Characterization of gains, losses, and regional amplification in testicular germ cell tumor cell lines by comparative genomic hybridization
Introduction
The most consistent cytogenetic abnormality described in adult testicular germ cell tumors (TGCT) is the gain of chromosome arm 12p. This has been demonstrated not only by classical cytogenetic methods [1], but also by molecular cytogenetic techniques including fluorescence in situ hybridization (FISH) [2], [3], [4], [5], comparative genomic hybridization (CGH) [6], [7], [8], and 24-color karyotyping [9]. Gain of 12p is observed across all histological subtypes of adult TGCT, including early lesions of carcinoma in situ [10], [11]. We and others have demonstrated that over-representation of 12p typically takes one of four distinct forms: 1) an i(12p); 2) increased copy number of apparently intact 12p, distributed as chromosome 12 or translocated to other chromosomes; 3) partial 12p material translocated to another chromosome; or 4) homogeneously-stained region–like amplified regions consisting of 12p [12], [13], [14], [15]. Germ cell tumors are often hyperdiploid, with triploid and tetraploid numbers of chromosomes in seminomas and hyperdiploid to hypertriploid counts common in nonseminomas. Structural chromosomal variations are observed from one tumor to another [1], [16].
Previously published FISH [3], [13], [15] and CGH data [7], [17] demonstrated that a proximal area of 12p (12p11.2∼p12.1) was over-represented in several TGCT cases, raising the possibility that a gene(s) located in this chromosomal area might be overexpressed [15]. Kraggerud et al. [18] recently reported regional amplification of 12p in 13 TGCT. Six tumors had amplification restricted to 12p12∼pter. We have previously demonstrated, by triple-color FISH analysis with 12p band–specific paint probes, that 4 of 9 cell lines and 2 of 49 surgical specimens examined carried regional amplification of 12p12∼p13 [14]. Taken together, these findings could indicate that genes in at least two areas of 12p might be necessary for oncogenesis, explaining the ubiquitous 12p over-representation found in all TGCT. To identify and compare chromosome genome-wide losses and gains in tumor cell lines and to evaluate the lines for regional amplification of 12p, we performed CGH analysis on 12 TGCT cell lines and one paraffin-embedded surgical specimen.
Section snippets
Specimens and DNA preparation
The following tumor cell lines were used in this study (Table 1): GCT72∗, GCT48, 1411H∗, N2102ep∗, GCT35∗, GCT44∗, GCT27C4∗, 9512-17, NT2D1∗, 833K∗, 64CP, Tera I, and Tera II. Of these 12 lines, 7 of them (marked by asterisks) were analyzed by triple-color FISH analysis [14]. A paraffin-embedded surgical sample of a teratoma, UH9512-17, was also analyzed. Tumor DNA was prepared from approximately one to three T75 flasks per cell line using a standard sodium dodecyl sulfate (SDS)/proteinase K
Results
This study reports the CGH results from 12 TGCT lines and one surgical specimen listed in Table 1. Analyses of the computer profiles indicated that chromosomes 1, 12p, and the X chromosome were primarily over-represented, whereas chromosomes 4, 9, 13, 14, and 18 were primarily under-represented. Chromosomes 3, 10, and 21 were least involved in numerical aberrations (Fig. 1).
The experimental results were then analyzed statistically to assess the significance of the potential gains and losses on
Discussion
Comparative genomic hybridization is a molecular cytogenetic method that detects the losses and gains of chromosomes across the entire genome [20], [21]. Using this method, 12 cell lines and one surgical specimen from testicular tumors were examined for chromosomal losses and gains. All lines were derived from nonseminoma tumors. The one surgical specimen was a teratoma. Statistical analysis of the results indicated that there was a gain of the X chromosome and chromosome arms 1q, 12p, and 20q,
Acknowledgements
This work was supported in part by funding from the Walther Cancer Institute (Indianapolis, IN).
References (30)
- et al.
Overrepresentation of the short arm of chromosome 12 in seminoma and nonseminoma groups of testicular germ cell tumors
Cancer Genet Cytogenet
(2002) - et al.
Use of fluorescence in situ hybridization and comparative genomic hybridization in the cytogenetic analysis of testicular germ cell tumors and uveal melanomas
Cancer Genet Cytogenet
(1997) - et al.
Fluorescence in situ hybridization-based approaches for detection of 12p overrepresentation, in particular i(12p), in cell lines of human testicular germ cell tumors of adults
Cancer Genet Cytogenet
(1996) - et al.
Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analysis
Cancer Genet Cytogenet
(2001) - et al.
Cytogenetics of the progression of adult testicular germ cell tumors
Cancer Genet Cytogenet
(1997) - et al.
Amplification of chromosome subregion 12p11.2-p12.1 in a metastasis of an i(12p)-negative seminoma: relationship to tumor progression?
Cancer Genet Cytogenet
(1994) - et al.
Reviews of chromosome studies in urological tumors. III. Cytogenetics and genes in testicular tumors
J Urology
(1996) - et al.
Comparative genomic hybridization of germ cell tumors of the adult testis: confirmation of karyotypic findings and identification of a 12p-amplicon
Cancer Genet Cytogenet
(1996) - et al.
Compilation of published comparative genomic hybridization studies
Cancer Genet Cytogenet
(2002) - et al.
Pathogenesis of adult testicular germ cell tumors: a cytogenetic model
Cancer Genet Cytogenet
(1990)
Cytogenetic analysis of 124 prospectively ascertained male germ cell tumors
Cancer Res
Identification of multiple chromosome 12 abnormalities in human testicular germ cell tumors by two-color fluorescence in situ hybridization (FISH)
Genes Chromosomes Cancer
Molecular cytogenetic analysis of adult testicular germ cell tumours and identification of regions of consensus copy number change
Br J Cancer
Detection of chromosomal DNA gains and losses in testicular germ cell tumors by comparative genomic hybridization
Genes Chromosomes Cancer
Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a nonseminoma
J Pathol
Cited by (16)
Biology of Germ Cell Tumors
2011, Hematology/Oncology Clinics of North AmericaCitation Excerpt :The remaining i(12p)-negative TGCTs have also been shown to contain gain of chromosome 12p sequences, either as tandem duplications located in situ or transposed elsewhere in the genome.21,22 In addition to gain of the complete 12p, amplification of subregions of chromosome 12p (12p11.2-p12.1 and 12p12∼13) has also been reported in some TGCTs.23–29 The 12p11.2-p12.1 amplification was first identified in a patient with metastatic seminoma and occurs almost exclusively in i(12p)-negative TGCTs.23–27
Molecular Pathology of the Genitourinary Tract: Molecular Pathology of Kidney and Testes
2009, Surgical Pathology ClinicsCitation Excerpt :With classical cytogenetic karyotyping and comparative genomic hybridization, numerous and more specific chromosomal imbalances and structural abnormalities have been identified.188,191,203–209 However, few have been consistently demonstrated except for isochromosome 12p.210,211 Isochromosome 12p is the most consistent and specific chromosomal change found in testicular GCTs with approximately 80% of adult NSGCTs and 50% or greater of pure seminomas possessing at least one i(12p) (Box 9; Figs. 4 and 5).211–214
Fluorescence in situ hybridization of 12p in germ cell tumors using a bacterial artificial chromosome clone 12p probe on paraffin-embedded tissue: clinical test validation
2008, Cancer Genetics and CytogeneticsCitation Excerpt :Despite the varied morphology common chromosomal aberrations of 12p sequences and response to cisplatinum-based therapy support a common origin for these tumors [3]. Numerous chromosomal anomalies have been reported for germ cell tumors, but isochromosome 12p [i(12p)] is the only consistently reported genetic finding [4,5]. Approximately 80–90% of germ cell tumors have i(12p) [6], and cases negative for i(12p) have 12p overrepresentation detected by fluorescence in situ hybridization [4,7].
Retinoic acid represses a cassette of candidate pluripotency chromosome 12p genes during induced loss of human embryonal carcinoma tumorigenicity
2005, Biochimica et Biophysica Acta - Gene Structure and ExpressionFrequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas
2020, Endocrine-Related CancerFluorescent in Situ Hybridization Analysis for 12p Alterations in Sarcomatoid Yolk Sac Tumors
2019, American Journal of Surgical Pathology