Restricted 12p Amplification and RAS Mutation in Human Germ Cell Tumors of the Adult Testis

Abstract

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional highlevel amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is ϳ8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients. (Am J Pathol

12p-Amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH

“…The transcription factor SOX5 is located at the center of the amplicon, a region consistently showing a less-amplified level than the flanking sequences in some of the TGCTs investigated. This result is in line with our previous observation that this gene is not overexpressed in TGCTs with a 12p amplification and is, therefore, not a prime candidate (Roelofs et al, 2000;Rodriguez et al, 2003). The only annotated gene mapped to the telomeric border of the amplicon is EKI1 (Zafarana et al, 2002).…”

“…Data regarding the physical description of these amplicons as well as the SRO are summarized in Table 2. The borders of the amplicon, as previously determined by FISH on tissue sections (Mostert et al, 1998;Roelofs et al, 2000;Zafarana et al, 2002), were found to be fully in line with the data obtained by array CGH. Since 20 out of the 68 BACs map within the SRO, the array data also allowed a detailed analysis of the amplicon structure for each individual tumor.…”

“…The short arm of this chromosome is about 40 000 Kb in length, but the SRO (Roelofs et al, 2000;Zafarana et al, 2002) is only about 3000 Kb and is located between 24 500 and 27 000 Kb from the telomere. Therefore, the array contains a more dense distribution of clones from this area.…”

“…Identification of these genes will also have direct implications on the diagnosis/subclassification of seminoma-and nonseminoma-like tumors found at other anatomical localizations, including the anterior mediastinum and midline of the brain, as well as the ovary, because these GCTs also show a consistent gain of 12p for review). About 10% of the testicular seminomas contain a restricted 12p amplification (Suijkerbuijk et al, 1994;Korn et al, 1996;Mostert et al, 1996Mostert et al, , 1998Roelofs et al, 2000;Zafarana et al, 2002). These tumors lack the most frequent cytogenetic anomaly leading to gain of 12p in (T)GCTs, the isochromosome 12p (i(12p)) (Sandberg et al, 1996).…”

“…We subsequently showed that the breakpoints of the amplicon cluster significantly (Roelofs et al, 2000;Zafarana et al, 2002), which could indicate that two relevant genes are present within the SRO, one at the centromeric and the other at the telomeric side. Here, we extend these studies by performing a high-resolution copy number analysis on a series of 17 testicular seminomas and one nonseminoma with known highlevel 12p amplifications.…”