Loss of the Y-chromosome in the primary metastasis of a male sex cord stromal tumor: pathogenetic implications.

The first published chromosomal pattern of the retroperitoneal lymph node metastasis of a malignant gonadal stroma cell tumor of the adult testis is presented. Karyotyping showed structural chromosomal abnormalities and loss of the Y-chromosome. This loss was confirmed in primary tumor and metastasis using fluorescence in situ hybridization (FISH). The characteristic chromosomal abnormality of adult testicular germ cell tumors, an i(12p), was not present. The results are compared with other data of testicular and ovarian sex cord stromal tumors. From the comparison of the male tumors, it is concluded that loss of the Y-chromosome might have a pathogenetic significance.

Cytogenetic analysis of a mature teratoma and a yolk sac tumor component of a late relapse of a disseminated testicular nonseminoma.

We report on the cytogenetics of a primary testicular nonseminoma, a residual mature teratoma after remission-induction chemotherapy, and a late relapse after 9 years of follow-up, in one patient. The late relapse was composed of a mature teratoma and a yolk sac tumor component. Cytogenetic comparison of the different tumors shows that progression of primary testicular nonseminoma to residual mature teratoma and to a late-relapse lesion is accompanied by net loss of chromosomes. In addition, our findings may suggest that transformation to viable cancer in a late-relapse lesion is accompanied by further chromosomal losses.

Chromosomal constitution of human spermatocytic seminomas: comparative genomic hybridization supported by conventional and interphase cytogenetics.

No data on the chromosomal constitution of spermatocytic seminomas are available thus far because of their rarity. Ploidy analysis performed on paraffin-embedded cases showed varying results from (near-) diploid to aneuploid. We applied comparative genomic hybridization on four snap-frozen primary spermatocytic seminomas of three different patients. Conventional cytogenetic analysis was successful in one, and "interphase cytogenetics" with centromeric region-specific probes was applied to another. The results from comparative genomic hybridization showed almost exclusively numerical chromosomal aberrations, in agreement with the data from karyotyping. Despite the limited number of cases studied, a nonrandom pattern of chromosome imbalances was detected: chromosome 9 was gained in all spermatocytic seminomas. This suggests that that this aberration plays a role in the development of this cancer. Interphase cytogenetics shows that the copy number of most chromosomes ranges from two to four, with an average of near trisomic. This constitutes the first report on the chromosomal constitution of spermatocytic seminomas.

Cytogenetics of a malignant ovarian germ-cell tumor.

Cytogenetic investigation of a malignant ovarian tumor diagnosed as a mixed germ-cell tumor, composed of extensive choriocarcinoma and foci of yolk-sac tumor, revealed a highly abnormal chromosomal pattern. We found a chromosome number in the hypertriploid/hypotetraploid range, and several clonal structural abnormalities, including 2 copies of an isochromosome 12p. We showed that the chromosomal pattern of this ovarian tumor is very similar to that of testicular germ-cell tumors. This finding, together with reported cytogenetic data of malignant ovarian germ-cell tumors, supports the hypothesis that ovarian and testicular germ-cell tumors are strongly related entities that may have a similar origin and pathogenetic pathway.

Identification of the critical region of 12p over-representation in testicular germ cell tumors of adolescents and adults.

Cytogenetically, testicular germ cell tumors of adolescents and adults (TGCTs) are characterized by gain of 12p-sequences, most often through isochromosome formation (i(12p)). Fluorescence in situ hybridization (FISH) has shown that i(12p))-negative TGCTs also cryptically contain extra 12p-sequences. The consistency of 12p-over-representation in all histological subtypes of TGCTs, including their preinvasive stage, suggests that gain of one or more genes on 12p is crucial in the development of this cancer. So far, studies aimed at the identification of the relevant gene(s) were based on the 'candidate-gene approach'. No convincing evidence in favor of or against a particular gene has been reported. We combined conventional karyotyping, comparative genomic hybridization, and FISH to identify TGCTs with amplifications of restricted regions of 12p. Out of 49 primary TGCTs (23 without i(12p), 13 with and 13 unknown), eight tumors (six without i(12p) and two unknown) showed amplifications corresponding to 12p11.1-p12.1. Using bicolour-FISH, physical mapping, and semi-quantitative polymerase chain reactions, the size of the shortest region of overlap of amplification (SROA) was estimated to be between 1750-3000 kb. In addition, we mapped a number of genes in and around this region. While fourteen known genes could be excluded as candidates based on their location outside this region, we demonstrate that KRAS2, JAW1 and SOX5 genes are localized within the SROA. While KRAS2 and JAW1 map to the proximal border of the SROA, SOX5 maps centrally in the SROA. KRAS2 and JAW1 are expressed in all TGCTs, whereas one 12p amplicon-positive TGCT lacks expression of SOX5. The critical region of 12p over-represented in TGCTs is less than 8% of the total length of the short arm of chromosome 12. It will be helpful in the identification of the gene(s) involved in TGCT-development.

Comparison of the chromosomal pattern of primary testicular nonseminomas and residual mature teratomas after chemotherapy.

About 70 to 75% of patients with nonseminomatous testicular germ cell tumors (NSs) present with metastases. When these metastases are treated with chemotherapy, often residual mature teratoma (RMT) is left. RMT is composed of fully differentiated somatic tissue. Untreated metastases of NSs rarely consist exclusively of mature somatic tissue. Apparently, after chemotherapy treatment there is a shift towards higher degrees of differentiation. Investigating tumor progression and the mechanism(s) involved in therapy-related differentiation, we compared the cytogenetically abnormal karyotypes of a series of 70 NSs with those of 31 RMTs. In NSs and RMTs, the modal total chromosome number does not differ and is in the triploid range. Both the frequency and the average copy number of i(12p) are the same, and the pattern of chromosomal over- and underrepresentation and distribution of breakpoints do not differ significantly in these series. So, we found the chromosomal pattern of RMTs as abnormal as those of primary NSs. Based on cytogenetics, we found no indication that specific chromosomal alterations parallel metastasis and therapy-related differentiation of the metastases. The cytogenetic data suggest that both induction of differentiation of (selected) cells or selection of cells with capacity to differentiate are possible mechanisms for the therapy-related differentiation of RMTs.

Cytogenetic support for early malignant change in a diffuse neurofibroma not associated with neurofibromatosis.

A 62-year-old woman presented with a solitary diffuse neurofibroma; a second recurrence showed features indicative of malignancy, but insufficient for a certain histologic diagnosis. Cytogenetic analysis revealed abnormalities previously described in malignant peripheral nerve sheath tumors and not in their benign counterparts, thus supporting the histologic suspicion of emerging malignancy.

Cytogenetics of primary testicular nonseminoma, residual mature teratoma, and growing teratoma lesion in individual patients.

Residual mature teratoma (RMT) is often left behind when metastases of primary nonseminomatous germ cell tumors (NSs) are treated with chemotherapy. RMT is composed of fully differentiated somatic tissue. A growing teratoma (GTE) lesion may occur after (incomplete) resection of RMT. To shed light on tumor progression or the mechanism(s) of therapy related differentiation we investigated the chromosomal pattern of the primary NSs and RMTs in twelve patients, of the primary NS, RMT, and GTE lesion in one patient, and of the RMT and GTE lesion in two patients. Although several chromosomal differences are observed between the RMT and NSs and between the GTE and RMTs in the same patient, we obtained no evidence that specific chromosomal alteration(s) play a role in metastasis or differentiation.

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.

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.

Overrepresentation of the short arm of chromosome 12 is frequently detected in human testicular germ cell tumors of adolescents and adults (TGCT). This overrepresentation mostly results from the formation of an isochromosome: i(12p). Whether the overrepresentation consistently involves the complete 12p arm including the centromere is still unclear. We studied five TGCT-derived cell lines (NT2, 2102Ep, H12.1, NCCIT, and S2), combining conventional chromosome banding, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) to investigate the suitability of each of these techniques to detect aberrations involving chromosome 12. Karyotyping showed one or more i(12p)s in NT2, 2102Ep, H12.1, and S2. However, FISH with a centromere-specific probe (p alpha 12H8), a 12p "paint" and a 12p11.2--p12.1 region-specific probe yeast artificial chromosome (YAC)#5 and CGH could not confirm the presence of an i(12p) in S2. Additional randomly distributed 12p sequences were detected by FISH in H12.1, NCCIT, and S2. In most of these cases, (a part of) the centromere was included. All overrepresented 12p regions, except for those in S2, showed hybridization with YAC#5. CGH showed increased copy numbers of the complete 12p arm in the cell lines with one or more i(12p)s but no overrepresentation was noted in the cell lines without i(12p). In metaphase spreads, the centromeric block of the i(12p)s differed in size as compared with those of normal chromosomes 12. This was rarely noted in interphase nuclei. A decrease in size of the centromeric block in 2102Ep and H12.1 caused a weak FISH signal, which was difficult to detect, especially in interphase nuclei. The ratio between p alpha 12H8- and YAC#5-derived signals reflected the presence or absence of one or more i(12p)s. Our results indicate that double FISH with a centromere- and a 12p-specific probe can be used to detect 12p overrepresentation [including i(12p)] in TGCT both in metaphase spreads and interphase nuclei. CGH confirmed the relative overrepresentation of 12p sequences as detected by FISH and showed that in these cell lines the complete 12p was involved.

Mixed testicular germ cell tumors: monoclonal or polyclonal.

Testicular germ cell tumors (TGCT) of adolescents and adults are, for clinical and pathological reasons, divided in seminomas (SE) and nonseminomatous germ cell tumors (NS). Whether and to what degree these two entities are pathogenetically related is still controversial and a matter of debate. TGCT may contain both SE and NS components. Cytogenetic studies of the SE and NS components of these mixed TGCT might shed light on the pathogenetic relationship between both components. Separate cytogenetic analysis was performed on both components of three cases of mixed TGCT with both SE and NS components. The karyotypes of both components were compared with each other. In one case, the SE and NS component share eight different structural chromosomal abnormalities, indicating that the SE and NS component are pathogenetically closely related and have a common neoplastic pathway for a considerable length. Both components of the other two cases have, respectively, i(12p) and no structural chromosomal abnormalities in common. Our results, together with data from the literature, indicate that in mixed TGCT with SE and NS components, both components may have either a monoclonal or a polyclonal origin.

Cytogenetic evidence that carcinoma in situ is the precursor lesion for invasive testicular germ cell tumors.

A cytogenetic study of two cases of carcinoma in situ of the testis (CIS) and their adjacent invasive tumors, one a nonseminomatous germ cell tumor (NS) and one a seminoma (SE), revealed similarities in chromosomal pattern between the CIS and the invasive lesion in the same patient. These findings present for the first time cytogenetic evidence that CIS of the testis and its adjacent germ cell tumor are clonally related, which suggests that the CIS is indeed the precursor lesion of the invasive tumor.

No recurrent structural abnormalities apart from i(12p) in primary germ cell tumors of the adult testis.

Malignant transformation may be caused by gene deregulation resulting from specific chromosomal rearrangements, by amplification, by mutations in proto-oncogenes, by loss of tumor suppressor genes, or a combination of these. We investigated the role of numerical and structural chromosomal abnormalities in 102 cytogenetically abnormal cases of primary testicular germ cell tumors of adolescents and adults (TGCT) [32 seminomas (SE) and 70 nonseminomatous germ cell tumors (NS)]. We confirmed that an isochromosome for 12p, i(12p), is the only consistent structural chromosomal abnormality in TGCT, present in about 70% of our cases. Both the frequency and the number of copies of i(12p) are higher in NS than in SE. This may suggest that i(12p) is involved in tumor progression. Besides i(12p), several clonal structural chromosomal abnormalities were found, but none appeared to be specific. SE and NS had chromosome numbers in the triploid range, with significantly higher numbers in SE than in NS (average modal chromosome numbers of 73.4 in SE and 65.0 in NS). Both in SE and NS, some chromosomes were significantly underrepresented (e.g., 11, 13, 18, and Y) and others overrepresented (e.g., 7, 8, 12, 21, and X). In SE, a significantly higher copy number of chromosomes 7, 15, 19, and 22 was found and a significantly lower number of chromosome 17, compared with NS. These chromosomes may play an important role in the differentiation of TGCT.

An important role for chromosome 17, band q25, in the histogenesis of alveolar soft part sarcoma.

A cytogenetic study of two cases of alveolar soft part sarcoma showed near-diploid karyotypes with multiple chromosomal rearrangements. An abnormality of the long arm of chromosome 17, involving band q25, is present in both cases and in 2 of 4 cases in the literature. This recurrent structural abnormality probably plays an important role in the histogenesis of this unusual neoplasm and therefore is important for further molecular investigation.

Lymphomas with testicular localisation show a consistent BCL-2 expression without a translocation (14;18): a molecular and immunohistochemical study.

The presence of the BCL-2 protein was studied in nine non-Hodgkin's lymphomas with testicular localisation. A consistent presence of the BCL-2 protein was found. The chromosomal translocation (14;18) was seen neither by cytogenetic analysis (n = 4) nor by polymerase chain reaction amplification and Southern blotting (n = 9). Therefore, this translocation is not responsible for the presence of the BCL-2 protein in non-Hodgkin's lymphomas with testicular localisation. We suggest that the presence of the BCL-2 protein in these lymphomas is related to the differentiation stage of the B-lymphocytes or may play a role in the pathogenesis of these lymphomas. The consistent finding of the BCL-2 protein in lymphomas with testicular localisation may support the clinical observation that these lymphomas are a separate entity.

Definition of a new entity of malignant extragonadal germ cell tumors.

Two malignant extragonadal germ cell tumors are reported, histologically classified as immature teratomas, having pseudodiploid karyotypes with complex structural rearrangements but lacking isochromosome 12p or other rearrangements involving 12p. The absence of 12p material in structural rearrangements was confirmed by chromosome painting. In the two tumors the following common chromosomal breakpoints were found: 6p21, 6p22, 6q23, and 11q13. Exactly the same chromosomal regions, 6p22::6q23 and 6p21::11q13, were involved in fusions. The two tumors belong to a new entity of extragonadal immature teratomas of adults which may be located in the retroperitoneum and posterior mediastinum and are prone to blood borne metastasis.

Cytogenetic analysis of a leiomyosarcoma of the kidney.

Nonepithelial malignant renal tumors are very rare, comprising approximately 2-3% of all malignant renal tumors. We were able to culture and subsequently karyotype a leiomyosarcoma (LMS) of the kidney that showed the following representative karyotype: 84,XY,add(X)(q25),-Y,add(1)(p11),dic(1;20) (p34;q13.3),-5,-6,dup(6)(q24),i(7)(p10),add(8)(p11),-9,-11,add(13)(p11), -15,-15 , add(17)(q24)x2,-18,-19,-21,-22,-22, +mar1(?hsr),+mar2,+2mar. A clonal add(1)(q11) was also evident. The DNA index was 1.9. Our results as compared with data from the literature suggest that LMS of the kidney has most cytogenetic aberrations reported to be characteristic of LMS, especially those located in the abdomen, and that different genetic mechanisms of initiation and progression appear to play a role in LMS of similar histology but from different anatomic sites.