Chromosome changes in germ cell tumors of the testis

Chromosome analysis was performed on short-term cultures established from samples of six tumors of the testis. Histologically, four tumors were embryonal cell carcinomas (three primary, one metastatic) and two of mixed histology with predominance of teratoma. The modal chromosome number was hypotriploid in four tumors, triploid in one, and hypertriploid in another. All tumors contained structurally abnormal chromosomes, ranging in number from 1 to 10 in different cases. A small metacentric marker chromosome, identified as an isochromosome of the short arm of chromosome #12 [i(12p)], was present in all tumors analyzed. Unlike other marker chromosomes, this one was invariably present in at least two copies per metaphase in all cases; all other chromosome markers were present in single copy in all tumors. Together with the previous reports on the presence of i(12p) in seminoma and teratoma of the testis, our findings suggest that this karyotypic abnormality is characteristic for all histologic varieties of germ cell tumors of the testis.     

Chromosomes 1 and 12 abnormalities in pediatric germ cell tumors by interphase fluorescence in situ hybridization

Chromosome studies of pediatric germ cell tumors (GCTs) show differences in abnormalities dependent on age, sex, tumor location, and histology. Previous studies suggest that loss of 1p is associated with a malignant phenotype, while amplification of 12p, a common finding in adult testicular GCTs, is uncommon in pediatric GCTs. Fifty-three pediatric GCTs were analyzed for 1p36 loss and 12p amplification by G-banding and dual-color interphase FISH with probes for the centromere and short arm of chromosomes 1 or 12. Twelve tumors with loss of 1p36 were identified. No deletion was detected in tumors with nonmalignant histology, such that there was a significant association of 1p loss with malignancy in these tumors (P = 0.00115). Five of 18 tumors from male patients had amplification of 12p, consistent with G-band results. Combined analysis of our data with those in the literature revealed a significant correlation of 12p amplification with patient age (P = 0.000196). Amplification of 12p was only seen in one of 35 tumors from female patients. Five female GCTs had numerical abnormalities of chromosome 12, and two tumors showed complete lack of 12p. This spectrum of abnormalities differs from what is seen in the male tumors, providing further evidence for different etiologies of GCTs between the sexes.     

Alpha-fetoprotein in germ cell tumors. An immunohistochemical study

Total 40 cases of testicular and ovarian germ cell tumors and one case of extragonadal germ cell tumor were studied for the presence of alphafetoprotein (AFP) by indirect immunoperoxidase technique. All seminomas (7 cases) and teratomas (13 cases) were negative for AFP; while 85% of the pure embryonal carcinomas, (E.C.) all pure yolk sac tumors (Y.S.T.) (7 cases) and all embryonal carcinoma and yolk sac components in mixed tumors were AFP positive. Immunostaining of tumor marker appeared to help only in differentiating seminomatous and nonseminomatous tumors and hence does not provide any additional information for classification of these tumors.     

Genomic profiling of pediatric ALK‐positive anaplastic large cell lymphoma: A Children's Cancer and Leukaemia Group Study

Anaplastic lymphoma kinase (ALK)‐positive anaplastic large cell lymphoma (ALCL) is a T‐cell malignancy in which ALK expression is a consequence of the t(2;5) or a variant translocation involving Chromosome 2. For the most part, this disease presents in the pediatric population and most, but not all, patients are successfully treated. Although the t(2;5) product nucleophosmin‐ALK has been extensively studied for its transforming properties, very little is known regarding cooperative genetic mutations that may contribute to lymphomagenesis and may predict survival outcome, specifically in a purely pediatric population. We set out to determine the frequency and positions of genomic imbalances in this relatively rare disease. We collected biopsy material from 15 UK‐resident children with ALK‐expressing ALCL. We performed array comparative genomic hybridization at a resolution of 1 MB using DNA isolated from tumor tissue. Some of the more common genomic gains were confirmed by quantitative PCR. Regions of genomic gain were far more common than losses and were most often detected on chromosomes 1–4, 5–12, 14, and 17, with Chromosome 11 being the most frequent site of genomic imbalances. Patients with 14 or fewer imbalances had a lower overall 3‐year survival (87.5–40%, P = 0.14) as did patients with gains in the regions of DDB1 or BIRC5. A range of genomic imbalances exist in ALK‐expressing ALCL of a pediatric origin, with a greater number associated with poorer overall survival. © 2009 Wiley‐Liss, Inc.     

Cytogenetic and molecular analysis of human male germ cell tumors: chromosome 12 abnormalities and gene amplification

We report karyotypic analysis of 24 male germ cell tumors (GCTs) with clonally abnormal karyotypes biopsied from testicular and extragonadal lesions from 20 patients belonging to the histologic categories seminoma, teratoma, embryonal carcinoma, choriocarcinoma, and endodermal sinus tumor. Chromosomes 1, 7, 9, 12, 17, 21, 22, and the X chromosome were nonrandomly gained in these tumors. Nonrandom structural changes affected most frequently chromosomes 1 and 12, the latter as i(12p) and/or del(12)(q13----q22). The i(12p) was seen in 90% of tumors which included all histologic subtypes and gonadal as well as extragonadal presentation. Our present results, along with those from published data on fresh GCT biopsies, establish that i(12p) is a highly nonrandom chromosome marker of all histologic as well as anatomic presentations of GCTs. in contrast, we found del(12)(q13----q22) exclusively in nonseminomatous GCTs (NSGCTs) and mixed GCTs (MGCTs) occurring in 44% of such lesions. Because successful cytogenetic analysis of fresh tumor specimens is not always possible, we developed a method based on DNA analysis to detect i(12p) as increased copy number of 12p. In addition to the changes affecting chromosome 12 identified above, we have detected, for the first time, cytological evidence of gene amplification in the form of homogeneously staining regions (HSRs) and double minute chromosomes (dmins) in treated as well as untreated primary extragonadal and metastatic GCTs and confirmed the presence of amplified DNA in one of these tumors at the molecular level by the in-gel renaturation method. Hybridization of DNA from cultured cells from an HSR-bearing tumor with a panel of probes for genes known to be amplified or otherwise perturbed in diverse tumor systems did not identify the amplified gene, suggesting amplification of a novel gene or genes. This study comprises the largest series of GCT cytogenetics attempted so far. Notably, it includes data on a series of primary mediastinal tumors, a group which previously has not been studied in any detail.     

Loss of Fhit expression in testicular germ cell tumors and intratubular germ cell neoplasia.

The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors. We studied the expression of Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells. All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion. The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.     

Associations between variants in KITLG, SPRY4, BAK1, and DMRT1 and pediatric germ cell tumors

Recent genome wide association studies have identified susceptibility loci for adult testicular germ cell tumors (GCT) near KITLG, SPRY4, BAK1, and DMRT1. We evaluated variants in these four genes to determine whether these are also susceptibility loci for pediatric GCTs. DNA was isolated from 52 pediatric GCTs (ages 0-21 years) obtained from the Cooperative Human Tissue Network. Control DNA was isolated from de-identified dried blood spots from 141 white newborns. Genotyping was conducted using TaqMan assays (rs4474514) or by PCR and sequencing (rs4324715, rs210138, and rs755383). Associations between variants and GCT were evaluated using logistic regression with adjustment for sex. We also evaluated whether the associations differed by age at GCT diagnosis (0-9 years, 10-21 years), sex, and tumor location (gonadal, non-gonadal). We observed a significant association for rs210138 (BAK1) and pediatric GCT overall (odds ratio (OR) = 1.80, 95% confidence interval (CI) 1.10-2.95, P = 0.02) with non-significant associations similar in magnitude in both the pediatric (P = 0.09) and adolescent (P = 0.06) age groups. The KITLG (rs4474514) and SPRY4 (rs4324715) variants were significantly associated with GCT only in the adolescent age group (rs4474514: OR = 2.28, 95% CI 1.09-4.79, P = 0.03 and rs4324715: OR = 2.40, 95% CI 1.19-4.83, P = 0.01). Associations were mostly similar when stratified by sex. This is the first study to suggest that these loci may also be important in susceptibility to GCTs in the adolescent (KITLG, SPRY4, and BAK1) and pediatric (BAK1) age groups.     

Chromosomes, genes, and development of testicular germ cell tumors

A literature review found 265 articles on testicular germ cell tumors (TGCTs) detailing the copy number of chromosomal regions and expression of 245 genes. An initial precursor stage, intratubular germ cell neoplasia (IGCN), is characterized by triploidization and an upregulation of KIT, ALPP, CCDN2, and ZNF354A, and a downregulation of CDKN2D. TGCT regularly have a series of chromosomal aberrations: a decrease in copy number at 4q21 approximately qter and 5q14 approximately qter; an increase at 7p21 approximately pter, 7q21 approximately q33, and 8q12 approximately q23 (especially high increase in seminoma); a decrease at 11p11 approximately p15 and 11q14 approximately q24; an increase at 12p11 approximately pter; a decrease at 13q14 approximately q31; an increase of 17q11 approximately q21 (only for nonseminoma); a decrease of 18q12 approximately qter; and an increase at 21q21 approximately qter, 22q11 approximately qter (only for seminoma), and Xq. Macroscopically overt TGCT is associated with a characteristic series of abnormalities in the retinoblastoma pathway including upregulation of cyclin D2 and p27 and downregulation of RB1 and the cyclin-dependent kinase inhibitors p16, p18, p19, and p21. TGCT thus has a synergistic pattern in gene expressions of the retinoblastoma pathway that is rare in other malignancies.     

Frequency of intratubular germ cell neoplasia with invasive testicular germ cell tumors. Histologic and immunocytochemical features

Intratubular germ cell neoplasia (ITGCN) has been regarded as the preinvasive stage of testicular germ cell tumors. We evaluated its frequency in 53 testicular germ cell tumors and determined whether immunohistochemical stains for alpha-fetoprotein, human chorionic gonadotropin, carcinoembryonic antigen, and ferritin demonstrated an advantage in its detection in comparison with hematoxylin-eosin and periodic acid-Schiff (PAS) stains. Residual seminiferous tubules were found at the periphery of the invasive neoplastic foci in 47 cases. Intratubular germ cell neoplasia was detected in several or multiple seminiferous tubules in 46 cases (98%). Exquisite localization of PAS-positive intracellular granules was present in all but one case of ITGCN. Focal immunocytochemical positivity for human chorionic gonadotropin, carcinoembryonic antigen, and ferritin was noted in 2.3% of cases. We conclude that ITGCN is present in most invasive germ cell tumors and the PAS stain is very reliable in its demonstration. Antigenic expression of the proteins that we examined is extremely limited in these primordial germ cells.     

SNRPN methylation patterns in germ cell tumors as a reflection of primordial germ cell development.

Studies examining altered imprinted gene expression in cancer compare the observed expression pattern to the normal expression pattern for a given tissue of origin, usually the somatic expression pattern for the imprinted gene. Germ cell tumors (GCTs), however, require a developmental stage-dependent comparison. To explore using methylation as an indicator of germ cell development, we determined the pattern of methylation at the 5' untranslated region of SNRPN in 89 GCTs from both children and adults. Fifty-one of 84 tumors (60.7%) (12/30 (40%) of cultured pediatric GCTs, 23/36 (63.9%) of frozen adult GCTs, and 16/23 (69.5%) of frozen pediatric GCTs, with five samples having results from both cultured and uncultured material) demonstrated a nonsomatic methylation pattern after dual digestion with XbaI, NotI, and Southern blot analysis. In contrast, only 2 of 18 (11%) control samples (16 non-GCTs and 2 normal ovaries) exhibited a nonsomatic pattern. In both cases, the result was shown to be due to copy number differences between maternal and paternal homologs, unlike the GCTs in which there was no evidence of an uneven homolog number. A comparison of the data for only the gonadal GCTs and the control data showed a highly significant difference in the proportion of tumors with methylation alterations at this locus (P = 0.0000539). Since there is no published evidence of the involvement of SNRPN methylation changes in the development of malignancy, the data suggest that the methylation pattern of SNRPN in GCTs reflects that of the primordial germ cell giving rise to the tumor.     

X-chromatin, sex chromosomes, and ploidy in 37 germ cell tumors of the testis.

X-chromatin was present in interphase cells from nine of 14 teratomas and all of three combined tumors, but only one of 20 seminomas (which tended to have higher chromosome numbers). Eight of the 37 tumors were karyotyped; seven, only one of which (a teratoma) was X-chromatin-positive, had two X chromosomes while one, the X-chromatin-positive seminoma, had three. A possible relationship between the presence of inactive, X-chromatin-forming, X chromosomes and the number of autosomes is suggested by the data on the eight karyotyped tumors; the ratio of the number of Xs to the number of autosomes was higher for the two X-chromatin-positive tumors than for the remainder. All eight had at least one Y chromosome, and eight further tumors had one to three Y-bodies in their interphase cells. It is uncertain whether retention of the Y is a characteristic of male germ cell tumors, as tumors lacking a Y have been described by other workers. Two characteristics of these tumors, however, are high ploidy (at least 55 chromosomes), perhaps signifying an origin from a triploid or tetraploid cell, and chromosome 12 aberrations, usually resulting in an i(12p).     

EPCAM–A novel molecular target for the treatment of pediatric and adult germ cell tumors

Germ cell tumors (GCTs) are thought to develop from totipotent primordial germ cells. Although the epithelial cell adhesion molecule (EPCAM) is expressed on embryonic stem cells as well as different tumor cells, it has not yet been extensively studied in GCTs. We analyzed EPCAM expression by quantitative RT‐PCR in 48 fresh‐frozen GCT specimens of different histology (10 mature teratoma, MT; 6 immature teratoma, IT; 7 dysgerminoma; 6 mixed malignant GCTs; 19 yolk sac tumor, YST) and in the GCT cell lines NCCIT, TE76.T, JAR and 2102Ep, and correlated its expression with AFP and hCG protein levels, histologic differentiation, and clinical follow‐up data. EPCAM protein was visualized by immunohistochemistry of selected corresponding paraffin embedded tumor tissues. EPCAM was expressed in malignant but not in benign GCTs irrespective of age, sex, site and clinical stage of tumor (P = 0.001). In primary teratomas, EPCAM expression increased with their grade of immaturity (mean 2^?ΔCt values: MT 0.23, IT 1.61, P = 0.007) and significantly correlated with serum AFP (P = 0.03) and hCG (P = 0.03) levels in malignant GCTs. Particularly high EPCAM levels were found in nonseminomatous GCTs such as YSTs (8.49) and choriocarcinoma (13.54). Immunohistochemical analysis verified gene expression data showing a distinct EPCAM staining in YST. Similarly in vitro, highest EPCAM expression was measured in GCT cell lines comprising yolk sac (2102Ep: 5.59) or choriocarcinoma (JAR: 10.65) components. This first comprehensive analysis of EPCAM in GCTs revealed high EPCAM expression in YSTs and choriocarcinomas. Thus, these nonseminomatous GCTs may be interesting targets for EPCAM immunotherapy, which has to be evaluated in further studies. © 2012 Wiley Periodicals, Inc.     

Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group

Ewing's sarcoma family tumors (ESFT) are characterized by the presence of EWSR1-ETS fusion genes. Secondary chromosome changes are frequently described, although their clinical significance is not clear. In this study, we have collected and reviewed abnormal karyotypes from 88 patients with primary ESFT and a rearrangement of 22q12. Secondary changes were identified in 80% (70/88) of tumors at diagnosis. Multivariate analysis showed a worse overall and relapse free survival (RFS) for those with a complex karyotype (overall survival, P = 0.005; RFS, P = 0.04), independent of metastatic disease. Univariate survival analysis showed that a chromosome number above 50 or a complex karyotype was associated with a worse overall survival (>50 chromosomes, P = 0.05; complex karyotype, P = 0.04). There was no association between type of cytogenetic abnormality and the presence of metastatic disease at diagnosis. Univariate and multivariate survival analysis of a small subgroup with trisomy 20 indicated that trisomy 20 was associated with a worse overall and RFS. There was no difference in outcome associated with other recurrent trisomies (2, 5, 7, 8, or 12) or the common recurrent secondary structural rearrangements (deletions of 1p36, 9p12, 17p13, and 16q, and gain of 1q), although numbers were small. These data demonstrate the continued value of cytogenetics as a genome-wide screen in ESFT and illustrates the potential importance of secondary chromosome changes for stratification of patients for risk. Specifically, karyotype complexity appears to be a powerful predictor of prognosis, and the presence of trisomy 20 may be a marker of a more aggressive subset of this group.     

Variants in BAK1, SPRY4, and GAB2 are associated with pediatric germ cell tumors: A report from the children's oncology group

Germ cell tumors (GCT) are a rare form of childhood cancer that originate from the primordial germ cell. Recent genome‐wide association studies (GWAS) have identified susceptibility alleles for adult testicular GCT (TGCT). We test whether these SNPs are associated with GCT in pediatric and adolescent populations. This case‐parent triad study includes individuals with GCT diagnosed between ages 0 and 19. We evaluated 26 SNPs from GWAS of adult TGCT and estimated main effects for pediatric GCT within complete trios (N = 366) using the transmission disequilibrium test. We used Estimation of Maternal, Imprinting and interaction effects using Multinomial modelling to evaluate maternal effects in non‐Hispanic white trios and dyads (N = 244). We accounted for multiple comparisons using a Bonferroni correction. A variant in SPRY4 (rs4624820) was associated with reduced risk of GCT (OR [95% CI]: 0.70 [0.57, 0.86]). A variant in BAK1 (rs210138) was positively associated with GCT (OR [95% CI]: 1.70 [1.32, 2.18]), with a strong estimated effect for testis tumors (OR [95% CI]: 3.31 [1.89, 5.79]). Finally, a SNP in GAB2 (rs948662) was associated with increased risk for GCT (OR [95% CI]: 1.56 [1.20, 2.03]). Nominal associations (P < 0.05) were noted for eight additional loci. A maternal effect was observed for KITLG SNP rs4474514 (OR [95% CI]: 1.66 [1.21, 2.28]) and a paternal parent‐of‐origin effect was observed for rs7221274 (P = 0.00007), near TEX14, RAD51C, and PPM1E. We observed associations between SNPs in SPRY4, BAK1, and GAB2 and GCTs. This analysis suggests there may be common genetic risk factors for GCT in all age groups.     

Chromosome abnormalities, sister chromatid exchanges, and cell cycle analysis in phytohemagglutinin-stimulated adult T cell leukemia lymphocytes

Chromosome abnormalities, sister chromatid exchanges (SCE), and cell cycle kinetics were studied in phytohemagglutinin-stimulated lymphocytes from 8 adult T cell leukemia (ATL) patients. In all these cases, chromosome abnormalities were observed in 5-day PHA-stimulated cultures. Four cases had characteristic marker chromosomes; two were due to a balanced translocation, t(9;21), and two to a simple deletion, 5p-. The other four cases, however, had rather complicated chromosome abnormalities, e.g., 1q+, 2q+, 5q+, 6q-, 14q+, 10p-. When chromosome abnormalities were analyzed in previously reported cases, the abnormalities were mostly distributed among chromosomes #1, #2, #5, #6, #14, and #21. These findings suggest that the abnormalities involving #1, #2, #5, #6, #14, and #21 are intimately related to ATL. The SCE frequency was in the normal range in ATL cells. Cell cycle analysis revealed that the duration of two cell cycles in cells labeled with bromodeoxyuridine (BrdU) required approximately 80 hr in ATL cells, whereas the time of two cell cycles in normal cells is 40 hr. These findings indicate that the ATL cell cycle time is about 40 hr, about double that of normal cells (20 hr), in PHA-stimulated cultures. ATL has been known to be a mature T cell leukemia and to respond poorly to chemotherapy. The latter may be due to the elongated cell cycle or to the mature characteristics of the leukemic cells. The association of ATL with cutaneous T cell lymphoma is also discussed.     

Immunohistochemical analysis of small cell tumors of the thyroid gland: an Eastern Cooperative Oncology Group study.

The majority of small cell anaplastic tumors of the thyroid gland are generally believed to be non-Hodgkin's lymphomas, including most of those formerly classified as small cell carcinomas. Using a panel of antibodies capable of detecting epithelial, neuroendocrine, and B and T cells in paraffin-embedded tissue sections, we studied 68 thyroid neoplasms in which the original diagnosis was small cell carcinoma or lymphoma. Sixty-three of the tumors were identified as lymphomas of B-cell origin on the basis of L26 reactivity used in conjunction with light chain restriction and MB2 immunostaining. Two additional tumors were classified as lymphomas of indeterminate phenotype. Immunophenotyping indicated an epithelial origin in the remaining three tumors. No cases of medullary carcinoma were detected by immunostaining. Histologic review revealed a predominance of large cell and immunoblastic lymphomas, with low-grade lymphomas of mucosa-associated lymphoid tissue histology accounting for only five cases. Our findings indicate that the majority of small cell anaplastic tumors of the thyroid are B-cell lymphomas. Although primary small cell carcinoma of the thyroid may rarely occur, this diagnosis should not be made without immunohistologic confirmation.     

Ploidy of primary germ cell tumors of the testis. Pathogenetic and clinical relevance

The ploidy of testicular germ cell tumors (GCT), a heterogeneous group of neoplasms, was studied by DNA flow cytometry. The DNA index for infantile yolk sac tumor (N = 10), seminomas (N = 20), and nonseminomas (N = 36), was: 1.91, 1.66, and 1.43, respectively. These values differed significantly one from another (p less than 0.01). The seminoma and nonseminoma components of combined tumors (N = 16) had a significantly different median DNA index of 1.61 and 1.40, respectively. Three of the 10 infantile yolk sac tumors, but only one of the 72 testicular GCT of adults were diploid. The consistent aneuploidy of testicular GCTs of adults might be helpful in the differential diagnosis of primary nongerm cell tumors of the testis, and in differentiating between metastases of testicular GCTs and primary extragonadal malignant GCTs. These data fit into a model of pathogenesis of testicular GCTs of adults in which all tumors, with the possible exception of spermatocytic seminoma, pass through a seminoma stage. Tumor evolution seems to result from net loss of chromosomes from a (near)tetraploid carcinoma in situ cell. The pathogenesis of infantile yolk sac tumor might be different from that of testicular GCTs of adults.