Characterization of gains, losses, and regional amplification in testicular germ cell tumor cell lines by comparative genomic hybridization

https://doi.org/10.1016/S0165-4608(03)00211-5Get rights and content

Abstract

We have performed comparative genomic hybridization on 12 testicular germ cell tumor (TGCT) cell lines and one paraffin-embedded surgical specimen to identify and characterize genome-wide gains and losses of chromosomes in these specimens. All specimens demonstrated overrepresentation of 12p. Other significant chromosomal gains, apart from 12p, included the X chromosome and chromosome arms 1q and 20q. Chromosomal losses were observed for chromosomes 4 and 18 and chromosome arms 2q, 9q, and 13q. Genomic differences were observed between an embryonal carcinoma component of a mixed tumor, 833K, and its cisplastin-resistant derivative line, 64CP, including losses of 6q23∼qter and 9p22∼q21. Five lines also demonstrated gain of 12p and additional 12p12∼p13 material. Similarly, two lines demonstrated gain of 12p and additional 12p11.2∼p12 material. The data supports the consistent gain of 12p in adult TGCT cell lines and additional regional amplification of 12p in some lines. This regional amplification has been observed in both primary tumor specimens and TGCT cell lines and may support a hypothesis that at least two different regions of 12p, one proximal and one distal, harbor genes important for the pathogenesis of testicular germ cell neoplasia.

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).

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