J. Wolter Oosterhuis

Testicular germ-cell tumours in a broader perspective

The germ-cell tumours are a fascinating group of neoplasms because of their unusual biology and the spectacular therapeutic results that have been obtained in these tumours. Traditionally, this group of neoplasms is presented in an organ-oriented approach. However, recent clinical and experimental data convincingly demonstrate that these neoplasms are one disease with separate entities that can manifest themselves in different anatomical sites. We propose five entities, in which the developmental potential is determined by the maturation stage and imprinting status of the originating germ cell. Recent progress begins to explain the apparent unpredictable development of germ-cell tumours and offers a basis for understanding their exquisite sensitivity to therapy.

Carcinoma in situ in the testis

Carcinoma in situ (CIS) of the testis is a common precursor of germ-cell tumours in adults and adolescents, with the exception of spermatocytic seminoma. This article reviews existing knowledge on the pathobiology, genetic aspects and epidemiology of CIS, discusses current hypotheses concerning pathogenesis and invasive progression of germ-cell neoplasms and provides guidelines for diagnosis and clinical management of CIS.

PATHOGENESIS OF ADULT TESTICULAR GERM-CELL TUMORS - A CYTOGENETIC MODEL

In essence, two models exist of the pathogenetic relationship between seminomas and nonseminomatous germ cell tumors (NSGCTs). In the first model, the histogenesis of seminomas is assumed to diverge from that of the other testicular germ cell tumors (TGCTs) at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with limited or no crossover. The second model suggests that seminomas and NSGCTs have a common origin with a single neoplastic pathway on which seminomas are an intermediate stage in development of NSGCTs. Our data on the cytogenetics and ploidy of seminomas, combined tumors, and NSGCTs lend support to the model of pathogenesis of seminomas and NSGCTs in which all TGCTs (with the possible exception of spermatocytic seminoma and infantile yolk sac tumor) have a single origin and neoplastic pathway, with seminomas representing an intermediate stage in development of NSGCT components, as opposed to the model in which seminomas and NSGCTs develop separately. The progression of TGCTs probably proceeds from high to lower numbers of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end result of loss of specific chromosomes, gain of some other chromosomes (or part of chromosomes), and development of structural abnormalities.

Minimally Invasive Autopsy: An Alternative to Conventional Autopsy?

PURPOSE To determine the diagnostic performance of minimally invasive autopsy (MIA) for detection of causes of death and to investigate the feasibility of MIA as an alternative to conventional autopsy (CA) in the clinical setting. MATERIALS AND METHODS The institutional review board approved the MIA procedure and study, and informed consent was obtained for all deceased patients from relatives. Thirty deceased patients (19 men, 11 women; age range, 46-79 years), for whom family permission for CA on medical grounds had already been obtained, underwent additional evaluation with MIA prior to CA. MIA consisted of whole-body 16-section computed tomography (CT) and 1.5-T magnetic resonance (MR) imaging, followed by ultrasonography-guided 12-gauge needle biopsy of heart, both lungs, liver, both kidneys, and spleen. Percentage agreement between MIA and CA on cause of death was evaluated. Sensitivity and corresponding 95% confidence intervals (CIs) of MIA for detection of overall (major plus minor) findings, with CA as the reference standard, were calculated. Specificity was calculated for overall findings. Sensitivity analysis was performed to explore the effect of the clustered nature of the data. RESULTS In 23 patients (77%), MIA and CA were in agreement on the cause of death. Sensitivity of MIA for detection of overall findings and detection of major findings was 93% (95% CI: 90%, 96%) and 94% (95% CI: 87%, 97%), respectively. Specificity was 99% (95% CI: 98%, 99%) for detection of overall findings. MIA failed to demonstrate acute myocardial infarction as the cause of death in four patients. Sensitivity analysis indicated a negligible correlation between observations within each patient. CT was superior to MR for detection of pneumothorax and calcifications. MR was superior to CT for detection of brain abnormalities and pulmonary embolus. With biopsy only, detection of disease in 55 organs was possible, which included 27 major findings. CONCLUSION MIA is a feasible procedure with high diagnostic performance for detection of common causes of death such as pneumonia and sepsis; MIA failed to demonstrate cardiac diseases, such as acute myocardial infarction and endocarditis, as underlying cause of death. SUPPLEMENTAL MATERIAL http://radiology.rsnajnls.org/cgi/content/full/250/3/897//DC1.

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.

Tumour banks: well-guarded treasures in the interest of patients.

In order for the genomics revolution to change how we diagnose, categorize and treat cancer, scientists and clinicians must have access to tumour samples. There has therefore never been a better time to create banks of tumour tissue. Collecting and storing tumour samples and their associated data, however, creates numerous methodological, ethical, legal and technical problems. How can we leap these hurdles in a responsible manner and still make full use of the wealth of information that can be obtained from them?

Microsatellite Instability, Mismatch Repair Deficiency, and BRAF Mutation in Treatment-Resistant Germ Cell Tumors

PURPOSE Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are associated with cisplatin resistance in human germ cell tumors (GCTs). BRAF mutation (V600E) is found in MSI colorectal cancers. The role of RAS/RAF pathway mutations in GCT treatment response is unknown. PATIENTS AND METHODS Two patient cohorts were investigated: 100 control GCTs (50 seminomas and 50 nonseminomas) and 35 cisplatin-based chemotherapy-resistant GCTs. MMR proteins were analyzed by immunohistochemistry, and eight microsatellite loci were examined for MSI. Tumors were assessed for specific BRAF and KRAS mutations. RESULTS Resistant tumors showed a higher incidence of MSI than controls: 26% versus 0% in two or more loci (P < .0001). All resistant tumors were wild-type KRAS, and two controls (2%) contained a KRAS mutation. There was a significantly higher incidence of BRAF V600E mutation in resistant tumors compared with controls: 26% versus 1% (P < .0001). BRAF mutations were highly correlated with MSI (P = .006), and MSI and mutated BRAF were correlated with weak or absent staining for hMLH1 (P = .017 and P = .008). Low or absent staining of hMLH1 was correlated with promoter hypermethylation (P < .001). Tumors lacking expression of hMLH1 or MSH6 were significantly more frequent in resistant GCTs than in controls (P = .001 and 0.0036, respectively). Within the subgroup of resistant tumors, patients with MSI showed a trend to longer progression-free survival (P = .068). CONCLUSION We report for the first time a correlation between a gene mutation--BRAF V600E--and cisplatin resistance in nonseminomatous GCTs. Furthermore, a correlation between MMR deficiency, MSI, and treatment failure is confirmed.

Role of gain of 12p in germ cell tumour development.

Within the human testis, three entities of germ cell tumours are distinguished: the teratomas and yolk sac tumors of newborn and infants, the seminomas and nonseminomas of adolescents and young adults, referred to as testicular germ cell tumours (TGCT), and the spermatocytic seminomas. Characteristic chromosomal anomalies have been reported for each group, supporting their distinct pathogenesis. TGCT are the most common cancer in young adult men. The initiating pathogenetic event of these tumours occurs during embryonal development, affecting a primordial germ cell or gonocyte. Despite this intra‐uterine initiation, the tumour will only be clinically manifest after puberty, with carcinoma in situ (IS) as the precursor. All invasive TGCT, both seminomas and nonseminomas, as well as CIS cells are aneuploid. The only consistent (structural) chromosomal abnormalities in invasive TGCT are gains of the short arm of chromosome 12, mostly due to isochromosome (i(12p)) formation. This suggests that an increase in copy number of a gene(s) on 12p is associated with the development of a clinically manifest TGCT. Despite the numerous (positional) candidate gene approaches that have been undertaken thus far, identification of a causative gene(s) has been hampered by the fact that most 12p gains involve rather large genomic intervals, containing unmanageable numbers of candidate genes. Several years ago, we initiated a search for 12p candidate genes using TGCT with a restricted 12p‐amplification, cytogenetically identified as 12p11.2–p12.1. This approach is mainly based on identification of candidate genes mapped within the shortest region of overlap of amplification (SROA). In this review, data will be presented, which support the model that gain of 12p‐sequences is associated with suppression of apoptosis and Sertoli cell‐independence of CIS cells. So far, DAD‐R is one of the most likely candidate genes involved in this process, possibly via N‐glycosylation. Preliminary results on high through‐put DNA‐ and cDNA array analyses of 12p‐sequences will be presented.

Role of P53 and MDM2 in treatment response of human germ cell tumors.

PURPOSE Testicular germ cell tumors (TGCTs) of adolescents and adults are very sensitive to systemic treatment. The exquisite chemosensitivity of these cancers has been attributed to a high level of wild-type P53. MATERIALS AND METHODS To clarify the role of P53 in treatment sensitivity and resistance of TGCTs, we performed immunohistochemistry and Western blotting analysis on a series of 39 fresh-frozen primary TGCTs before therapy (unselected series). In a series of formalin-fixed paraffin-embedded TGCTs of patients with fully documented clinical course, including treatment-sensitive (n = 17) and -resistant (n = 18) tumors, P53 status was assessed by immunohistochemistry and mutation analysis. In addition, the involvement of MDM2, a P53 antagonist, was investigated by immunohistochemistry, reverse transcriptase polymerase chain reaction, and in situ hybridization. RESULTS Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%, and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the positive TGCTs, most tumors, ie, 49%, 41%, and 33%, showed 1% to 10% positive nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis revealed only one silent P53 mutation in one of the responding patients. All embryonal carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA, while a heterogeneous pattern was found for the other histologic components. Amplification of MDM2 was detected in one out of 12 embryonal carcinomas. CONCLUSION Although our results are in line with previous findings of the presence of wild-type P53 in TGCTs, they show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and inactivation of P53 is not a common event in the development of cisplatin resistance.

Sequence analysis of the protein kinase gene family in human testicular germ-cell tumors of adolescents and adults

The protein kinase gene family is the most frequently mutated in human cancer. Previous work has documented activating mutations in the KIT receptor tyrosine kinase in testicular germ‐cell tumors (TGCT). To investigate further the potential role of mutated protein kinases in the development of TGCT and to characterize the prevalence and patterns of point mutations in these tumors, we have sequenced the coding exons and splice junctions of the annotated protein kinase family of 518 genes in a series of seven seminomas and six nonseminomas. Our results show a remarkably low mutation frequency, with only a single somatic point mutation, a K277E mutation in the STK10 gene, being identified in a total of more than 15 megabases of sequence analyzed. Sequencing of STK10 in an additional 40 TGCTs revealed no further mutations. Comparative genomic hybridization and LOH analysis using SNP arrays demonstrated that the 13 TGCTs mutationally screened through the 518 protein kinase genes were uniformly aneuploid with consistent chromosomal gains on 12p, 8q, 7, and X and losses on 13q, 18q, 11q, and 4q. Our results do not provide evidence for a mutated protein kinase implicated in the development of TGCT other than KIT. Moreover, they demonstrate that the general prevalence of point mutations in TGCT is low, in contrast to the high frequency of copy number changes.