Molecular characterization of sarcomatous change in a granulosa cell tumor.

Sarcomatous transformation of a granulosa cell tumor (GCT) is a rare event. We describe the development of a rapidly progressive sarcomatous change in a woman who had initially presented with a classical GCT. A first recurrence occurred 23 months after the initial diagnosis when she was treated with external beam radiotherapy to her pelvis. A second recurrence 76 months following her initial surgery was consistent with a GCT. At 92 months, she presented with a further recurrence, outside of the radiotherapy field. This last recurrence had a different histologic appearance with features of sarcomatous change. Molecular analysis, using both reverse transcription-polymerase chain reaction and complementary DNA microarrays, has been used to analyze tissue obtained before and after the observed change in the tumor. The data show that GCT-specific genes, such as inhibin alpha, estrogen receptor, and follicle-stimulating hormone receptor, have been downregulated in the sarcomatous change. Significant upregulation of genes associated with an inflammatory response was also noted in the sarcoma, and this was consistent with the presence of a marked inflammatory infiltrate seen on histopathology. This study represents the novel application of microarray technology and demonstrates the unexpected finding of expression of the fibroblast activation protein gene in normal ovary. Although tumors such as this may be targets for the novel fibroblast activating protein-directed chemotherapeutic monoclonal antibody sibrotuzumab, the finding of expression in the normal ovary suggests the need for caution.

An altered antioxidant balance occurs in Down syndrome fetal organs: implications for the "gene dosage effect" hypothesis.

Down syndrome (DS) is the congenital birth defect responsible for the greatest number of individuals with mental retardation. It arises due to trisomy of human chromosome 21 (HSA21) or part thereof. To date there have been limited studies of HSA21 gene expression in trisomy 21 conceptuses. In this study we investigate the expression of the HSA21 antioxidant gene, Cu/Zn-superoxide dismutase-1 (SOD1) in various organs of control and DS aborted conceptuses. We show that SOD1 mRNA levels are elevated in DS brain, lung, heart and thymus. DS livers show decreased SOD1 mRNA expression compared with controls. Since non-HSA21 antioxidant genes are reported to be concomitantly upregulated in certain DS tissues, we examined the expression of glutathione peroxidase-1 (GPX1) in control and DS fetal organs. Interestingly, GPX1 expression was unchanged in the majority of DS organs and decreased in DS livers. We examined the SOD1 to GPX1 mRNA ratio in individual organs, as both enzymes form part of the body's defense against oxidative stress, and because a disproportionate increase of SOD1 to GPX1 results in noxious hydroxyl radical damage. All organs investigated show an approximately 2-fold increase in the SOD1 to GPX1 mRNA ratio. We propose that it is the altered antioxidant ratio that contributes to certain aspects of the DS phenotype.

The inhibins and ovarian cancer.

Interest in inhibin as a marker of ovarian malignancy was stimulated by the description of elevated immunoreactive inhibin levels in the sera of patients with granulosa cell tumours. Several groups have confirmed the value of serum inhibin in the diagnosis and follow-up of patients with this uncommon malignancy. Immunoreactive inhibin levels are also frequently elevated in patients with mucinous cystadenocarcinoma and less frequently in other forms of ovarian tumour. Assay of sera using the specific dimeric inhibin assays has shown that ovarian tumours are able to secrete dimeric inhibin particularly inhibin B. The less specific alpha-subunit directed assays, however, most frequently show elevated concentrations. Used in combination with CA125 as a dual tumour marker, it appears in principle that inhibin can be a useful diagnostic agent. Immunohistochemistry for the inhibin subunits has been reported with increasing frequency as a helpful method to assess suspected ovarian stromal cell tumours. Its diagnostic accuracy for other types of ovarian adenocarcinoma appears less reliable. Expression of the inhibin subunit mRNAs has been demonstrated in a variety of ovarian malignancies. The observation that inhibin levels are elevated in ovarian cancer has stimulated studies of their relevance to the molecular pathogenesis of these malignancies. Findings to date have been largely negative with no evidence for activating mutations of the FSH receptor or of the post-receptor signalling pathway proteins.

[Inhibin and ovarian cancer]. / Ingibin i rak iaichnikov.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumors and in the majority of postmenopausal women with mucinous ovarian cancers. The present report confirms these findings in a larger group of post-menopausal women. Immunohistochemistry for the inhibin alpha. beta A and beta B sununits shows predominantly epithelial staining in granulosa cell tumors and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha i-2 or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Microvessel density and vascular basement membrane immunostaining in tumours of the breast.

There is a well established correlation between increased breast tumour microvessel density (MVD) and reduced prognosis. The aims of this study were to investigate (1) if MVD is elevated in regions other than 'hotspots' of node positive versus node negative breast tumours, and (2) to quantitate the percentage of vessels without vascular basement membrane (VBM) components in high vascular density (HVD) and average vascular density (AVD) regions of node positive and node negative breast tumours. Serial sections were immunostained for CD31 and double-stained for CD31 and collagen IV (CollIV), laminin (LAM) or heparan sulphate proteoglycan (HSPG). Microvessel counts were obtained from HVD and AVD regions and the number of VBM positive vessels were expressed as a percentage of total CD31 positive vessels. MVD was significantly higher in both the HVD and AVD regions of node positive compared with node negative breast tumours (t-test; P < 0.03). The average percent vessels positive for CollIV, LAM or HSPG ranged from 18%-45% and did not differ between node positive and negative breast tumours (t-test; P > 0.05). No differences were observed in VBM immunostaining between regions of HVD and AVD (t-test; P > 0.05). These results demonstrate that vascular density is elevated throughout node positive breast tumours, rather than just in 'hotspots', and show that there is no apparent difference in the percentage of VBM-naked vessels in node positive versus node negative breast tumours.

Inhibin and ovarian cancer.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumours and in the majority of postmenopausal women with mucinous ovarian cancers. The present manuscript confirms these findings in a larger group of postmenopausal women. Immunohistochemistry for the inhibin alpha, betaA and betaB subunits shows predominantly epithelial staining in granulosa cell tumours and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha(i-2) or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Insulin gene expression in adult-onset nesidioblastosis.

Nesidioblastosis is a well-recognized cause of persistent hypoglycaemia in neonates. We describe the case of a 24-year-old woman who presented with hyperinsulinaemic hypoglycaemia in whom an insulinoma could not be identified at operation which resulted in her undergoing a subtotal distal pancreatectomy. Histological examination revealed the presence of nesidioblastosis. The finding of nesidioblastosis in adults has been reported previously, albeit rarely. We report the use of in situ hybridization to characterize the patterns of insulin and proglucagon gene expression in the resected pancreas. Insulin expression was observed in both the islets and also the isolated nesidioblasts. The alpha-cells of the islets had lost their usual peripheral distribution suggesting that not only is insulin gene expression dysregulated but that the islets are structurally abnormal.

Histologic assessment of equivocal cytologic smears.

OBJECTIVE: To determine if the conversion of equivocal cytologic smears into histologic sections would provide additional diagnostic information in those cases in which it was difficult to obtain additional cytologic samples. STUDY DESIGN: Over a year, eight equivocal cytologic smears were converted to histologic sections by removing the coverslips, rehydrating the smears, scraping off the smears into centrifuge tubes, making cell blocks, and sectioning and staining the blocks. RESULTS: The histologic sections enabled cell patterns to be studied and special staining (including immunoperoxidase studies) to be performed. In the eight cases studied, the additional information provided by histology led to a definitive diagnosis in six (75%). CONCLUSION: When cytologic findings are equivocal and it is difficult to obtain additional cytologic samples, conversion of the smears into histologic sections may provide additional information for diagnosis.

Problems in the histological assessment of hydatidiform moles: a study on consensus diagnosis and ploidy status by fluorescent in situ hybridisation.

Hydropic villi in products of conception continue to pose a diagnostic problem for the anatomical pathologist. It is important to distinguish between complete hydatidiform mole (CM), partial hydatidiform mole (PM) and hydropic degeneration (HD), as hydatidiform moles (especially CM) have a tendency to develop persistent trophoblastic disease. Several studies have demonstrated interobserver variability in the diagnosis of the three conditions, but there have been no studies testing the accuracy of the consensus diagnosis of pathologists experienced in the field. In this study four anatomical pathologists with experience in diagnosing hydatidiform moles selected five cases of HD, seven cases of PM and ten cases of CM on the basis of consensus diagnosis using established criteria. Ploidy studies were done on these 22 cases using fluorescent in situ hybridisation. The 15 cases of HD and CM were diploid, confirming the histological diagnosis. However only five of the seven cases of PM were triploid, the other two being diploid. Review of these two diploid cases showed a mixture of small and large villi with moderate to marked trophoblastic proliferation. On the basis of the significant trophoblastic proliferation and the DNA information, the two cases were reclassified as early complete moles. This study demonstrates that even pathologists experienced in the field have difficulty separating PM from CM. The findings suggest that, in the absence of DNA information, a lesion with hydropic villi showing moderate to marked trophoblastic proliferation should be classified as a complete mole, even if there is a mixture of small and large villi. Ploidy studies are an important adjunct to histological diagnosis, especially when there is an overlap of features.

Uterine glandular area during the menstrual cycle and the effects of different in-vitro fertilization related hormonal treatments.

The human uterine glandular epithelium undergoes a sequence of well characterized changes during the menstrual cycle that presumably play an important role in preparation for blastocyst implantation. The aim of this study was to measure objectively glandular volume over the entire menstrual cycle and compare the results with eight different clinical superovulation or hormone replacement therapy (HRT) subject groups. Endometrial biopsies were taken from control normal menstrual cycle subjects (n = 96), and eight other smaller groups of women who had received different in-vitro fertilization (IVF) related treatments. The total area of glandular epithelium was objectively measured from routine histological slides using computerized image analysis. Control menstrual cycle results showed a significantly greater gland area in the early secretory stage of the cycle than at any time between the early proliferative through to the mid-late proliferative stages (P < 0.05). IVF patients receiving clomiphene citrate and human menopausal gonadotrophin had a significantly smaller glandular area than those in the control groups at equivalent stages of the menstrual cycle. The use of progesterone supplementation removed this significant difference. Patients on the ¿Flare' regime had the highest gland area, although this was not significantly different from controls. Buserelin down-regulation gave a gland area that was closest to the normal cycle controls. The three HRT groups showed high variability in gland volume between patients. The results from this study demonstrate that superovulation can cause significant alterations in endometrial gland volume, but that these do not necessarily preclude implantation.

Aberrant FGF-2, FGF-3, FGF-4 and C-erb-B2 gene copy number in human ovarian, breast and endometrial tumours.

The important role of oncogene amplification and tumour suppressor gene deletion in human tumours is becoming increasingly apparent. However, extensive screening of human tumours is required before the prognostic significance of such genetic abnormalities can be fully appreciated. The present investigation describes a rapid non-radioactive and largely automated procedure for the analysis of aberrant gene copy number in large numbers of tissue samples of different human tumours. This procedure is based on the sequential use of the polymerase chain reaction (PCR) and high performance ion exchange liquid chromatography (HPIEX). Using this rapid PCR/HPIEX technique, we have identified amplification and deletion of the FGF-2 gene and the FGF-3, FGF-4 and c-erb-B2 oncogenes in human tumours of the breast, ovary and endometrium. Comparison of the data with tumour pathology has revealed possible associations between aberrant gene copy number and tumour type, invasiveness and metastases.

Immunolocalization of endothelin and neutral endopeptidase in the endometrium of users of subdermally implanted levonorgestrel (Norplant).

Subdermally implanted slow-release levonorgestrel (Norplant), a widely used effective contraceptive, has a high rate of discontinuation due to unacceptable menstrual bleeding disturbances. Endothelin (ET), a potent vasoconstrictor, varies across the menstrual cycle in normal endometrium. It has been proposed that ET has a potential paracrine role in the regulation of uterine blood flow. Neutral endopeptidase (NEP), a membrane-bound ecto-enzyme, can inactivate ET and is localized principally in endometrial stroma. We have compared the immuno-localization of ET and NEP in endometrial biopsies from Indonesian women using Norplant with normal controls. Differences were observed in the glandular and luminal epithelium of Norplant-treated subjects, where ET immunostaining was low while NEP immunoreactivity was increased. The latter may represent a local increase in enzyme activity, potentially explaining the reduced ET immunoreactivity. There was no correlation of ET immuno-reactivity with the duration of implant use or total number of bleeding days. The marked differences in the ET immunostaining pattern in Norplant users, with their increased risk of abnormal uterine bleeding, suggest that ET may be important in controlling menstrual bleeding. Whether endometrial epithelial cell ET has a role as a mitogen in endometrial repair and regeneration, or as a vasoconstrictor important in the cessation of bleeding following menstruation, remains to be determined.

PIP: In Indonesia, 107 women requesting insertion of the contraceptive implant system Norplant at the Klinik Raden Saleh in Jakarta kept a daily menstrual diary for 90 days before endometrial biopsy and allowed venous blood samples to be taken six times in the two-week period also before endometrial biopsy so researchers could examine the roles of endothelin (ET) and neutral endopeptidase (NEP) in menstrual bleeding changes in women using Norplant. They compared the degree of immunostaining in the endometrial biopsy samples of these women with those of 55 controls, most of which came from women in Melbourne, Australia. The endometrium of the Norplant group exhibited low glandular and luminal epithelial ET immunostaining as did the normal endometrium during the proliferative phase. Glandular epithelial ET immunostaining in the Norplant group fell considerably during the secretory or menstrual phases, however. NEP immunoreactivity was greater in the endometrium of Norplant users than in that of controls, suggesting that enzyme activity may have been increased locally among Norplant users. This increase may have accounted for the low ET reactivity. ET immunoreactivity was not related to duration of implant use or total number of bleeding days. These findings indicate that, since Norplant users are at an increased risk of abnormal uterine bleeding, ET may control menstrual bleeding. It is not known whether ET acts as a mitogen in endometrial repair and regeneration or as a vasoconstrictor to stop bleeding after menstruation.

Reproductive and other factors and risk of epithelial ovarian cancer: an Australian case-control study. Survey of Women's Health Study Group.

Of the few factors known to be associated with epithelial ovarian cancer, the most consistently observed relate to women's reproductive function, although even here uncertainties remain. We have undertaken a case-control study involving personal interviews with over 1,600 women, the largest of its kind to date, to investigate further the associations between women's reproductive histories and other factors and the development of ovarian cancer. Cases were drawn from women diagnosed with epithelial ovarian cancer in 3 Australian states, Queensland, New South Wales and Victoria, between August 1990 and December 1993, and controls were drawn at random from the electoral roll, stratified by age and geographic region. Trained interviewers administered standard questionnaires to obtain detailed information about women's reproductive and contraceptive histories and other factors of interest, such as smoking and family history of ovarian or other cancer. Findings were based on data from 824 cases and 860 controls and confirmed the reduced risk of ovarian cancer associated with increasing parity and duration of use of the oral contraceptive pill (OCP), hysterectomy and tubal ligation. The strongest association of all was seen with use of the OCP for 10 years or more. An inverse association between ovarian cancer and age at first birth was observed, but this was not statistically significant. There were no associations between development of ovarian cancer and number of incomplete pregnancies, use of hormone replacement therapy or menstrual history. Among other factors considered, education after leaving school was negatively associated and high body mass index, family history of ovarian cancer, use of talc in the abdominal or perineal region and smoking were positively associated with occurrence of ovarian cancer.

Twin-twin transfusion syndrome: intraamniotic pressure measurement in the assessment of volume reduction at serial amniocenteses.

Two cases of severe twin-twin transfusion syndrome are described. In both, serial amniocenteses were followed by resolution of the disordered inter-twin haemodynamics with 4 intact term survivors. In all reports to date of aggressive reduction with or without successful outcome, volume reduction has been dictated by subjective or semiquantitative ultrasonic estimates of liquor volume. With the use of intraamniotic pressure estimation we describe a more rational basis for the removal of these large volumes of amniotic fluid.

Cerebral sparganosis in an East Timorese refugee.

OBJECTIVE: To report the first case of cerebral sparganosis diagnosed in Australia. CLINICAL FEATURES: A 23-year-old East Timorese refugee, whose diet before migration included raw snakes and frogs, presented with a generalised tonic-clonic seizure and a nine-month history of episodic left hemianaesthesia. Computerised axial tomography of the brain showed a right frontal lesion, which was excised, and histological examination demonstrated changes typical of sparganosis. INTERVENTION AND OUTCOME: Excision of the lesion resulted in cure. Postoperative eosinophilia and a subcutaneous nodule presumed to be due to disseminated sparganosis resolved following a course of praziquantel. CONCLUSION: Clinicians should consider the possibility of unusual parasitic infections in refugees who present with intracranial space-occupying lesions, especially those from developing countries. A dietary history may aid the diagnosis.

Activating point mutations of the Neu oncogene in schwannomas induced by ethylnitrosourea exposure to day-15 and day-18 fetal rats.

Single transplacental exposure of day 15 fetal rats to the carcinogen ethylnitrosourea (ENU) primarily induces tumours of neuroectodermal origin, whereas exposure to ENU at neonatal and adult stages results in tumours arising predominantly from secretory epithelial tissue. Expression of the neu gene is found in fetal neural tissue up until day 16 of gestation, but predominantly only in secretory epithelium after this time. The presence of an activating mutation in the neu oncogene has been associated with these ENU induced neuroectodermal tumours, and on this basis it has been proposed that only the transcriptionally active neu gene is susceptible to ENU induced mutation. In this study we compare the spectrum of tumourigenesis in rats exposed to ENU on days 15 and 18 of gestation. Neuroectodermal rumours were produced at high incidence; mostly schwannomas derived from the peripheral nervous system and some gliomas of the central nervous system. PCR analysis of DNA from these tumours reveals that schwannomas predominantly (29/37=78%) contain a mutated neu gene. This consistent T-->A transversion at codon 671 is known to convert neu into a potent oncogene. Conversely gliomas and various carcinomas do not contain such a mutation (0/13=0%). Our data reveals little difference either in tumour type or activation of the neu oncogene in rats exposed to ENU at these two stages of development. However, we do find that the c-neu gene is expressed at both days 15 and 18 in fetal rat neural tissue; albeit at lower levels by day 18.

Concomitant mucinous tumors of appendix and ovary. Result of a neoplastic field change?

BACKGROUND: Mucinous tumors of the appendix and ovary are known to occur together in association with pseudomyxoma peritonei. It has been postulated that this association may be attributable to the development of independent tumors or to metastasis from one site to another. METHODS AND RESULTS: This article reports two patients with concomitant mucinous ovarian and appendiceal tumors in the absence of pseudomyxoma peritonei. CONCLUSIONS: The evidence suggests that these tumors are independent primary neoplasms that develop as a result of neoplastic field change that affects colonic-type epithelium.

Endothelial cell migratory signal produced by human endometrium during the menstrual cycle.

Human endometrial explants were cultured in a three-dimensional collagen/bovine aortic endothelial cell (BAEC) matrix to measure angiogenic activity, as represented by migration of BAEC towards the explants. The 57 endometrial biopsies were classified by histological appearance into nine stages of the menstrual cycle; five proliferative groupings, three secretory groupings and one menstrual. The BAEC migratory score was taken as the average for 12 explants assayed from each biopsy. The results showed two significant peaks of BAEC migratory activity, one during the early proliferative phase and one during the mid--late proliferative phase. There was a significant drop in the BAEC migratory signal from early--mid-proliferative endometrial explants compared to most other stages of the cycle. The results also show a non-significant rise in BAEC migratory activity in the mid-secretory phase of the cycle. Overall, the results support the concept of two or three different endometrial angiogenic events during the human menstrual cycle, a post-menstrual repair, a mid--late proliferative growth and a lesser mid-secretory activity that may be associated with spiral arteriole growth. Each of these events occurs under different hormonal environments and will need to be investigated separately in terms of local mechanisms controlling angiogenesis.

Correlation of endometrial histology, morphometry, and ultrasound appearance after different stimulation protocols for in vitro fertilization.

The role of endometrial factors in controlling embryo implantation is poorly understood. In the present study, histopathology and morphometry were used to investigate differences in endometrial appearance seen by ultrasound (US) in 107 in vitro fertilization patients receiving different superovulation regimens. Seventy-seven patients received clomiphene citrate (CC)/human menopausal gonadotropin (hMG) and 30 buserelin acetate down regulation/hMG. All patients received an endometrial US at the time of embryo transfer (ET). Endometrial biopsies were taken from 17 women (12 CC/hMG, 5 buserelin acetate/hMG) with fertilization failure at the time when ET would normally have occurred. The morphometry results showed that endometrial glandular volume 2 days after oocyte retrieval was significantly reduced after CC/hMG compared with buserelin acetate/hMG, despite the fact that histopathological dating was similar for both groups. In addition, significant differences in endometrial thickness and echogenicity between CC/hMG and buserelin acetate/hMG were evident by US.

The prognostic significance of grading in borderline mucinous tumors of the ovary.

Fifty-three stage I borderline mucinous tumors of the ovary were placed into four histologic grades according to a simple system of grading based on degree of cell layering, nuclear characteristics, and mitotic count. Three patients died of recurrence and spread of their tumors. All three patients had grade 4 tumors, suggesting that there may be some prognostic value in grading borderline mucinous ovarian tumors.