R.F. Suijkerbuijk

Overrepresentation of chromosome 12p sequences and karyotypic evolution in i(12p)-negative testicular germ-cell tumors revealed by fluorescence in situ hybridization

Human testicular germ-cell tumors (TGCTs) comprise a heterogeneous group of solid neoplasms. These tumors are characterized by the presence of a highly specific chromosomal abnormality, i.e., an isochromosome of the short arm of chromosome 12. At present, this i(12p) chromosome is found in more than 80% of TGCTs. Isochromosome 12p has also been observed in some ovarian and extragonadal germ cell tumors. In the remaining so-called i(12p)-negative TGCTs other abnormalities involving chromosome 12, mainly 12p, can be found. In order to establish whether 12p abnormalities other than i(12p) are a common phenomenon in TGCTs, a panel of 11 i(12p)-negative tumors was investigated using multicolor fluorescence in situ hybridization. All TGCTs examined appeared to contain chromosomal abnormalities involving 12p, resulting in a distinct overrepresentation of short arm sequences. In addition, indications were obtained for a clonal evolution in one of the tumors. Our data suggest that the occurrence of 12p abnormalities is a common phenomenon in i(12p)-negative TGCTs and that these abnormalities, analogous to i(12p), may contribute to the process of tumor development.

Comparative genomic hybridization of germ cell tumors of the adult testis: Confirmation of karyotypic findings and identification of a 12p- amplicon

Comparative genomic hybridization (CGH) was carried out on 15 primary testicular germ cell tumors (TGCT) of adolescents and adults and two metastatic residual tumors after chemotherapeutic treatment. The results were compared with karyotypic data obtained form the same tumor specimens after direct harvesting of metaphases or short-term in vitro culture. Both techniques revealed that the most consistent abnormality in primary TGCT is gain of 12p-sequences. Although in most cases over-representation of the complete short arm was observed, CGH revealed a specific amplification of 12p11.1-p12.1 region in two independent primary tumors. In addition, loss of (parts of) chromosome 13 (always involving q31-qter), and gain of (parts of) chromosome 7 (mostly involving q11), (parts of) chromosome 8, and the X chromosome were detected in more than 25% of the tumors by this latter technique. Loss of 6q15-q21 in both residual tumors analyzed may suggest a role for this anomaly in acquired resistance to chemotherapeutic treatment. Overall, the CGH analyses confirmed gains and losses of certain chromosomal regions in TGCT as observed by karyotyping, and thus support their role in the development of these neoplasms. The amplification of a restricted region of 12p in primary TGCT confirms and extends our previous observations and, as such, represents an important step forward in the identification of gene(s) on 12p relevant for the pathogenesis of these tumors.

Detection of chromosomal DNA gains and losses in testicular germ cell tumors by comparative genomic hybridization

To extend the results of conventional cytogenetic analysis of testicular germ cell tumors (TGCTs), we applied the new molecular cytogenetic method of comparative genomic hybridization (CGH), which enables the detection of chromosomal imbalances without the need for dividing cells. DNA from II TGCTs was studied by CGH. In all tumors examined, gain of 12p, mostly of the whole p arm, could be demonstrated. However, in three tumors, an amplification of 12p material restricted to the chromosomal bands 12p11.2‐p12.1 was found. Further fluorescence in situ hybridization (FISH) analysis using a yeast artificial chromosome (YAC) that was previously mapped to that region revealed multiple copies of that chromosomal segment in interphase nuclei of these tumors. This finding is an important clue to the localization of candidate protooncogenes at 12p involved in TGCTs. Gains of small chromosomal regions at 2p, 4q, 6p, and 19p were also detected recurrently. Furthermore, gains of chromosomes 8, 14, 21, and X as well as loss of chromosome 13 were frequent findings. In conclusion, CGH provides new insights into genetic alterations of TGCTs. By using CGH, chromosomal subregions could be identified that may harbor genes involved in the pathogenesis of this malignancy. Genes Chromosom Cancer 17:78–87 (1996).

Distinct Xp11.2 breakpoint regions in synovial sarcoma revealed by metaphase and interphase FISH: relationship to histologic subtypes.

Fluorescence in situ hybridization (FISH) and molecular analyses of synovial sarcomas with cytogenetically similar (X;18)(p11.2;q11.2) translocations have revealed two alternative breakpoint regions in Xp11.2, one residing in the ornithine aminotransferase-like 1 (OATL1) region and the other one in the related but distinct OATL2 region. As these results were obtained by different groups, we set out to evaluate an extended series of tumors with special emphasis on the two possible X-related breakpoint regions. Together, seven synovial sarcomas were identified with a break in the OATL1 region and six with a break near OATL2, thereby confirming the actual existence of the two alternative Xp breakpoint regions. We speculate that there seems to be a relationship between the occurrence of these breakpoint regions and the histologic phenotype of the tumors, with a predominance of OATL1-related breakpoints in the classical biphasic tumors and of OATL2-related breakpoints in the monophasic fibrous tumors.

Verification of isochromosome 12p and identification of other chromosome 12 aberrations in gonadal and extragonadal human germ cell tumors by bicolor double fluorescence in situ hybridization.

A diverse group of gonadal and extragonadal human germ cell tumors (GCT) and GCT-derived cell lines was examined for the presence of an i(12p) marker chromosome and/or other abnormalities involving chromosome 12, especially 12p, by bicolor double fluorescence in situ hybridization (FISH). For this purpose three probes, pBS-12, M28, and p alpha 12H8, were used, allowing specific identification of the entire chromosome 12, its short arm, and its pericentromeric region, respectively. The presence of one or more copies of a genuine i(12p) chromosome could be demonstrated in three GCT of the testis, in one ovarian GCT, in one dysgenetic GCT, and in one extragonadal intracranial GCT. Moreover, additional aberrations involving chromosome 12 were shown to be present not only in i(12p) minus but also in i(12p) positive GCT. These data suggest that the occurrence of such aberrations may be a common, although less clearly perceptible and frequent, phenomenon in human GCT.

Molecular cytogenetics of human germ cell tumours: i(12p) and related chromosomal anomalies

Human testicular germ cell tumours (TGCTs) comprise a heterogeneous group of solid neoplasms. These tumours are characterized by a highly specific chromosomal anomaly, i.e. an isochromosome of the short arm of chromosome 12. At present, this i(12p) chromosome has been observed in about 80% of TGCTs. Also in dysgerminomas of the ovary and in some extragonadal germ cell tumours i(12p) has been observed. In the remaining so-called i(12p)-negative tumours other cytogenetic abnormalities can be found. In addition, TGCTs are usually highly aneuploid. The exact nature and role of these different anomalies in tumour development are as yet undefined. Here we present a molecular cytogenetic analysis of a diverse group of gonadal and extragonadal germ cell tumours. Our results indicate that all tumours examined exhibit anomalies involving 12p [i(12p) and/or others], resulting in a distinct overrepresentation of short arm sequences. Thus, we argue that the occurrence of 12p abnormalities may be a characteristic of both i(12p)-positive and -negative TGCTs and that these abnormalities may, through similar mechanisms, contribute to the process of TGCT development. This notion is substantiated by our finding that in all cases the supernumerary 12p sequences are of uniparental origin.

Generation of a panel of somatic cell hybrids containing fragments of human chromosome 12P by X-ray irradiation and cell fusion

We have employed an irradiation and fusion procedure to generate somatic cell hybrids containing various fragments of the short arm of human chromosome 12 using a 12p-only hybrid (M28) as starting material. For the initial identification of hybrids retaining human DNA, nonradioactive in situ hybridization was performed. Seventeen cell lines appeared to contain detectable amounts of human material. Detailed characterization of these hybrids by Southern blot analysis and chromosomal in situ suppression hybridization (chromosome painting), using hybrid DNAs as probes after Alu element-mediated PCR, resulted in a hybrid panel encompassing the entire chromosome 12p arm. This panel will provide a valuable resource for the rapid isolation of region-specific DNA markers. In addition, this panel may be useful for the characterization of chromosome 12 aberrations in, e.g., human germ cell tumors.

Molecular characterization of a recurring complex chromosomal translocation in two human extragonadal germ cell tumors.

The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6q23, and 11q13 in two independent but similar extragonadal human germ cell tumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques. By using a series of specific probes from the 11q13 region, the translocation breakpoint in this chromosomal band could be located within a long-range restriction enzyme map in between the markers D11S457 and D11S546. In addition, aberrantly hybridizing restriction fragments were revealed by PFGE in both tumors, indicating that the breakpoint region must be located within a distance of at maximum 200 kilobase pairs (kbp) from the nearest DNA marker (D11S546).

A 46,XY female with mixed gonadal dysgenesis and a 48,XY, +7, +i(12p) chromosome pattern in a primary gonadal tumor.

Cytogenetic analysis of a primary germ-cell tumor originating from the streak gonad of a 20-year-old phenotypic female with a 46,XY karyotype and mixed gonadal dysgenesis revealed a 48,XY, +7, +i(12p) chromosomal pattern. Germ-cell tumors originating from gonads of normal males are usually highly aneuploid. An isochromosome 12p as well as an overrepresentation of chromosome 7 material are among the specific changes most consistently observed. The present case shows that tumors of dysgenetic gonads, albeit being near-diploid, may exhibit similar chromosomal changes. This observation lends additional support to the hypothesis that these specific cytogenetic anomalies may play an important role in the pathogenesis of human germ-cell tumors.

MOLECULAR CYTOGENETIC ANALYSIS OF A FAMILIAL PERICENTRIC-INVERSION OF CHROMOSOME-12.

Speleman F, Van Roy N, De Vos E, Hilliker C, Suijkerbuijk RFS, Leroy JG. Molecular cytogenetic analysis of a familial pericentric inversion of chromosome 12