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

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2-p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500 Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T/R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.     

Aneuploidy of human testicular germ cell tumors is associated with amplification of centrosomes

Testicular germ cell tumors occur in three age groups. Seminomas and nonseminomas of adults, including mature teratomas, and the precursor carcinoma in situ (CIS) are aneuploid. This also holds true for yolk sac tumors of newborn and infants, while the mature teratomas of this age are diploid. In contrast, spermatocytic seminomas occurring in the elderly contain both diploid and polyploid cells. Aneuploidy has been associated with centrosome aberrations, sometimes related to overexpression of STK15. Aneuploidy of non-neoplastic germ cells has been demonstrated in the context of male infertility, a risk factor for the development of seminoma/nonseminoma. We investigated aneuploidy, centrosome aberrations and the role of STK15 in different types of testicular germ cell tumors as well as in normal and disturbed spermatogenesis. The aneuploid seminomas and nonseminomas tumors (including CIS) showed increased numbers of centrosomes, without STK15 amplification or overexpression. Four out of six infantile teratomas had normal centrosomes, the remaining two and an infantile yolk sac tumor showed a heterogeneous pattern of cells with normal or amplified centrosomes. Spermatocytic seminomas had two, four or eight centrosomes. Germ cells in seminiferous tubules with disturbed spermatogenesis shared both aneuploidy and centrosome abnormalities with seminomas/nonseminomas and showed a more intense STK15 staining than those with normal spermatogenesis and CIS. Therefore, aneuploidy of testicular germ cell tumors is associated with amplified centrosomes probably unrelated to STK15.     

Chromosomal imbalances associated with carcinoma in situ and associated testicular germ cell tumours of adolescents and adults.

Carcinoma in situ (CIS) or intratubular germ cell neoplasia is generally considered the precursor lesion of adult testicular germ cell tumours (TGCT). The chromosomal imbalances associated with CIS and the corresponding seminoma (SE) or nonseminoma (NS) have been determined by comparative genomic hybridization (CGH) analysis of microdissected material from seven cases. Significantly, the CIS showed no gain of 12p material whereas in the invasive components of all cases gain of 12p was found, in 2 cases associated with amplification of the 12p11.2-12.1 region. Interphase fluorescence in situ analysis was consistent with this and provided evidence for the i(12p) or 12p11.2-12.1 amplification in the SE and NS but not in the corresponding CIS. This suggests a role for these changes in progression of CIS to invasive testicular cancer or progression of the invasive disease. Other imbalances such as gain of material from chromosomes 1, 5, 7, 8, 12q and X and loss of material from chromosome 18 were frequently identified (> 40% of cases) in the CIS associated with both SE and NS as well as in the invasive components. Loss of material from chromosome 4 and 13 and gain of 2p were more frequently found in the invasive components. The results shed light on the genetic relationship between the non-invasive and invasive components of testicular cancer and the stage at which particular chromosomal changes may be important.     

Amplification and overexpression of the KIT gene is associated with progression in the seminoma subtype of testicular germ cell tumors of adolescents and adults

We have previously identified amplification at 4q12 in testicular germ cell tumors of adolescents and adults centered around the KIT gene encoding a tyrosine kinase transmembrane receptor. Analysis of primary testicular germ cell tumors totaling 190 cases revealed 21% of the seminoma subtype with an increased copy number of KIT whereas this change was rarely found in the nonseminomas. In most cases, gain of KIT did not include the immediately flanking noncoding DNA or the flanking genes KDR and PDGFRA. Increased copy number of KIT was not found in the putative precursor lesion, carcinoma in situ (CIS), adjacent to tumor with this change. KIT overexpression was found independent of gain and KIT immunostaining was stronger in selected cases with gain of KIT compared to those without. Taken together with activating mutations of KIT in exon 17 identified in 13% of seminomas, this suggests that the KIT gene product plays a role in the progression of CIS towards seminoma, the further understanding of which may lead to novel less toxic therapeutic approaches.     

Testicular microlithiasis predicts concurrent testicular germ cell tumors and intratubular germ cell neoplasia of unclassified type in adults

BACKGROUND:

There is an increasing body of literature associating testicular microlithiasis (TM), a common finding on testicular ultrasound, with testicular germ cell tumor (TGCT) and intratubular germ cell neoplasia of unclassified type (ITGCNU). Determining these associations is pertinent both clinically and biologically. To the authors' knowledge, no previous systematic review or meta‐analysis has been performed.

METHODS:

A comprehensive systematic literature review was performed without language restrictions through July 2009 and included an exhaustive search of electronic databases and article references. Two reviewers extracted data independently. Studies were categorized according to the clinical context in which sonography was performed. The primary study outcomes were concurrent diagnoses of TGCT or ITGCNU, with TM. In addition, studies with prospective follow‐up of patients with TM were reviewed.

RESULTS:

Thirty‐three studies met inclusion criteria. TM was not associated with an increased risk of TGCT in asymptomatic men. However, in referral populations, TM was associated overall with a risk ratio of 8.5 (95% confidence interval [CI], 4.5‐16.1; P < .001) for a concurrent diagnosis of TGCT and 10.5 (95% CI, 5.3‐20.8; P < .0001) for ITGCNU. Seventeen observational studies were identified in which the interval development of TGCT in patients with TM was reported; however, the majority of those studies did not report the follow‐up of a control arm and could not be summarized.

CONCLUSIONS:

In the presence of risk factors, TM was associated with a substantially elevated risk of a concurrent diagnosis of TGCT and ITGCNU. The authors suggest modifications to recently proposed guidelines for the management of TM. Cancer 2010. © 2010 American Cancer Society.     

Serum human chorionic gonadotropin is associated with angiogenesis in germ cell testicular tumors

Background

Germ cell testicular tumors have survival rate that diminishes with high tumor marker levels, such as human chorionic gonadotropin (hCG). hCG may regulate vascular neoformation through vascular endothelial growth factor (VEGF). Our purpose was to determine the relationship between hCG serum levels, angiogenesis, and VEGF expression in germ cell testicular tumors.

Methods

We conducted a retrospective study of 101 patients. Serum levels of hCG, alpha-fetoprotein (AFP), and lactate dehydrogenase were measured prior to surgery. Vascular density (VD) and VEGF tissue expression were determined by immunohistochemistry and underwent double-blind analysis.

Results

Histologically, 46% were seminomas and 54%, non-seminomas. Median follow-up was 43 ± 27 months. Relapse was present in 7.5% and mortality in 11.5%. Factors associated with high VD included non-seminoma type (p = 0.016), AFP ≥ 14.7 ng/mL (p = 0.0001), and hCG ≥ 25 mIU/mL (p = 0.0001). In multivariate analysis, the only significant VD-associated factor was hCG level (p = 0.04). When hCG levels were stratified, concentrations ≥ 25 mIU/mL were related with increased neovascularization (p < 0.0001). VEGF expression was not associated with VD or hCG serum levels.

Conclusion

This is the first study that relates increased serum hCG levels with vascularization in testicular germ cell tumors. Hence, its expression might play a role in tumor angiogenesis, independent of VEGF expression, and may explain its association with poor prognosis. hCG might represent a molecular target for therapy.     

Genetic variation in hormone metabolizing genes and risk of testicular germ cell tumors

Testicular germ cell tumors (TGCT) that arise in young men are composed of two histologic types, seminomas and nonseminomas. Risk patterns for the two types appear to be similar and may be related to either endogenous or exogenous hormonal exposures in utero. Why similar risk patterns would result in different histologic types is unclear, but could be related to varying genetic susceptibility profiles. Genetic variation in hormone metabolizing genes could potentially modify hormonal exposures, and thereby affect which histologic type a man develops. To examine this hypothesis, 33 single nucleotide polymorphisms (SNPs) in four hormone metabolism candidate genes (CYP1A1, CYP17A1, HSD17B1, HSD17B4) and the androgen receptor gene (AR) were genotyped. Associations with TGCT were evaluated among 577 TGCT cases (254 seminoma, 323 nonseminoma) and 707 controls from the US Servicemen’s Testicular Tumor Environmental and Endocrine Determinants (STEED) study. There were no significant associations with TGCT overall based on a test using an additive model. However, compared to homozygotes of the most common allele, two nonredundant SNPs in CYP1A1 were inversely associated with nonseminoma: CYP1A1 promoter SNP rs4886605 OR = 0.75 (95% CI = 0.54–1.04) among the heterozygotes and OR = 0.37, 95% CI = 0.12–1.11 among the homozygotes with a p-value for trend = 0.02; rs2606345 intron 1 SNP, OR = 0.69 (95% CI = 0.51–0.93) among heterozygotes and OR = 0.70 (95% CI = 0.42–1.17) among homozygotes, with a p-value for trend = 0.02. Caution in interpretation is warranted until findings are replicated in other studies; however, the results suggest that genetic variation in CYP1A1 may be associated with nonseminoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subfertility and the risk of testicular germ cell tumors (United States)

Objectives: Previous studies have reported an association between subfertility and the risk of testicular germ cell tumors. We examined fertility, measured by number of children fathered and prior diagnosis of infertility, as a risk factor for testicular cancer, while accounting for the influence of occult cancer and cryptorchidism. Methods: Tumor registry data were used to identify 329 cases of testicular cancer in white men aged 20 to 69 years, diagnosed in western Washington State from 1977 to 1983; 672 cancer-free controls were identified by random-digit dialing. Telephone interviews ascertained reproductive histories and basic demographic information. Logistic regression was used to estimate the relative risk of testicular cancer associated with fertility. Results: Testicular cancer risk was decreased among men who had previously fathered a child (age-adjusted odds ratio (OR) 0.76, 95% confidence interval (CI): 0.54–1.06). Inverse associations were seen for seminomas and non-seminomas, and only slight attenuations in the ORs were observed when men with a history of cryptorchidism were excluded. Prior diagnosis of infertility was associated with an increased risk of testicular cancer (OR 2.40, 95% CI: 1.00–5.77). Conclusions: These results are consistent with an increased risk of testicular cancer among men with reduced fertility that goes beyond the effects of cryptorchidism.     

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

Background

Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs.

Materials and methods.

Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method.

Results

LOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component.

Conclusions

The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior.     

Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors

Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.     

Risk of testicular germ cell tumors and polymorphisms in the insulin-like growth factor genes.

Because taller men are at increased risk of developing testicular germ cell tumors (TGCT), it is conceivable that factors that influence adult height could be related to risk of TGCT. Because common genetic variation in genes of the insulin-like growth factor (IGF) pathway could influence somatic growth, 43 single nucleotide polymorphisms in four IGF genes (IGF-1, IGF-1R, IGF-2, and IGFALS) were genotyped in 577 case and 707 control participants from the U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study to assess relationships with TGCT risk; additionally, associations between polymorphisms and adult height were examined. Relationships between polymorphisms and adult height were assessed using adjusted linear regression models, and associations between polymorphisms and TGCT risk were determined by adjusted logistic regression models estimating odds ratios. Although four IGF-1R polymorphisms (rs907806, rs3743258, rs229765, and rs9282714) were associated with height (P trend < 0.05), there were no relationships with any other polymorphism. Overall, there were no associations among polymorphisms or haplotypes in the IGF genes and TGCT risk, with odds ratios ranging from 0.55 to 1.50. Similarly, there was no association among the polymorphisms and risk of specific TGCT histologies (seminoma and nonseminoma). There was a suggestion, however, that adult height may modify the relationship between an IGF-1 haplotype and TGCT risk. These results suggest that, in aggregate, genetic variation in IGF loci is not associated with TGCT risk.     

Expression of the hst-1 and c-kit protooncogenes in human testicular germ cell tumors.

Seventy testicular germ cell tumors were analyzed at the DNA and RNA levels for the c-kit, hst-1, and int-2 oncogenes using Northern and Southern blot analyses, respectively. There were significant differences in oncogene expression between seminomas and nonseminomas with c-kit being expressed in 24 of 30 (80%) seminomas but in only 3 of 40 (7%) nonseminomatous tumors (P = 0.0001, chi 2 test) and hst-1 being expressed in 24 of 38 (63%) nonseminomas but only 1 of 24 (4%) of seminomas (P = 0.0001, chi 2 test), demonstrating an inverse relationship in the expression pattern of these 2 oncogenes in human testicular germ cell tumors. A significant association between tumor stage and hst-1 expression in the nonseminoma group was found (P = 0.0002, chi 2 test). No gross alterations in the c-kit, hst-1, and int-2 loci were found at the DNA level and no int-2 mRNA expression was detected in any of the germ cell tumors examined.     

Synchronous bilateral testicular germ cell tumors of different histologic type. Pathogenetic and practical implications of bilaterality in testicular germ cell tumors.

Patients with a germ cell tumor (GCT) in one testicle are prone to develop cancer in the contralateral testicle. Metachronous development of a GCT in the remaining testicle occurs in 3% of patients. The synchronous development of bilateral GCT occurs in 0.5% of patients. Bilateral testicular neoplasms are almost always identical histologically. Only five cases of synchronous bilateral GCT of different histologic types have been reported in the literature. The authors report the sixth case of this uncommon phenomenon and discuss its clinical and pathogenetic implications.     

Recent advances in management of patients with platinum-refractory testicular germ cell tumors

With the use of cisplatin-based combination chemotherapy, metastatic testicular germ cell tumors can be cured in 70-80% of patients, but patients refractory to cisplatin-based chemotherapy continue to have a very poor prognosis. Various chemotherapeutic agents have been evaluated in intensively pretreated or cisplatin-refractory patients, but as single agents, only orally administered etoposide, paclitaxel, gemcitabine, and, most recently, oxaliplatin have been shown to be active with selected patients achieving complete remissions. This has for the first time lead to clinical evaluation of combination chemotherapy regimens such as gemcitabine-paclitaxel or oxaliplatin-gemcitabine, demonstrating the feasibility of combination therapy in these heavily pretreated patients. High response rates of up to 45% were observed in particular with the latter combination. Salvage surgery remains a very important treatment option for patients with resectable disease. The molecular mechanisms of cisplatin resistance have been intensively studied, and several mechanisms have been discussed such as a decreased intracellular concentration of the drug, increased repair of the drug-induced damage, or an altered apoptotic response to this damage. This increasing knowledge may now allow design of new therapeutic options. Ongoing studies in refractory germ cell tumors are evaluating 3-drug regimens such as gemcitabine-paclitaxel-oxaliplatin but also biologic approaches such as inhibitors of the epidermal growth factor receptor or the vascular endothelial growth factor. This research may eventually allow the development of a noncross-resistant multidrug combination regimen that can be evaluated in an earlier line of therapy.     

Defining minimum genomic regions of imbalance involved in testicular germ cell tumors of adolescents and adults through genome wide microarray analysis of cDNA clones

Identifying changes in DNA copy number can pinpoint genes that may be involved in tumor development. Here we have defined the smallest overlapping regions of imbalance (SORI) in testicular germ cell tumors other than the 12p region, which has been previously investigated. Definition of the regions was achieved through comparative genomic hybridization (CGH) analysis of a 4559 cDNA clone microarray. A total of 14 SORI were identified, which involved at least five of the 11 samples analysed. Many of these refined regions were previously reported using chromosomal or allelic imbalance studies. The SORI included gain of material from the regions 4q12, 17q21.3, 22q11.23 and Xq22, and loss from 5q33, 11q12.1, 16q22.3 and 22q11. Comparison with parallel chromosomal CGH data supported involvement of most regions. The various SORI span between one and 20 genes and highlight potential oncogenes/tumor suppressor genes to be investigated further.     

Childhood height and risk of testicular germ cell tumors in adulthood

Increased adult stature has been associated with risk of testicular germ cell tumors (TGCT) in a number of studies. Whether childhood stature is also associated with TGCT is unclear as no studies of measured childhood height and TGCT have been reported. Thus, associations between TGCT in adulthood and childhood height and growth between ages 7 and 13 years were examined in a cohort from the Copenhagen School Health Records Register. Analyses included 162,607 boys born during the years 1930–1989. Development of TGCT was determined via linkage to the Danish Cancer Registry. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazards regression. Between 1968 and 2014, 782 TGCT were diagnosed. Childhood height, per one unit increase in z‐score, was associated with risk of TGCT, with HRs ranging from 1.11 (95%CI 1.03–1.20) at age 7 to 1.09 (95%CI = 1.01–1.18) at age 13. In a categorical analysis, the shortest boys were at the lowest risk of developing TGCT. Results varied little by TGCT histology (seminoma and nonseminoma). Growth between ages 7 and 13 years was not associated with risk. These findings suggest that risk of TGCT in adulthood was already determined by age 7 years. Although the mechanism requires further investigation, these results provide additional evidence that risk of TGCT is determined at a young age, thus suggesting that additional investigation of early life factors is warranted.     

Frequent epigenetic inactivation of p53 target genes in seminomatous and nonseminomatous germ cell tumors

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. This study was designed to examine the methylation profiles of a selected group of p53 target genes (APAF-1, CASP-8, DAPK-1, IGFBP-3) and to correlate the findings with the histopathological characterization of testicular germ cell tumors (TGCT). Promoter methylation status was analysed by highly sensitive real-time methylation-specific PCR in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples. APAF-1 methylation was detected in all of the seminomatous and nonseminomatous TGCTs as well as in 60% of normal testicular tissue. Methylation of DAPK-1 was frequent in seminomas (50%) and nonseminomas (20%), but not in normal testicular tissue (6%). The degree of DAPK-1 methylation correlated with the clinical stage of the disease (P=0.05) and was useful in differentiating seminomatous from nonseminomatous, and malignant from nonmalignant testicular tissue (P=0.04 and 0.02, respectively). The APAF-1 methylation index achieved a highly significant differentiation between seminomatous or nonseminomatous tissue and nonmalignant testicular tissue (P=0.0001). In testicular tumorigenesis, promoter methylation of specific p53 target genes occurs at early stage but to varying degrees. Methylation also occurs in normal testicular tissue, which is in contrast to findings in other urogenital malignancies. Further studies will be necessary to determine whether the methylation level may be used as marker for risk estimation, especially in clinical stage I disease.     

Clinical usefulness of serum ferritin measurements in patients with testicular germ cell tumors

In 50 patients with seminoma and in 50 with nonseminomatous germ cell tumors of the testis, serum levels of conventional markers (CEA, AFP, hCG) and ferritin were measured at the time of admission and during management. The conventional markers behaved as reported previously. After orchiectomy, elevated levels of ferritin were found in the presence as well as in the absence of tumor; the extent of these elevations was highly variable. Serial determinations of serum ferritin showed two patterns of variation. First, in patients treated with retroperitoneal lymph node dissection, irradiation, and chemotherapy regimens without platinum, decreasing levels of the conventional markers and serum ferritin were associated with response to therapy and increasing levels with relapse of tumor. Second, in patients treated with chemotherapy regimens containing cis-diamminedichloroplatinum, the conventional markers returned to normal values while the ferritin level doubled or tripled and returned to pretreatment or normal values only several weeks after therapy. Thus, it appears that hyperferritinemia was a sensitive index of platinum toxicity. We conclude that the serum ferritin level has no value in staging after orchiectomy but is a useful index of response to therapy during treatment with retroperitoneal lymph node dissection, irradiation or chemotherapy without platinum or relapse of tumor. During treatment with platinum, elevated levels might be explained as a possible toxic side effect of this drug.