ER Immunoreactivity Research Articles

Sweat-gland carcinoma with neuroendocrine differentiation (SCAND): a clinicopathologic study of 13 cases with genetic analysis

Low-grade neuroendocrine carcinoma of the skin (LGNECS) was proposed in 2017 as a new primary cutaneous neoplasm with neuroendocrine differentiation; however, it is not yet well known due to its rarity. Herein, we perform a detailed clinicopathologic analysis of 13 cases as well as panel DNA sequencing in three cases. The study included 12 males and 1 female with a median age of 71 (43–85) years. All lesions occurred on the ventral trunk. The mean tumor size was 2.2 (0.8–11.0) cm. The histopathology resembled that of well-differentiated neuroendocrine tumors (NETs) in other organs, but intraepidermal pagetoid spreading was seen in 8 (61.5%) cases and stromal mucin deposits in 4 (30.8%). Immunoreactivity for CK7, CK19, EMA, BerEP4, CEA, chromogranin A, synaptophysin, INSM1, GCDFP15, GATA3, ER, and bcl-2 were present in varying degrees in all tested cases. PTEN c.165-1G>A splice site mutation was detected by panel sequencing in one case, and GATA3 P409fs*99 and SETD2 R1708fs*4 in another case. Lymph node metastasis was seen significantly in cases with tumor size >2.0 cm [8/8 (100%) vs. 1/5 (20%)]. All three cases with size >3.0 cm were in unresectable advanced-stage [3/3 (100%) vs. 1/10 (10%)], and two of the three patients succumbed to the disease. The two cases of death revealed mild nuclear atypia (mitosis: 1/10 HPFs) and moderate nuclear atypia (2/10 HPFs). Thus, tumor size would be a better prognostic factor than nuclear atypia, mitotic count, and Ki67 index, unlike in NETs. These clinicopathologic and immunohistochemical features would represent the characteristics as skin adnexal tumors with apocrine/eccrine differentiation rather than NETs; therefore, we rename it as sweat-gland carcinoma with neuroendocrine differentiation (SCAND).

TSC2-mutant uterine sarcomas with JAZF1-SUZ12 fusions demonstrate hybrid features of endometrial stromal sarcoma and PEComa and are responsive to mTOR inhibition

Uterine perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm that occasionally shares morphologic and immunohistochemical overlap with low- and high-grade endometrial stromal sarcoma (LGESS and HGESS). In this study, we sought to characterize the clinical, morphologic, genetic, and epigenetic features of five uterine sarcomas that display histologic features of LGESS, HGESS, and PEComa. All tumors demonstrated epithelioid cells often associated with a low-grade spindled component resembling LGESS, with both regions expressing CD10, ER, PR, variable HMB45, and Melan-A immunoreactivity, and strong cathepsin K and pS6 expression. Targeted massively parallel sequencing analysis revealed the presence of somatic TSC2 mutations in all five cases, of which four harbored concurrent or consecutive JAZF1-SUZ12 gene fusions. Unsupervised hierarchical clustering analysis of methylation profiles of TSC2-mutant uterine sarcomas (n = 4), LGESS (n = 10), and HGESS (n = 12) demonstrated two clusters consisting of (1) all LGESS and TSC2-mutant uterine sarcomas and (2) all HGESS. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, calcium, and Rap1 signaling. TSC2-mutant uterine sarcomas were responsive to hormone suppression, and mTOR inhibition demonstrated clinical benefit in four patients with these neoplasms. Our results suggest that these tumors represent histologically distinctive LGESS with TSC2 mutations. TSC2 mutations and JAZF1-SUZ12 fusion may help diagnose these tumors and possibly direct effective treatment.

Mixed endometrial carcinomas with a “low-grade serous”–like component: a clinicopathologic, immunohistochemical, and molecular genetic study

Recently, we reported 2 mixed endometrioid endometrial carcinomas with a "low-grade serous"-like component, which does not fit into any of the 4 molecular groups described by The Cancer Genome Atlas. To understand the nature of these tumors, we have done an immunohistochemical and molecular genetic study of these 2 cases and added a third case. Immunoreactivity for p53, ER, Ki67, WT1, MLH1, PMS2, MSH2, and MSH6 was assessed. Targeted next-generation sequencing for somatic mutations, including genes commonly implicated in carcinogenesis including TP53, KRAS, and PIK3CA, and Sanger sequencing for PTEN and POLE were also performed. All patients were nulliparous and had morbid obesity. Their tumors showed a micropapillary component that resembled that of ovarian low-grade serous carcinoma and merged with villoglandular endometrioid carcinoma. The invasive tumor glands exhibited a microcystic, elongated, or fragmented pattern and contained psammoma bodies. Two tumors showed aberrant p53 expression, and all 3 were positive for ER. All showed KRAS mutations, and TP53 mutations were found in 2 cases. One patient developed peritoneal carcinomatosis, one patient is alive with disease, and another died of a brain tumor. The third patient, whose tumor was confined to the uterus (stage IA), is alive without evidence of disease, but she has been followed for only 6 months. Mixed endometrial carcinomas with a "low-grade" serous-like component exhibit a morphologic spectrum of endometrioid and serous differentiation with microcystic, elongated, or fragmented features; ER expression; KRAS and TP53 mutations; and aggressive behavior.

Primary peritoneal low-grade serous carcinoma in a man: A case report and review of the literature

Primary peritoneal serous carcinoma (PPSC) almost always occurs in women, though rare cases have been reported in men. To date, only three cases of PPSC occurring in male patients have been published in the English literature. Here we describe the fourth case of primary peritoneal serous carcinoma in a male patient and the first showing morphologically and immunophenotypically confirmed low-grade serous histology. In addition to the classic morphologic features of low-grade serous carcinoma, the tumor cells show strong immunoreactivity for CK7, PAX8, WT-1 and ER, and very focal weak immunoreactivity for calretinin and CK5/6, supporting Müllerian serous differentiation. Mild to moderate cytological atypia and a wild-type staining pattern for p53 support a low-grade designation. Absence of a clinically detectable testicular or paratesticular primary tumor supports a primary peritoneal origin. The patient died shortly after his diagnosis; an autopsy was not performed.

Clinical validity of tumor-infiltrating lymphocytes analysis in patients with triple-negative breast cancer

Although tumor-infiltrating lymphocytes (TILs) have been associated with a favorable prognosis in triple-negative breast cancer (TNBC) patients, this marker is not currently considered robust enough for entering the clinical practice. In the present study, we assessed the clinical validity of the guidelines recently issued by the International TIL Working Group in a large retrospective series of well-annotated TNBC patients. TILs were evaluated in all the full-face H&E sections from 897 consecutive TNBC (i.e. tumors with <1% of ER and PgR immunoreactivity and absence of HER2 overexpression or amplification) patients diagnosed and treated at the European Institute of Oncology between 1995 and 2010 (median follow-up 8.2 years, range 6 months to 18 years). All mononuclear cells were evaluated in the stromal area within the borders of the invasive tumor, reported as a percentage value and treated as a continuous variable in survival analysis. The median percentage of TILs was 20%, and 21.9% of the cases had ≥50% (lymphocyte predominant breast cancer, LPBC) TILs. At univariable survival analysis, TILs were a significant predictor of better disease-free survival (DFS), distant disease-free survival (DDFS) and overall survival (OS) (P < 0.0001). Multivariable analysis confirmed that each 10% increase in TILs strongly predicted better survival, independent of patients' age, lymph node status, tumor size, histological grade, peritumoral vascular invasion and Ki-67 labeling index. Patients with LPBC had a 10-year survival rate of 71%, 84% and 96% for DFS, DDFS and OS, respectively. Stratified analysis revealed a positive correlation between TILs and OS across all the subgroups analyzed. Our data support the analytical validity of the recently issued TILs evaluation guidelines in the clinical practice.

Nek2A contributes to tumorigenic growth and possibly functions as potential therapeutic target for human breast cancer

Nek2A (NIMA-related kinases 2A) has been known as an important centrosome regulatory factor. The aim of this study was to investigate the expression of Nek2A and the role it played in different stages of breast cancer. We detected the expression of Nek2A in both mRNA and protein levels in MCF10 cell lines including MCF-10A, MCF-10DCIS.com, MCF-10CA1a and in human breast samples which contained normal breast tissue (NBT), breast ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Our study revealed that the mRNA and protein expression of Nek2A were significantly up-regulated in MCF-10DCIS.com and MCF-10CA1a cell lines as well as in human primary breast cancer tissue (DCIS and IDC). Our study also presented a correlation between Nek2A mRNA expression and some clinic pathological factors. We found that Nek2A mRNA expression was associated with molecular subtypes, ER, PR and Ki-67 immunoreactivity (P<0.05) in DCIS and associated with histological grade, lymph node metastasis, molecular subtypes, c-erbB-2, and Ki-67 expression (P<0.05) in IDC. In addition, we observed that ectopic expression of Nek2A in "normal" immortalized MCF-10A breast epithelial cell resulted in increased Nek2A which lead to abnormal centrosomes. Furthermore, knockdown of Nek2A in MCF-10DCIS.com could remarkably inhibit cell proliferation and induce cell cycle arrest in MCF-10DCIS.com cell line. These data suggested that Nek2A might bear a close relationship with development and progression of breast carcinoma, and highlighted its role as a novel potential biomarker for diagnosis and a possible therapeutic target for human breast cancer especially for DCIS.

Steroidogenic Pathway Proteins Change with Estrous Cycle and Photoperiod, but Are Not Affected by Inhibition of Matrix Metalloproteinases in Siberian Hamster Ovaries.

Restoration of ovarian function as a result of photostimulation corresponds with differential regulation of a family of proteases called matrix metalloproteinases (MMPs) in Siberian hamsters. Blocking MMP action with the in vivo inhibitor GM6001 impedes recrudescence: unlike vehicle treated controls, photostimulation of regressed animals treated with GM6001 fails to increase antral follicle numbers, ovulation, and plasma estradiol concentrations. Because normal recrudescence requires a return of estradiol synthesis and because inhibiting MMPs prevented the normal increase in plasma estradiol, we hypothesized that MMPs may be important in restoring steroidogenesis during photostimulated recrudescence. To test this hypothesis, we examined potential changes in steroidogenic enzymes 3β hydroxysteoid dehydrogenase (3βHSD), 17β hydroxysteoid dehydrogenase (17βHSD), aromatase, as well as estrogen receptor α in: stages of normal cycling hamsters (proestrus-P, estrus-E, diestrus I-DI, diestrus II-DII), hamsters exposed to long-days (LD; 16L:8D), regressed hamsters exposed to short days (SD; 8L:16D) for 14 wks, and regressed hamsters transferred to LD for 2 wks to stimulate recrudescence (PT). Hamsters in LD and PT groups were administered vehicle, IP GM6001, or no treatment daily for 2 wks. Immunoreactivity for 3β HSD protein was observed throughout the estrous cycle, with declines in extent, but not intensity of staining occurring at DII as compared to P, E, and DI (P < 0.05). No changes in 3βHSD immunostaining were noted in LD, SD, or PT ovaries, regardless of treatment with vehicle or GM6001 (P > 0.05). Both extent and intensity of 17β HSD immunostaining declined in DII as compared to other estrous cycle stages (P < 0.05). No changes in 17βHSD immunostaining were noted among LD ovaries, regardless of treatment; however, SD photoperiod decreased extent of immunoreactivity as compared to all groups regardless of treatment (P < 0.05). Aromatase immunostaining was noted throughout the estrous cycle, with declines in intensity and extent in DII as compared to other stages (P < 0.05). Aromatase was present in LD ovaries, and administration of vehicle or GM6001 did not alter intensity/extent of immunostaining (P > 0.05). In contrast, extent of aromatase immunoreactivity declined with SD exposure as compared to all other photoperiod groups (P < 0.05), although administration of vehicle or GM6001 did not affect a return of aromatase immunostaining in PT ovaries (P > 0.05). Finally, extent of immunostaining for ERα peaked in P as compared to DII (P < 0.05). Immunoreactivity for ER was observed in LD at low levels, regardless of treatment. In contrast to steroidogenic enzymes, intensity and extent of ER immunostaining peaked in SD, although administration of vehicle or GM6001 did not alter immunodetection. Our findings confirm and extend the cyclic nature of steroidogenic proteins, with most peaking in the active stages of the estrous cycle. Additionally, the significant decline in plasma estradiol that occurs with SD exposure is concomitant with decreases in later enzymes (17βHSD, aromatase) of steroidogenesis. Finally, although MMPs influence photostimulated return of ovarian function, they do not directly affect the return of ovarian steroidogenesis, as inhibiting MMP action did not affect these steroidogenic enzymes in either cycling LD females, or females undergoing recrudescence. Research supported by NIH 1SC3GM089611 (K.A.Y.) and CSUPERB-Howell Award (J.J.S.). (poster)

Abstract P4-08-01: ESR1 Gene Aberrations Correlate with ER Protein Levels Measured by DCC and IHC

Abstract Background: The Estrogen receptor alfa (ER) is an established biomarker that has been studied in details on the protein and RNA level. Recently, the existence of amplifications and deletions of the ESR1 gene have been documented (1-3), although the frequency of the aberrations has been extensively debated. Here we hypothesize that a positive correlation exists between ESR1 gene copy number and ER protein content measured by both a biochemical ligand assay, dextran coated charcoal (DCC), and immunohistochemistry (IHC). Material and Methods: From 289 primary high-risk breast cancer patients, randomized in the DBCG 77C trial between August 1977 and November 1982, ER data from DCC analyses was available. An ER positive tumor was defined as ≥10 fmol ER/mg protein (4). Archival tumor tissue was available from 257 patients. ESR1 copy number was analyzed with Dako Histology FISH Accessory Kit (K5599, DAKO, Glostrup, Denmark) using a probe covering the ESR1 gene at 6q25 and a centromere 6 reference probe (3). IHC analysis for ER was applied on archival paraffin embedded tissue using the antibody ER1D5 (DAKO), 1:200 with a positive cut off value of 10% ER positive tumor cells. Results: ESR1 FISH analysis was performed successfully in 215 (84%) patients. Amplification (ratio ESR1/CEN-6≥2) was observed in 47 of 215 patients (22%) and ESR1 deletion (ratio ESR1/CEN-6&amp;lt;0.8) was observed in 69 (32%). A positive correlation of ER-DCC with both FISH ESR1 and ER-IHC was found (P&amp;lt;0.0001). The ESR1 amplified tumors had higher average ER-DDC values compared to ESR1 normal tumors (ratio ESR1/ CEN6: 0,80-1,29) and tumors with ESR1 gain (ratio ESR1/CEN6: 1,30-1,99), while deleted tumors had lower ER-DCC values as illustrated in Figure 1. A significant difference p=0.005 was found for the ESR1 deleted tumors compared to the ESR1 amplified tumors. Fig. 1 Box plut showing the ER content according to ESR1/CEN-6 status Discussion: Amplification of the ESR1 gene is associated with higher ER protein content by ER-DCC and more intense immunoreactivity by IHC while ESR1 deletions are associated with decreased content and immunoreactivity compared to tumors with normal ESR1 gene copy numbers. Major variations in ER content and immunoreactivity are however observed within tumors with a normal ESR1 copy number, and other mechanisms than gene aberrations seem to contribute.

Benign mixed tumor of the skin, hypercellular variant: a case report

A 71-year-old man presented with a slowly growing 2.0x2.0x1.0 cm scalp lesion that was surgically removed. Microscopic examination showed a well-circumscribed dermally located tumor composed of ductal elements lined by double to multiple cell layers of bland cuboidal inner cells and elongated spindled outer cells with areas showing cribriform and solid growth patterns. Some cells showed prominent cytoplasmic clearing. A few mitotic figures are noted ranging from 1-2 mitotic figure/10 hpf. There are also foci of squamous differentiation as well as occasional mature adipocytes. The background stroma was predominantly sclerotic with only small area of myxoid background (confirmed by Hale's colloidal iron). Immunohistochemical studies revealed positive immunoreactivity for EMA, CEA, CD117, HWMK, LWMK, CK7, Androgen receptor and S100 in the ductal (epithelial) cells and positive immunereactivity for calponin, SMA, CK 5/6 and p63 in the myoepithelial component. No immunoreactivity for Brst-2, ER, PR and CK20 was noted. MIB-1 showed mildly increased proliferrative index highlighting 5% of the nuclei. The overall morphology and immunohistochemical profile are that of a benign cutanoues mixed tumor (chondroid syringoma). Given the unusual striking celluarlity, we suggest to subclassify this as a hyper-cellular variant.

Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions

BackgroundBreast cancer is the most frequently diagnosed cancer in women. Intraepithelial lesions (IELs), such as usual ductal hyperplasia (UH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors that predict a woman's chance of developing invasive breast cancer. Therefore, a comparative study that establishes an animal model of pre-invasive lesions is needed for the development of preventative measures and effective treatment for both mammary IELs and tumors. The purpose of this study was to characterize the histologic and molecular features of feline mammary IELs and compare them with those in women.MethodsFormalin-fixed, paraffin-embedded specimens (n = 205) from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN), and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine (Sassari, Italy). Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were compared to those of humans. Immunohistochemistry for estrogen receptor (ER-alpha), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu) and Ki-67 was performed in IELs and adjacent tumor tissues.ResultsIntraepithelial lesions were found in 57 of 203 (28%) feline mammary specimens and were categorized as UH (27%), ADH (29%), and DCIS (44%). Most IELs with atypia (ADH and DCIS) were associated with mammary cancer (91%), whereas UH was associated with benign lesions in 53% of cases. Feline IELs were remarkably similar to human IELs. No ER or PR immunoreactivity was detected in intermediate-grade or high-grade DCIS or their associated malignant tumors. HER-2 protein overexpression was found in 27% of IELs.ConclusionThe remarkable similarity of feline mammary IELs to those of humans, with the tendency to lose hormone receptor expression in atypical IELs, supports the cat as a possible model to study ER- and PR-negative breast lesions.

Multifocal matrix-producing mammary carcinoma mimicking lobular carcinoma on core biopsy – A case with long survival reported

We report the clinico-pathologic features of a patient with extensive multifocal matrix-producing mammary carcinoma which mimicked invasive pleomorphic lobular carcinoma on biopsy. Neoplastic cells both in areas with and without matrix were positive for EMA and vimentin. Cytokeratin antibodies were negative, and there was no immunoreactivity for ER, PR, HER2, CD10, GCDF15, p63, or E-cadherin. The proliferative activity was low (<5%) in areas both with and without matrix production. Despite significant tumor volume and high-grade histologic features, an 8-year follow-up has been uneventful.

Papillary lesions of the breast—accuracy of core biopsy

To assess the accuracy of diagnosing papillary breast lesions in core needle biopsy. One hundred biopsy specimens of papillary breast lesions were reviewed and compared with the final excisional diagnoses. The discordant biopsy specimens were stained for oestrogen receptor (ER), cytokeratin (CK) 14 and p63, and these specimens were reclassified based on these results. The overall core biopsy accuracy, false-positive and false-negative rate were 79%, 5% and 16%, respectively. A benign core biopsy specimen diagnosis gave a false-negative rate of 10%, and malignant core biopsy specimen diagnosis did not give any false-positive results. Using homogeneous ER (epithelial), positive CK14 (epithelial) and p63 (myoepithelial) immunoreactivity as benign criteria, the discordant rate was reduced by 30% and 69% when using all three or two of these three criteria for diagnosis. However, false-positive and -negative cases could not be totally eliminated. Immunohistochemistry is helpful in core biopsy diagnosis of papillary breast lesions, but some cases remained misdiagnosed.

Mixed epithelial and stromal tumour (MEST) of the kidney: report of 14 cases with male and PEComatous variants and proposed histopathogenesis

This article adds new cases and variants of MEST with discussion of the histopathogenesis. Fourteen MEST were originally diagnosed as cystic nephroma which represents an incidence of 1.6% of renal neoplasms in adults. In females, the stromal component showed areas of müllerian differentiation with positive immunoreactivity for oestrogen (ER) and progesterone receptors (PR) and CD10. Immunoreactivity for HMB45 was identified in a single case having a leiomyomatous appearance. The epithelial component displayed features of müllerian epithelium and reactive renal tubular cells. In two male cases, MEST consisted of fibrous and smooth muscle stroma and cysts lined only by reactive renal tubular cells. Immunoreactivity for ER and PR was focal and weak. MEST represents a tumour developing from müllerian-like stromal cells in the kidney. The neoplastic stroma encroaches on the renal tubules and has the potential to stimulate the growth of the renal tubules by contact, with development into cysts. Furthermore, the müllerian stroma likely induces the renal tubules to differentiate into müllerian-like epithelium. Melanocytic differentiation of the stroma may occur which represents the PEComatous variant. MESTs in males were histopathologically slightly different from those in females due to the different hormonal milieu.

Oyster estrogen receptor: cDNA cloning and immunolocalization

A cDNA encoding the Pacific oyster, Crassostrea gigas, estrogen receptor (cgER) was cloned using degenerate PCR primers. The open reading frame predicted 485 amino acid residues. Comparisons of the amino acid sequence of cgER with other mollusk ERs show high similarities of the C domain (95–97%), and the E domain (56–66%). The amino acid sequence of the C domain of cgER shows 86 and 89% identity with the respective sequences of human ER-α and ER-β. The amino acid sequence of the E domain of cgER shows 45% identity with those of human ER-α and ER-β. The phylogenetic analysis indicated that the cgER is an ortholog of the other mollusk ERs. In the C domain, the positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. The cgER mRNA was expressed in various tissues including the ovary. Reporter gene assay revealed that cgER is unresponsive to estrogen. This result is similar to those of other mollusk ERs. ER immunoreactivity was localized mainly in the nuclei of follicle cells, the site of vitellogenin synthesis, and in oocytes. This result suggests that cgER could work as a nuclear receptor.

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

Expression of Oestrogen Receptors α and β and of Aromatase in the Testis of Immature and Mature Boars

The boar testis secretes high amounts of oestrogens. In order to test for a likely local significance, we investigated the expression of oestrogen receptors (ER) in immature and mature boar testes using immunohistochemistry (IHC), in vitro and in situ reverse transcription-polymerase chain reaction (RT-PCR). Samples were from 25 boars castrated at ages of 50, 100, 150, 200 and 250 days. Mouse monoclonal primary antibodies against porcine ERalpha (clone HT227), human ERbeta1 (clone PPG5/10) and human P450 aromatase (clone SM1671P) were used. Expression of the mRNA was tested utilizing primers specific for the respective porcine mRNA sequences. ER immunoreactivity was exclusively localized to the nuclei. In immature boars, 90.6 +/- 1.2% of prespermatogonia and 71.0 +/- 2.6% of the Leydig cells showed a strong staining for ERalpha; 95.5 +/- 3.5% of the prespermatogonia but none of the Leydig and Sertoli cells were ERbeta-positive. In mature boars a strong staining for the ERbeta was observed in virtually all Sertoli, Leydig and germ cells, except for the elongating/ed spermatids, which were clearly negative; for the ERalpha, strong immunoreaction signals were restricted to spermatogonia and primary spermatocytes with 93.6 +/- 2.7% of these cells being positive; distinctly less intensive signals were observed in 51.4 +/- 0.27% of the secondary spermatocytes, round spermatids and Leydig cells. In vitro RT-PCR was positive for both receptors and results of in situ RT-PCR matched those obtained by IHC. P450 aromatase immunoreaction was restricted to the cytoplasm of Leydig cells. These findings suggest that testicular ER may be important factors contributing to onset and maintenance of spermatogenesis in the boar.

Expression of EBAG9/RCAS1 is associated with advanced disease in human epithelial ovarian cancer.

Oestrogen receptor-binding fragment associated gene 9, EBAG9, is an oestrogen-responsive gene that was identified in MCF-7 human breast carcinoma cell line. It is identical to RCAS 1, a cancer cell surface antigen possibly involved in immune escape. In the present study, we examined the expression of EBAG9/RCAS1 in human epithelial ovarian cancer using immunohistochemistry, immunoblotting and reverse transcription–polymerase chain reaction (RT–PCR). A total of 90 epithelial ovarian cancer cases were examined immunohistochemically by means of the antibodies for EBAG9 and ERα. The correlation between EBAG9 immunoreactivity and clinicopathological parameters was examined. mRNA expression of EBAG9 and ERα were evaluated by RT–PCR in 22 cases. The expression for EBAG9 and ERα was examined by immunoblotting in 12 ovarian cancer cell lines. EBAG9 immunoreactivity was detected in the surface and cytoplasm of carcinoma cells in 46 out of 90 cases (51.1%). EBAG9 expression was significantly higher in serous histology (P=0.0402) and advanced disease (P=0.0206). No significant relationship was detected between EBAG9 immunoreactivity and overall survival (P=0.689). There was a highly significant correlation between EBAG9 and ER immunoreactivity (P<0.0001). The EBAG9 mRNA was detected in 20 out of 22 cases. In all of the cases that were positive for ERα mRNA, they were also positive for EBAG9 mRNA. Immunoreactive band corresponding to EBAG9 was detected in 11 out of 12 of ovarian cancer cell lines, and was consistent with ERα expression. In conclusion, the wide distribution of EBAG9 and its relation to advanced disease suggest that this protein may play important roles in epithelial ovarian cancer.

Parallel changes between the percentage of fetal pituitary cells immunoreactive to oestrogen receptor α and the concentration of 17β-oestradiol in fetal and maternal plasma during gestation in sheep

The present study was designed to investigate the relationship between fetal sheep pituitary oestrogen receptor (ER) alpha expression and changes in fetal and maternal plasma 17beta-oestradiol (E2) concentrations during gestation. The results revealed that immunoreactivity for ER was located in the nuclei and distributed throughout the fetal pituitary gland during gestation. The percentage of ERalpha-positive cells was approximately 2% of the total cell population in female fetuses at Day 60 of gestation, increased to approximately 7% and 13% of the total cell population at Days 90 and 120 of gestation, respectively, and then declined to approximately 10% at birth. The fetal plasma E2 concentrations were approximately 19 and 71 pg mL(-1) at Days 90 and 120 of gestation, respectively, and decreased to 22 pg mL(-1) after birth. In male fetuses, plasma E2 concentrations and the percentage of ERalpha-positive cells were similar to values in female fetuses throughout gestation, except on Day 120 when the plasma E2 level in female fetuses was significantly higher than in male fetuses. These data demonstrate that changes in the percentage of fetal pituitary ERalpha-positive cells parallel fetal plasma E2 concentrations throughout gestation.

Dysregulated PTEN-PKB and negative receptor status in human breast cancer.

Recent studies demonstrate that abnormalities in PTEN may be one of the most frequent genetic events observed in human cancers. PTEN dysfunction leads to tumorigenesis through unopposed survival signals mediated via activated protein kinase B (PKB), which may also be associated with hormone-independence. We therefore investigated the relationship between PTEN-PKB and receptor status in human breast cancer. Several molecular variables, including immunohistochemical staining for PTEN, PKB (phosphorylated on ser473), p53 and p21 were evaluated. The p53 gene was sequenced from exons 2-11. Seventy-eight participants in a randomised breast cancer trial served as the cohort for our study. Twenty-eight of 77 (36%) patients' tumours demonstrated absent or reduced PTEN expression; 17 of 78 (22%) tumours over-expressed P-PKB. A significant inverse relationship was observed between reduced PTEN and increased P-PKB expression. Reduced PTEN also correlated with reduced ER or PR expression. None of the molecular variables correlated with survival. ER and PR negative tumours, however, experienced a significantly inferior disease-free survival than other ER/PR status tumours. Immunohistochemical analyses of ER expression in mammary carcinomas arising in PTEN heterozygous knockout mice did not demonstrate a reduction in ER immunoreactivity, in comparison to wild-type mice. Our data demonstrate that the PTEN-PKB pathway is abnormal in approximately 1/3 of lymph node negative breast cancer. Dysregulated PTEN-PKB was also associated with reduced ER/PR expression, but this does not appear to be a simple direct causal relationship. These observations support the contention that dysregulation in PTEN-PKB contributes to disease progression and hormone resistance of human breast cancer.

Adenocarcinomas arising from uterine adenomyosis: a report of four cases.

Adenocarcinomas arising from adenomyosis uteri are rare. This study reports four such cases and characterizes them clinically and microscopically. In all four patients, the endometrial cytology was negative, and MR imaging and ultrasound sonography did not detect the tumors preoperatively. The histological subtypes of the four tumors were endometrioid (one grade 1, one grade 3), serous, and clear cell. In three cases, the adenocarcinomas were present exclusively in the myometrium, and a transition between the carcinomas and the adenomyotic glands was observed in all cases. The eutopic endometrium was normal except in one case in which there was a small focus of invasive carcinoma. In two of four cases, pelvic or paraaortic lymph node metastases were present. In the carcinomas, ER immunoreactivity was not found in any tumor and PR positivity was found in only one tumor. In contrast, p53 immunopositivity was found in three of four carcinomas. Adenocarcinomas arising from adenomyosis are difficult to diagnose preoperatively, and their aggressive behavior in some cases seems to be related to the histological subtype.