specific for the common aberrant chromosomes, such as the Philadelphia chromosome, can be generated and made widely available. These may find particular use in the analysis of complex or masked chromosomal translocations.

Involvement of 3q21 in Nodular Fasciitis

Cytogenetic data on nodular fasciitis are sparse. We present a case of this lesion and discuss our results in view of previously reported findings in nodular fasciitis and other benign mesenchymal lesions.

Chromosomes 1 and 12 abnormalities in pediatric germ cell tumors by interphase fluorescence in situ hybridization

Chromosome studies of pediatric germ cell tumors (GCTs) show differences in abnormalities dependent on age, sex, tumor location, and histology. Previous studies suggest that loss of 1p is associated with a malignant phenotype, while amplification of 12p, a common finding in adult testicular GCTs, is uncommon in pediatric GCTs. Fifty-three pediatric GCTs were analyzed for 1p36 loss and 12p amplification by G-banding and dual-color interphase FISH with probes for the centromere and short arm of chromosomes 1 or 12. Twelve tumors with loss of 1p36 were identified. No deletion was detected in tumors with nonmalignant histology, such that there was a significant association of 1p loss with malignancy in these tumors (P = 0.00115). Five of 18 tumors from male patients had amplification of 12p, consistent with G-band results. Combined analysis of our data with those in the literature revealed a significant correlation of 12p amplification with patient age (P = 0.000196). Amplification of 12p was only seen in one of 35 tumors from female patients. Five female GCTs had numerical abnormalities of chromosome 12, and two tumors showed complete lack of 12p. This spectrum of abnormalities differs from what is seen in the male tumors, providing further evidence for different etiologies of GCTs between the sexes.

Isolation of Osteosarcoma-Associated Amplified DNA Sequences Using Representational Difference Analysis

Comparative genomic hybridization analysis of a primary osteosarcoma and its metastasis revealed two regions of DNA amplification, one at 17p11.2‐12 and one at 19q12‐13. Subsequent representational difference analysis of the primary tumor resulted in the isolation of two distinct tumor‐amplified DNA fragments originating from chromosome 19. A YAC clone corresponding to one of the two isolated DNA fragments was used for fluorescence in situ hybridization on normal human lymphocyte metaphases and tumor‐derived nuclei. This resulted in the localization of this YAC to 19q12‐13.1 and confirmed the amplification status of the isolated fragment in the tumors. The availability of such RDA‐isolated sequences may be instrumental in the search for genes relevant for tumor development. Genes Chromosomes Cancer 20:196–200, 1997.

A novel case of infantile sacral teratoma and a constitutional t(12;15)(q13;q25) pat.

Cytogenetic analysis of peripheral lymphocytes of an infantile patient with a sacral teratoma revealed a constitutional translocation (12;15)(q13;q25) pat. The same translocation was found in four additional relatives. Loss of heterozygosity analysis of the patients tumor material showed retention of both translocation-derived chromosomes. Since allelic loss in the 12q13 region has been observed in germ cell tumors, we hypothesize that disregulation of genes located at or near the 12q13 breakpoint may be related to the development of this sacral teratoma. As a first step towards the identification of these genes, a 12q13 genomic contig that spans the breakpoint has been constructed.