Oosterhuis, J.W.

Delayed recognition of disorders of sex development (DSD): A missed opportunity for early diagnosis of malignant germ cell tumors (Article)

2012-02-03T00:00:00Z

Disorders of sex development (DSD) are defined as a congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical. DSD patients with gonadal dysgenesis or hypovirilization, containing part of the Y chromosome (GBY), have an increased risk for malignant type II germ cell tumors (GCTs: seminomas and nonseminomas). DSD may be diagnosed in newborns (e.g., ambiguous genitalia), or later in life, even at or after puberty. Here we describe three independent male patients with a GCT; two were retrospectively recognized as DSD, based on the histological identification of both carcinoma in situ and gonadoblastoma in a single gonad as the cancer precursor. Hypospadias and cryptorchidism in their history are consistent with this conclusion. The power of recognition of these parameters is demonstrated by the third patient, in which the precursor lesion was diagnosed before progression to invasiveness. Early recognition based on these clinical parameters could have prevented development of (metastatic) cancer, to be treated by systemic therapy. All three patients showed a normal male 46,XY karyotype, without obvious genetic rearrangements by high-resolution whole-genome copy number analysis. These cases demonstrate overlap between DSD and the so-called testicular dysgenesis syndrome (TDS), of significant relevance for identification of individuals at increased risk for development of a malignant GCT. Copyright

Gonadal pathology and tumor risk in relation to clinical characteristics in patients with 45,X/46,XY mosaicism (Article)

2011-07-01T00:00:00Z

Context: Gonadectomy is avoided whenever possible in boys with 45,X/46,XY. However, no clinical markers are currently available to guide clinicians in predicting gonadal tumor risk or hormone production. Objective: The objective of the study was to test the hypothesis that gonadal histology and risk for development of a malignant germ cell tumor are reflected by the clinical presentation of a 45,X/46,XY individual. Design: The design of the study was the correlation of clinical data [external masculinization score (EMS), pubertal outcome] with pathology data (gonadal phenotype, tumor risk). Setting: This was a multicenter study involving two multidisciplinary disorder of sex development teams. Patients: Patients included genetically proven 45,X/46,XY (and variants) cases, ofwhomat leastone gonadal biopsy or gonadectomy specimen was available, together with clinical details. Interventions: Patients (n = 48) were divided into three groups, based on the EMS. Gonadal histology and tumor risk were assessedonparaffin-embedded samples (n = 87) by morphology and immunohistochemistry on the basis of established criteria. Main Outcome Measures: Gonadal differentiation and tumor risk in the three clinical groups were measured. Clinical outcome in patients with at least one preserved gonad was also measured. Results: Tumor risk in the three groups was significantly related to the gonadal differentiation pattern (P < 0.001). In boys, hormone production was sufficient and was not predicted by the EMS. Conclusions: The EMS reflects gonadal differentiation and tumor risk in patients with 45,X/46,XY. In boys, testosterone production is often sufficient, but strict follow-up is warranted because of malignancy risk, which appears inversely related to EMS. In girls, tumor risk is limited but gonads are not functional, makinggonadectomythemostreasonable option. Copyright

Gonadal development and tumor formation at the crossroads of male and female sex determination (Article)

2011-07-01T00:00:00Z

Malignant germ cell tumor (GCT) formation is a well-known complication in the management of patients with a disorder of sex development (DSD). DSDs are defined as congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. DSD patients in whom the karyotype - at least at the gonadal level - contains (a part of) the Y chromosome are at increased risk for neoplastic transformation of germ cells, leading to the development of the so-called 'type II germ cell tumors'. However, tumor risk in the various forms of DSD varies considerably between the different diagnostic groups. This contribution integrates our actual knowledge on the pathophysiology of tumor development in DSDs, recent findings on gonadal (mal)development in DSD patients, and possible correlations between the patient's phenotype and his/her risk for germ cell tumor development. Copyright

Diagnosis of testicular carcinoma in situ'(intratubular and microinvasive)' seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections (Article)

2011-02-01T00:00:00Z

Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumour components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mainly unavoidable. The aim was to develop an intraoperative assessment technique using alkaline phosphatase as a marker. Methods and results: A total of 4093 snap-frozen samples and matched paraffin-embedded tissue of 1500 patients were included. Besides standard haematoxylin and eosin (H&E) staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15min) was applied on frozen sections, while H&E and immunohistochemistry for detection of OCT3/4, α-fetoprotein, human chorionic gonadotrophin (hCG) and cytokeratin was performed on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. Positive staining was found in all CIS (n=965), seminoma (n=1035) and embryonal carcinoma (n=584), either intratubular, microinvasive or invasive. Differentiated non-seminomas (n=1238) showed variable staining. No staining was identified in spermatocytic seminomas (n=5), testicular lymphomas (n=42), testicular rhabdomyosarcomas (n=7), Leydig cell tumours (n=31), Sertoli-cell-only nodules (n=4), (epi) dermoid cyst (n=16), normal testicular parenchyma (n=116), testicular torsion (n=32) and inflammation of the epididymis (n=19). The dAP test results matched H&E-stained parallel sections, as well paraffin-embedded tissue analysis, including immunohistochemistry. Conclusions: The dAP test is an informative, reproducible and easy tool to diagnose CIS, (intratubular and microinvasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.

Tumor risk in disorders of sex development (Article)

2010-09-01T00:00:00Z

Certain patients with disorders of sex development (DSD), who bear Y chromosome material in their karyotype, are at increased risk for the development of type II germ cell tumors (GCT), which arise from early fetal germ cells. DSD gonads frequently harbor immature germ cells which express early fetal germ cell markers. Some of them (e.g. OCT3/4 and NANOG) seem to be of pathogenetic relevance in GCT development providing cells with the ability of pluripotency, proliferation and apoptosis suppression. Also TSPY (testis-specific protein Y-encoded), the main candidate for the so-called gonadoblastoma locus on Y chromosome, is overexpressed in germ cells of DSD patients and possibly contributes to their survival and proliferation. Nowadays, the use of immunohistochemical methods is highly relevant in identifying DSD gonads at risk. The risk for GCT development varies. While the prevalence of GCT is 15% in patients with partial androgen insensitivity, it may reach more than 30% in patients with gonadal dysgenesis. Patients with complete androgen insensitivity and ovotesticular DSD develop malignancies in 0.8% and 2.6% of cases, respectively. However, these data may be biased for various reasons. To better estimate the risk in individual groups of DSD, further investigations on large patient series are needed. Copyright

Cervical lymph node dissection for metastatic testicular cancer (Article)

2010-06-01T00:00:00Z

Introduction. Despite high response rates to systemic chemotherapy, 30% of patients with advanced stage testicular carcinoma will have extra- retroperitoneal residual masses that require resection. Most often, these are located in the lungs and mediastinum and neck. Limited data are available concerning the incidence, surgical management, and follow-up of neck metastasis arising from a testicular primary tumor. Methods. We retrospectively reviewed all 665 patients who were referred to a tertiary referral center with the diagnosis of testicular cancer from January 1997 to June 2009 for the presence of cervical metastases. Patients who underwent concomitant surgical therapy were identified and analyzed. Clinical and pathological data were collected from patient records, including radiology and pathology reports. Furthermore, data on primary treatment strategy, chemotherapeutic regimens, timing of surgical procedures, complications, disease recurrence, and follow-up were collected. Results. Twenty-six patients (4%) had cervical lymph node metastasis. The majority (n = 19) had multiple ERP sites. Nine patients (35%) underwent selective neck dissection: in six patients, this was indicated because of residual masses after chemotherapy, and in three patients, cervical masses represented a late and distant relapse of previously treated disease. Viable cancer cells were present in the resected specimen only in these three patients. Seven patients are currently without evidence of disease. Two patients died of disseminated disease. Conclusions. Cervical lymph node metastases originating from testicular cancer are rare but are more commonly observed in patients with advanced stage disease. Selective neck dissection can be safely performed both after chemotherapy and in the case of recurrent disease.

Combined optical coherence tomography and intravascular ultrasound radio frequency data analysis for plaque characterization. Classification accuracy of human coronary plaques in vitro (Article)

2010-04-15T00:00:00Z

This study was performed to characterize coronary plaque types by optical coherence tomography (OCT) and intravascular ultrasound (IVUS) radiofrequency (RF) data analysis, and to investigate the possibility of error reduction by combining these techniques. Intracoronary imaging methods have greatly enhanced the diagnostic capabilities for the detection of high-risk atherosclerotic plaques. IVUS RF data analysis and OCT are two techniques focusing on plaque morphology and composition. Regions of interest were selected and imaged with OCT and IVUS in 50 sections, from 14 human coronary arteries, sectioned post-mortem from 14 hearts of patients dying of non-cardiovascular causes. Plaques were classified based on IVUS RF data analysis (VH-IVUSTM), OCT and the combination of those. Histology was the benchmark. Imaging with both modalities and coregistered histology was successful in 36 sections. OCT correctly classified 24; VH-IVUS 25, and VH-IVUS/OCT combined, 27 out of 36 cross-sections. Systematic misclassifications in OCT were intimal thickening classified as fibroatheroma in 8 cross-sections. Misclassifications in VH-IVUS were mainly fibroatheroma as intimal thickening in 5 cross-sections. Typical image artifacts were found to affect the interpretation of OCT data, misclassifying intimal thickening as fibroatheroma or thin-cap fibroatheroma. Adding VH-IVUS to OCT reduced the error rate in this study.

Gonadal tumours and DSD (Article)

2010-04-01T00:00:00Z

Disorders of sex development (DSD), previously referred to as intersex, has been recognised as one of the main risk factors for development of type II germ cell tumours (GCTs), that is, seminomas/dysgerminomas and non-seminomas (e.g., embryonal carcinoma, yolk sac tumour, choriocarcinoma and teratoma). Within the testis, this type of cancer is the most frequent malignancy in adolescent and young adult Caucasian males. Although these males are not known to have dysgenetic gonads, the similarities in the resulting tumours suggest a common aetiological mechanism(s), -genetically, environmentally or a combination of both. Within the group of DSD patients, being in fact congenital conditions, the risk of malignant transformation of germ cells is highly heterogeneous, depending on a number of parameters, some of which have only recently been identified. Understanding of these recent insights will stimulate further research, with the final aim to develop an informative clinical decision tree for DSD patients, which includes optimal (early) diagnosis without overtreatment, such as prophylactic gonadectomy in the case of a low tumour risk.

Evaluation of testicular biopsies for carcinoma in situ: Immunohistochemistry is mandatory (Article)

2009-12-01T00:00:00Z

Carcinoma in situ (CIS) is the common precursor of all type II testicular germ cell tumors (TGCTs), i.e. seminomas and non-seminomas, which can be diagnosed using a surgical biopsy. The objective of this study was to investigate the additional value of immunohistochemistry for the diagnosis of CIS in assessing testicular biopsies taken in the context of infertility. A series of 21 infertile patients were retrieved from the Dutch pathological database (PALGA), being diagnosed with an invasive TGCT, while a matched previously obtained testicular biopsy was diagnosed as non-malignant. From 20 patients, both the invasive tumors as well as the biopsies were revised using morphology and immunohistochemistry for OCT3/4, placental-like alkaline phosphatase and c-KIT, all known established markers for CIS. The presence of CIS or invasive malignancies was scored. There are no interventions. Morphological criteria alone allowed an experienced pathologist in TGCTs to diagnose CIS in five and an invasive tumor in two cases (total n = 7, 35%). Application of immunohistochemistry resulted in the identification of an additional four cases of CIS (total n = 11, 55%, additional value of 20%). The initial correct diagnosis of CIS could have prevented a second gonadectomy in four patients (20%). This study, for the first time, really shows that time of progression from CIS to seminoma is longer than to non-seminoma. Our study demonstrates that immunohistochemistry should be performed for the diagnosis of CIS of the testis on single biopsies obtained because of infertility, resulting in an extra diagnostic yield of at least 20%. Application of this protocol will allow early diagnosis, and therefore prevent any adverse anti-cancer treatment sequelae including gonadectomy, and requiring life long androgen supplementation in some patients.

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma (Article)

2009-06-12T00:00:00Z

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.

Minimally invasive autopsy: An alternative to conventional autopsy? (Article)

2009-03-01T00:00:00Z

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 ultra-sonography-guided 12-guage 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.

New insights into type II germ cell tumor pathogenesis based on studies of patients with various forms of disorders of sex development (DSD) (Article)

2008-09-10T00:00:00Z

Disorders of sex development (DSD), previously known as intersex, refer to congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. Patients with specific variants of this disorder have an elevated risk for the development of so-called type II germ cell cancers, i.e., the seminomatous and nonseminatous tumors, referred to as germ cell tumors (GCTs). Specifically DSD patients with gonadal dysgenesis or hypovirilization are at risk. A prerequisite for type II GCT formation is the presence of a specific part of the Y chromosome (referred to as the GBY region), with the TSPY gene being the most likely candidate. Also the octamer binding transcription factor OCT3/4 is consistently expressed in all type II GCTs with pluripotent potential, as well as in the precursor lesions carcinoma in situ (CIS) in case of a testis and gonadoblastoma (GB) in the DSD gonad. The actual risk for malignant transformation in individual DSD patients is hard to predict, because of confusing terminology referring to the different forms of DSD, and unclear criteria for identification of the presence of malignant germ cells, especially in young patients. This is specifically due to the phenomenon of delay of germ cell maturation, which might result in over diagnosis. This review will give novel insight into the pathogenesis of the type II GCTs through the study of patients with various forms of DSD for which the underlying molecular defect is known. To allow optimal understanding of the pathogenesis of this type of cancers, first normal gonadal development, especially regarding the germ cell lineage, will be discussed, after which type II GCTs will be introduced. Subsequently, the relationship between type II GCTs and DSD will be described, resulting in a number of new insights into the development of the precursor lesions of these tumors.

Stem cell factor as a novel diagnostic marker for early malignant germ cells (Article)

2008-09-01T00:00:00Z

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay.

Oral Manifestations of Oxalosis: A Case Report and Review of the Literature (Article)

2008-09-01T00:00:00Z

A Pure Triphasic Testicular Wilms Tumor of Primordial Germ Cell Origin (Article)

2008-07-01T00:00:00Z

Noninvasive Detection of Testicular Carcinoma In Situ in Semen Using OCT3/4 (Article)

2008-07-01T00:00:00Z

Objective: Carcinoma in situ (CIS) is accepted as the precursor of the germ cell tumors of the adult testis. CIS cells are located within the seminiferous tubules and can be exfoliated into semen. We performed a study to detect CIS cells in semen using the highly specific immunohistochemical marker OCT3/4, potentially a method for noninvasive diagnosis. Material and methods: In 2006, 41 men at risk for CIS of the testis were found eligible for this study. Indications for inclusions were a suspicious lesion on scrotal ultrasound investigation (n = 14), patients on surveillance after a history of a testicular tumor (n = 14), and 13 patients with bilateral testicular microlithiasis (TM). Results: Three of the 13 men (23%) who underwent testicular biopsies for bilateral TM were histologically diagnosed with CIS (two bilateral), and their semen showed OCT3/4-positive cells in all cases. Twelve of the 14 patients (86%) with a solid mass were diagnosed with a TGCT with adjacent CIS in the parenchyma, and in 9 cases (75%) OCT3/4-positive cells were present in the semen. No OCT3/4-positive cells were found in patients with biopsies who did not show any evidence of malignancy. Conclusion: This study demonstrates that OCT3/4-positive cells can be found in semen from the majority of patients with CIS. The observations indicate that there is probably a time window in which the CIS cells are exfoliated, which gives an opportunity for early detection of CIS cells in semen of men at risk for TGCT.

FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD) (Article)

2008-05-01T00:00:00Z

The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter-sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre-)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well-developed seminiferous tubules, but double staining demonstrated that it was never strongly co-expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non-seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB. Copyright

Further characterization of the first seminoma cell line TCam-2 (Article)

2008-03-01T00:00:00Z

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

Heterogeneous distribution of ITGCNU in an adult testis: Consequences for biopsy-based diagnosis (Article)

2008-01-01T00:00:00Z

Carcinoma in situ (CIS) of the testis, also referred to as intratubular germ cell neoplasia unclassified (ITGCNU), is currently accepted as the common precursor for all malignant germ cell tumors of adolescents and adults- that is, the seminomatous and nonseminoma cancers. These preinvasive cells have specific cellular characteristics, which can be used for the early diagnosis-routinely done by morphological analysis, sometimes supported by immunohistochemistry-of tissue obtained by an open surgical biopsy. False-negative biopsy results can occur mostly because of the nonrandom distribution of ITGCNU within the testis, misdiagnosis, or suboptimal tissue treatment and analysis. In this article, we demonstrate the potential pitfalls in the diagnosis of ITGCNU. The results support the use of the highly specific and sensitive immunohistochemical marker OCT3/4 for the diagnosis of ITGCNU and provide evidence for the nonrandom distribution of ITGCNU, which is a significant limitation in the diagnosis of this preinvasive lesion.

A 40-year-old woman with a progressive periventricular white matter lesion (Article)

2008-01-01T00:00:00Z

A 40-year-old woman presented with blurred vision and diplopia, followed by slowly progressive left-sided motor and sensory disturbances. She also suffered from memory loss and had mild spatial and temporal disorientation. A T2-weighted MRI showed a large area of high signal intensity in the periventricular white matter of the right more than the left occipital region and the corpus callosum, without enhancement on T1-weighted images after gadolinium administration and without mass effect. A stereotacticbiopsy of the intracerebral lesion showed blast-like neoplastic cells within a mononuclear infiltrate. No diagnosis could be made based on morphology and immunohistochemistry using a large series of markers. However, based on positive OCT3/4 nuclear staining, the tumor was diagnosed as a germinoma (seminoma of the brain). The patient was treated accordingly and her condition improved, although focal deficits remained.

High-throughput microRNAome analysis in human germ cell tumours (Article)

2007-11-01T00:00:00Z

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow intersample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines (Article)

2007-10-01T00:00:00Z

Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the UI33Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity.

Tumor risk in disorders of sex development (DSD) (Article)

2007-09-01T00:00:00Z

Disorders of sex development (DSD), previously referred to as intersex disorders, comprise a variety of anomalies defined by congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Besides issues such as gender assignment, clinical and diagnostic evaluation, surgical and psychosocial management, and sex steroid replacement, the significantly increased risk for developing specific types of malignancies is both clinically and biologically relevant. This relates to germ-cell tumors specifically in DSD patients with hypovirilization or gonadal dysgenesis. The presence of a well-defined part of the Y chromosome (known as the GBY region) is a prerequisite for malignant transformation, for which the testis-specific protein on the Y chromosome (TSPY) is a likely candidate gene. The precursor lesions of these cancers are carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU) in testicular tissue and gonadoblastoma in those without obvious testicular differentiation. Most recently, undifferentiated gonadal tissue (UGT) has been identified as the likely precursor for gonadoblastoma. The availability of markers for the different developmental stages of germ cells allows detailed investigation of the characteristics of normal and (pre)malignant germ cells. Although informative in a diagnostic setting for adult male patients, these markers - such as OCT3/4 - cannot easily distinguish (pre)malignant germ cells from germ cells showing delayed maturation. This latter phenomenon is frequently found in gonads of DSD patients, and may be related to the risk of malignant transformation. Thus, the mere application of these markers might result in over-diagnosis and unnecessary gonadectomy. It is proposed that morphological and histological evaluation of gonadal tissue, in combination with OCT3/4 and TSPY double immunohistochemistry and clinical parameters, is most informative in estimating the risk for germ-cell tumor development in the individual patient, and might in future be used to develop a decision tree for optimal management of patients with DSD.

JKT-1 is not a human seminoma cell line (Article)

2007-08-01T00:00:00Z

The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

Morphological and immunohistochemical differences between gonadal maturation delay and early germ cell neoplasia in patients with undervirilization syndromes. (Article)

2005-09-01T00:00:00Z

CONTEXT: Maturation delay of germ cells and their progression into carcinoma in situ (CIS) frequently occurs in intersex patients. A developmentally delayed germ cell resembles a CIS cell and displays prolonged expression of immunohistochemical markers used for the diagnosis of CIS. This questions their applicability in young children. OBJECTIVE: The objective of the study was the elaboration of tools to distinguish germ cells with maturation delay and CIS. DESIGN: The design was a qualitative and quantitative analysis of the expression of diagnostic markers for CIS in gonads of young patients with undervirilization syndromes. Setting: The study was conducted in the pathology department of a university center, specializing in germ cell tumor pathogenesis. PATIENTS: Fifty-eight formalin-fixed, paraffin-embedded testicular tissue samples of 30 undervirilized patients (1 month to 23 yr of age) were analyzed. Interventions: Interventions included hematoxylin-eosin staining, immunohistochemistry for octamer binding transcription factor (OCT)3/4, gene encoding the stem cell factor receptor that has tyrosine kinase activity c-KIT, placental/germ alkaline phosphatase (PLAP), testis-specific protein Y encoded (TSPY), and VASA, double staining for OCT3/4 and VASA, with ploidy determination by fluorescent in situ hybridization. MAIN OUTCOME MEASURE: Maturation delay and CIS are characterized by the staining patterns of the immunohistochemical markers. RESULTS: CIS was diagnosed in three of 30 patients (10%) and four of 58 gonads (6.9%). Patient age, distribution of OCT3/4-positive cells throughout the gonad, and their position within the seminiferous tubule differ between maturation delay and CIS. Abnormal OCT3/4 and testis-specific protein Y encoded expression appear to be of pathogenetic relevance in the development of these lesions. CONCLUSION: The dimorphic expression of OCT3/4 allows distinction between maturation delay and CIS. Studies in larger patient series are essential before a biopsy to evaluate the neoplastic risk can eventually be proposed as an alternative for gonadectomy.

Molecular determinants of treatment response in human germ cell tumors (Article)

2003-01-01T00:00:00Z

PURPOSE: Germ cell tumors (GCTs) are highly sensitive to cisplatin-based chemotherapy. This feature is unexplained, as is the intrinsic chemotherapy resistance of mature teratomas and the resistant phenotype of a minority of refractory GCTs. Various cellular pathways may influence the efficacy of chemotherapy. Their impact has not been investigated in a comprehensive study of tumor samples from clinically defined subgroups of GCT patients. EXPERIMENTAL DESIGN: We investigated proteins involved in regulation of apoptosis (p53, BAX, BCL-2, and BCL-X(L)), cell cycle control [p21 and retinoblastoma protein (RB)], and drug export and inactivation [P-glycoprotein, multidrug resistance-associated protein (MRP) 1, MRP2, breast cancer resistance protein, lung resistance protein, metallothionein, and glutathione S-transferase pi] immunohistochemically in samples of unselected GCT patients (n = 20), patients with advanced metastatic disease in continuous remission after first-line chemotherapy (n = 12), and chemotherapy-refractory patients (n = 24). Mature teratoma components (n = 10) within tumor samples from all groups were analyzed separately. The apoptotic index was studied by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: Invasive GCTs of all groups showed a correlation between wild-type p53 and apoptotic index (r(s) = 0.66; P < 0.001). The levels of the antiapoptotic proteins BCL-2 and BCL-X(L) were generally low. p21 was hardly detectable and did not correlate with p53 (r(s) = 0.29; P = 0.07). No significant differences among the three patient groups were identified regarding any of the investigated parameters (all Ps were >0.08), even though only individual samples from chemotherapy-resistant cases showed a strong staining for MRP2 and GSTpi. In contrast to other components, mature teratomas showed an intense p21 and RB staining and were mostly positive for MRP2, lung resistance protein, and GSTpi. CONCLUSIONS: Our results indicate a multifactorial basis for the chemosensitivity of GCTs with lack of transporters for cisplatin, of antiapoptotic BCL-2 family members, of p21 induction by p53, and of RB and an intact apoptotic cascade downstream of p53. These findings suggest a preference for apoptosis over cell cycle arrest after up-regulation of p53. None of the examined parameters offers a general explanation for the chemotherapy-resistant phenotype of refractory tumors. The up-regulation of various factors interfering with chemotherapy efficacy and ability for a p21-induced cell cycle arrest may explain the intrinsic chemotherapy resistance of mature teratomas.

POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors (Article)

2003-01-01T00:00:00Z

Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonseminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblastoma, seminomas/germinoma/dysgerminoma, and embryonal carcinoma but not in the various types of differentiated nonseminomas. Multitumor tissue microarray analysis covering >100 different tumor categories and 3600 individual cancers verified that POU5F1 expression is specific for particular subtypes of GCT of adults. No protein was observed in GCT of newborn and infants, spermatocytic seminomas, and the various tumors of nongerm cell origin. In addition, no difference in staining pattern was found in chemosensitive and chemoresistant GCT of adults. These results indicate preservation of the link between POU5F1 and pluripotency, as reported during normal development, after malignant transformation. Therefore, POU5F1 immunohistochemistry is an informative diagnostic tool for pluripotent GCT and offers new insights into the histological heterogeneity of this cancer.

Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors (Article)

2003-01-01T00:00:00Z

Testicular germ-cell tumors (TGCTs) of adolescents and adults originate from intratubular germ cell neoplasia (ITGCN), which is composed of the malignant counterparts of embryonal germ cells. ITGCN cells are characterized, among others, by the presence of stem cell factor receptor c-KIT. Once established, ITGCN will always progress to invasiveness. Approximately 2.5-5% of patients with a TGCT will develop bilateral disease and require complete castration, resulting in infertility, a need for lifelong androgen replacement, and psychological stress. To date, the only way to predict a contralateral tumor is surgical biopsy of the contralateral testis to demonstrate ITGCN. We did a retrospective study of 224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three independently collected series in Europe) for the presence of activating c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In the two wild-type bilateral tumors for which ITGCN was available, the preinvasive cells contained the mutation. The mutations were somatic in origin and identical in both tumors. We conclude that somatic activating codon 816 c-KIT mutations are associated with development of bilateral TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies patients at risk for bilateral disease. These patients may undergo tailored treatment to prevent the development of bilateral disease, with retention of testicular hormonal function.

Coamplification of DAD-R, SOX5, and EKI1 in human testicular seminomas, with specific overexpression of DAD-R, correlates with reduced levels of apoptosis and earlier clinical manifestation (Article)

2002-01-01T00:00:00Z

Seminomas and nonseminomas represent the invasive stages of testicular (TGCTs) of adolescents and adults. Although TGCTs are characterized by extra copies of the short arm of chromosome 12, the genetic basis for gain of 12p in the pathogenesis of this cancer is not yet understood. We have demonstrated that gain of 12p is related to invasive growth and that amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates with reduced apoptosis in the tumors. Here we show that three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs. In addition, our results also imply differences in expression regulation of DAD-R between seminomas and nonseminomas.

Microsatellite instability of germ cell tumors is associated with resistance to systemic treatment (Article)

2002-01-01T00:00:00Z

Systemic cisplatin-based chemotherapy cures > or =90% of patients with metastatic germ cell tumors (GCTs). The biological basis of this exquisite chemo-sensitivity and the resistant phenotype encountered in 10-15% of patients with GCT is yet unclear. A defective mismatch repair pathway leading to microsatellite instability (MSI) has been related to resistance to cytotoxic drugs. We investigated 100 unselected GCTs and 11 clinically defined chemotherapy-resistant GCTs for MSI using 8 mono- or dinucleotide markers and the presence of the mismatch repair factors MLH1, MSH2, and MSH6 by immunohistochemistry. The resistant tumors, both chemo-naive (n = 8) and pretreated (n = 3), showed a significantly higher incidence of MSI compared with the unselected series (45 versus 6% in at least one locus and 36 versus 0% in > or =2 of 8 loci, both P < or = 0.001). In 5 of all 11 unstable tumors, MSI correlated with immunohistochemical findings. This study demonstrates for the first time a positive correlation between MSI and treatment resistance in GCT.

Best cases from the AFIP. Maffucci syndrome: radiologic and pathologic findings. Armed Forces Institutes of Pathology (Article)

2001-01-01T00:00:00Z

Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis (Article)

2000-01-01T00:00:00Z

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 high-level 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 approximately 8% 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.

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas (Article)

1999-01-01T00:00:00Z

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.

Detection of human endogenous retrovirus type K-specific transcripts in testicular parenchyma and testicular germ cell tumors of adolescents and adults: clinical and biological implications (Article)

1998-01-01T00:00:00Z

Testicular germ cell tumors (TGCTs) of adolescents and adults have been shown to contain proteins of the human endogenous retrovirus type K family. In a recent study, expression of these retroviral sequences was confirmed using in situ hybridization, which also showed expression in carcinoma in situ, the precursor of all TGCTs. Because of the clinical significance of a test for early diagnosis of TGCTs, we studied whether expression of human endogenous retrovirus type K genes could be an informative parameter. Therefore, we investigated TGCTs of various histologies and testicular parenchyma with and without carcinoma in situ using reverse transcription-polymerase chain reaction for expression of the gag, env, and prt genes. The gag and prt genes were expressed in all samples tested. The env transcripts were not found in TGCTs showing somatic differentiation only but could be detected in most normal testicular parenchyma samples. Therefore, detection of human endogenous retrovirus type K transcripts cannot be used for early diagnosis of TGCTs. Simultaneous expression of multiple gag sequences was found both in normal parenchyma and TGCTs, and we demonstrated that expression of gag sequences with an extra G, necessary to generate a functional protein, was not limited to TGCTs.

Kiemceltumoren: "meer dan meer van hetzelfde" (Inaugural Lecture)

1993-01-01T00:00:00Z