Prevalence of c-KIT mutations in gonadoblastoma and dysgerminomas of patients with disorders of sex development (DSD) and ovarian dysgerminomas

Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent similar to 3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY

Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase–polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM

Late Onset Myasthenia Gravis Is Associated with HLA DRB1*15:01 in the Norwegian Population

BACKGROUND: Acquired myasthenia gravis (MG) is a rare antibody-mediated autoimmune disease caused by impaired neuromuscular transmission, leading to abnormal muscle fatigability. The aetiology is complex, including genetic risk factors of the human leukocyte antigen (HLA) complex and unknown environmental factors. Although associations between the HLA complex and MG are well established, not all involved components of the HLA predisposition to this heterogeneous disease have been revealed. Well-powered and comprehensive HLA analyses of subgroups in MG are warranted, especially in late onset MG. METHODOLOGY/PRINCIPAL FINDINGS: This case-control association study is of a large population-based Norwegian cohort of 369 MG patients and 651 healthy controls. We performed comprehensive genotyping of four classical HLA loci (HLA-A, -B, -C and -DRB1) and showed that the DRB1*15:01 allele conferred the strongest risk in late onset MG (LOMG; onset ≥ 60 years) (OR 2.38, p(c)7.4 × 10(-5)). DRB1*13:01 was found to be a protective allele for both early onset MG (EOMG) and LOMG (OR 0.31, p(c) 4.71 × 10(-4)), a finding not previously described. No significant association was found to the DRB1*07:01 allele (p(nc) = 0.18) in a subset of nonthymomatous anti-titin antibody positive LOMG as reported by others. HLA-B*08 was mapped to give the strongest contribution to EOMG, supporting previous studies. CONCLUSION: The results from this study provide important new information concerning the susceptibility of HLA alleles in Caucasian MG, with highlights on DRB1*15:01 as being a major risk allele in LOMG

Back Complaints in the Elders (BACE); design of cohort studies in primary care: an international consortium

Background: Although back complaints are common among older people, limited information is available in the literature about the clinical course of back pain in older people and the identification of older persons at risk for the transition from acute back complaints to chronic back pain. The aim of this study is to assess the course of back complaints and identify prognostic factors for the transition from acute back complaints to chronic back complaints in older people who visit a primary health care physician. Methods/design. The design is a prospective cohort study with one-year follow-up. There will be no interference with usual care. Patients older than 55 years who consult a primary health care physician with a new episode of back complaints will be included in this study. Data will be collected using a questionnaire, physical examination and X-ray at baseline, and follow-up questionnaires afte

Myasthenia gravis

Myasthenia gravis (MG) is a rare, autoimmune neuromuscular junction disorder. Contemporary prevalence rates approach 1/5,000. MG presents with painless, fluctuating, fatigable weakness involving specific muscle groups. Ocular weakness with asymmetric ptosis and binocular diplopia is the most typical initial presentation, while early or isolated oropharyngeal or limb weakness is less common. The course is variable, and most patients with initial ocular weakness develop bulbar or limb weakness within three years of initial symptom onset. MG results from antibody-mediated, T cell-dependent immunologic attack on the endplate region of the postsynaptic membrane. In patients with fatigable muscle weakness, the diagnosis of MG is supported by: 1. pharmacologic testing with edrophonium chloride that elicits unequivocal improvement in strength; 2. electrophysiologic testing with repetitive nerve stimulation (RNS) studies and/or single-fiber electromyography (SFEMG) that demonstrates a primary postsynaptic neuromuscular junctional disorder; and 3. serologic demonstration of acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) antibodies. Differential diagnosis includes congenital myasthenic syndromes, Lambert Eaton syndrome, botulism, organophosphate intoxication, mitochondrial disorders involving progressive external ophthalmoplegia, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), motor neuron disease, and brainstem ischemia. Treatment must be individualized, and may include symptomatic treatment with cholinesterase inhibitors and immune modulation with corticosteroids, azathioprine, cyclosporine, and mycophenolate mofetil. Rapid, temporary improvement may be achieved for myasthenic crises and exacerbations with plasma exchange (PEX) or intravenous immunoglobulin (IVIg). Owing to improved diagnostic testing, immunotherapy, and intensive care, the contemporary prognosis is favorable with less than five percent mortality and nearly normal life expectancy

Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Background: Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods: We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results: Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions: Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency

Intratubular germ cell neoplasia of the human testis:heterogeneous protein expression and relation to invasive potential

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas