Cools, M.B.C.M.

Requirements for a multicentric multidisciplinary registry on patients with disorders of sex development (Article)

2012-10-10T00:00:00Z

Disorders of Sexual Development (DSDs) are a group of rare to very rare congenital anomalies of the genito-urinary tract of genetic and endocrine causes. Recently, an international database I-DSD was successfully implemented to register patients with DSD and to provide the basis for epidemiologic, genetic, and clinical research. This tool needs to be adjusted and supplemented with additional modules in order to better assess the anatomical basis of DSD as well as to monitor risk factors such as gonadal histology. A proposal for the additional information to be obtained is discussed.

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development (Article)

2012-03-01T00:00:00Z

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development.

Partial deletion of the NR5A1 (SF1) gene detected by synthetic probe MLPA in a patient with XY gonadal disorder of sex development (Article)

2011-07-01T00:00:00Z

Steroidogenic factor 1 (SF1, officially NR5A1) is a nuclear receptor involved in adrenal and gonadal development. NR5A1 mutations have been identified in patients with various forms of 46,XY disorders of sex development (DSD), including complete gonadal dysgenesis with or without adrenal insufficiency, mild testicular dysgenesis with ambiguous external genitalia or female external genitalia with clitoromegaly, and penoscrotal hypospadias. We developed a synthetic probe set for MLPA analysis of the NR5A1 gene covering its 7 exons and analyzed 20 patients with 46,XY gonadal DSD in whom analyses failed to identify a genetic cause. We identified a partial NR5A1 deletion affecting exons 2 and 3, leading to NR5A1 haploinsufficiency in 1 patient presenting with female external genitalia with clitoromegaly, absence of a uterus, and mildly dysgenetic testes. This is the first partial NR5A1 gene deletion identified by MLPA in a patient with 46,XY gonadal DSD. This finding stresses the importance of investigating copy number changes, even at the exon level, in genes involved in gonadal DSD. As NR5A1 mutations can cause a wide spectrum of DSD with relatively high frequency, the analysis of the NR5A1 gene by MLPA is quite important and should be extended to larger groups of patients. 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

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

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.

Disorders of sex development: Update on the genetic background, terminology and risk for the development of germ cell tumors (Article)

2009-07-27T00:00:00Z

Background: Considerable progress has been made on genetic mechanisms involved in disorders of sex development and on tumor formation in dysgenetic gonads. Clinical and psychological outcome of patients are, as far as evaluated, unsatisfactory at present. Guidelines are emerging in order to optimize long-term outcome in the future. Data sources: The information obtained in this review is based on recent original publications and on the experience of our multidisciplinary clinical and research group. Results: This review offers an update on our knowledge concerning gene mutations involving in disorders of sex development, on the renewed nomenclature and classification system, and on the mechanisms of tumor development in patients. Conclusions: The consensus meeting on disorders of sex development has renewed our interest in clinical studies and long-term outcome of patients. Psychological research emphasizes the importance to consider male gender identity wherever possible in cases of severe undervirilization. Patient advocacy groups demand a more conservative approach regarding gonadectomy. Medical doctors, scientists and governmental instances are increasingly interested in the set-up of international research collaborations. As a consequence, it is expected that new guidelines for the optimal care of patients will be proposed in the coming years.

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.

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.

Impact of the Y-containing cell line on histological differentiation patterns in dysgenetic gonads (Article)

2007-08-01T00:00:00Z

Objective: Gonadal karyotyping is considered a tool for increasing our knowledge of disturbed gonadal development in patients with gonadal dysgenesis and for estimating more accurately the risk for gonadoblastoma formation. The objective was to gain insight into the role of Y chromosome distribution in the histological heterogeneity of gonads of patients with gonadal dysgenesis. Design: Investigation of the possible relationship between peripheral blood karyotype, gonadal karyotype, morphological differentiation patterns of dysgenetic gonads and tumour formation. Patients: In total 22 gonadal samples from 19 patients with gonadal dysgenesis (45,X/46,XY and variants n = 14; 46,XY: n = 3; 46,XX: n = 2) were examined. Measurements: Morphological examination and immunohistochemical staining for testis specific protein, Y encoded (TSPY) and fluorescent and nonfluorescent in situ hybridization directly on gonadal tissue. Results: No correlation was observed between peripheral blood karyotype and gonadal karyotype or between gonadal karyotype and the corresponding differentiation pattern. A Y-containing cell line in Sertoli cells was encountered no more frequently than were other cell types. Conclusions: The distribution of the Y-containing cell line in peripheral blood is not a suitable indicator for predicting the histological differentiation pattern found in the gonads of patients with gonadal dysgenesis. The analysis of Y-containing cell lines in the gonads of such patients could be informative with regard to the specific characteristics of gonadal development in humans as compared to chimeric mouse models. Moreover, it is essential to understand the mechanisms underlying disturbed gonadogenesis in these patients. As the gonadal karyotype is not related to the encountered gonadal differentiation pattern, it does not allow prediction of the risk for gonadoblastoma formation.

Germ cell tumors in patients with disorders of sex development: Risk factors, initial developmental stages and targets for early diagnosis (Doctoral Thesis)

2006-05-24T00:00:00Z

This thesis describes the origin, identification and correct diagnosis of the earliest stages of malignant germ cell tumors, i.e. intra tubular germ cell neoplasia unclassified (ITGNU) and gonadoblastoma, in patients with disorders of sex development. Special attention is given to the possible pathogenetic mechanisms underlying the development of these lesions, in order to identify high-risk patients. Ultimately, this study aims to contribute to the actual process of re-thinking the clinical management of patients with disorders of sex development, in accordance with the intense aspirations of these patients to evolve towards a safe, evidence-based, but more conservative approach regarding gonadectomy and other surgical procedures in the future wherever possible. Chapter 1 situates the ITGNU and gonadoblastoma lesions within the group of malignant germ cell tumors. It offers an overview of our actual knowledge on normal gonadal and sexual development and describes the recent hypotheses on how this process can be disturbed by environmental influences. The different phenotypes in the various disorders of sex development are described from a developmental point of view, with emphasis on the biology and functioning of the malformed gonad. In chapter 2, the normal gonadal development is studied in female embryos: the sequential expression of protein markers for germ cells, detectable by immunohistochemisty, in relation to the ultrastructure and cellular organization within the developing ovary is described. Together with a similar study on male gonadal development (Honecker et al, J Pathol 2004, 203 (3): 849-57), this study forms the basis for further research on the process of maturation delay of germ cells, as described in the following chapters. Chapter 3 examines if and to what extent maturation delay of germ cells is present in patients with trisomy 21, a condition unrelated to disorders of sex development but also characterized by an increased risk for germ cell tumors in males. Accordingly, a delay in germ cell maturation was mainly found in the male trisomy 21 population. This adds evidence to the “testicular dysgenesis syndrome” hypothesis, suggesting that a disturbed gonadal development is a common underlying mechanism in various, apparently unrelated conditions that are characterized by male subfertility and increased risk for germ cell cancer. The origin and progression of ITGNU lesions in patients with undervirilization syndromes are studied in chapter 4. Aberrant expression of immunohistochemical germ cell markers, a typical finding in young patients with undervirilization has for a long time been interpreted as “a pre-pubertal carcinoma in situ pattern” (i.e. ITGNU), but is now placed in the context of maturation delay of germ cells. This study presents diagnostic tools to discriminate between the benign condition of developmental delay of germ cells and the malignant ITGNU and offers a model for the stepwise progression from the first condition into the second. Chapter 5 focuses on the origin of gonadoblastoma lesions in patients with gonadal dysgenesis. Within these gonads, gonadal differentiation patterns and germ cells which are most prone to malignant transformation are sought, with the aim of identifying high-risk patients. This approach led to the identification of the hitherto undescribed “undifferentiated gonadal tissue” (UGT), a pattern only found in dysgenetic gonads. In this chapter it is hypothesized that gonadoblastomas originate from surviving germ cells residing in UGT. A model for the differential development of the two types of in situ malignant lesions (ITGNU and gonadoblastoma) in patients with disorders of sex development is presented. Chapter 6 describes the progression from the in situ malignant gonadoblastoma cells towards the invasive dysgerminoma. It underscores the heterogeneity of the germ cells regarding their maturation status within a gonadoblastoma lesion, and identifies those germ cells that will escape proliferation-regulating mechanisms and will become invasive. The value and possible pathogenetic relevance of the diagnostic markers OCT3/4 and TSPY in this context becomes apparent from this study. In chapter 7 it is examined to what extent gonadal development and germ cell tumor formation are influenced by the presence of the Y chromosome in patients with gonadal dysgenesis. The results indicate that the peripheral blood and gonadal karyotypes are not a suitable indicator to predict the differentiation patterns present in the dysgenetic gonad, nor do they identify high-risk patients. Chapter 8 offers an extensive and updated review of the literature in order to gain insight in the risk for germ cell cancer in the various types of disorders of sex deelopment. This goal cannot be achieved at present and specific problems in this context are discussed. The results from the studies in the present thesis are interpreted in the light of overcoming these problems in the future. This thesis concludes with the presentation of a new classification system for patients with disorders of sex development, opening perspectives for a better estimation of the risk for germ cell tumor development in the individual patient and thus providing a basis for optimised medical care in accordance with aspirations from patient advocacy groups in the future.

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.