Effect of 3α-dihydroprogesterone and 5α-dihydroprogesterone on DCIS cells and possible impact for postmenopausal women.

OBJECTIVE: Progesterone metabolites 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αP) have opposite effects on proliferation, apoptosis and metastasis in the breast. Evidence regarding their influence on ductal carcinoma in situ (DCIS) lesions is lacking. METHODS: MCF10DCIS.com cells were cultured in a 3D culture system and treated with 5αP or 3αP. After 5 and 12 days of treatment, polymerase chain reaction (PCR) of proliferation, invasion/metastasis, anti-apoptotic or other markers was performed. Cells treated with the tumor-promoting 5αP were observed under the light and confocal microscopes to reveal possible morphological changes that could indicate a transition from an in situ to an invasive phenotype. As a control, the morphology of the MDA-MB-231 invasive cell line was examined. The invasive potential after exposure to 5αP was also assessed using a detachment assay. RESULTS: The PCR analysis of the chosen markers showed no statistically significant difference between naive cells and cells treated with 5αP or 3αP. DCIS spheroids retained their in situ morphology after treatment with 5αP. The detachment assay showed no increased potential for invasion after exposure to 5αP. Progesterone metabolites 5αP and 3αP do not facilitate or prohibit tumor promotion/invasion in MCF10DCIS.com cells, respectively. CONCLUSION: As oral micronized progesterone has been proved effective for hot flushes in postmenopausal women, first in vitro data propose that progesterone-only therapy could possibly be considered for women after DCIS suffering from hot flushes.

Fertility preservation and management of pregnancy in melanoma patients requiring systemic therapy.

Melanoma is one of the most common cancers in adolescents and adults at fertile age, especially in women. With novel and more effective systemic therapies that began to profoundly change the dismal outcome of melanoma by prolonging overall survival, the wish for fertility preservation or even parenthood has to be considered for a growing portion of melanoma patients-from the patients' as well as from the physicians' perspective. The dual blockade of the mitogen-activated protein kinase pathway by B-Raf proto-oncogene serine/threonine kinase and mitogen-activated protein kinase inhibitors and the immune checkpoint inhibition by anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated protein-4 monoclonal antibodies constitute the current standard systemic approaches to combat locally advanced or metastatic melanoma. Here, the preclinical data and clinical evidence of these systemic therapies are reviewed in terms of their potential gonadotoxicity, teratogenicity, embryotoxicity and fetotoxicity. Recommendations for routine fertility and contraception counseling of melanoma patients at fertile age are provided in line with interdisciplinary recommendations for the diagnostic work-up of these patients and for fertility-protective measures. Differentiated recommendations for the systemic therapy in both the adjuvant and the advanced, metastatic treatment situation are given. In addition, the challenges of pregnancy during systemic melanoma therapy are discussed.

The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro.

STUDY QUESTION: What are the effects of plant-derived antioxidant compounds urolithin A (UA) and B (UB) on the growth and pathogenetic properties of an in vitro endometriosis model? SUMMARY ANSWER: Both urolithins showed inhibitory effects on cell behavior related to the development of endometriosis by differentially affecting growth, adhesion, motility, and invasion of endometriotic cells in vitro. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. As current pharmacological therapies are associated with side effects interfering with fertility, we aimed at finding alternative therapeutics using natural compounds that can be administered for prolonged periods with a favorable side effects profile. STUDY DESIGN, SIZE, DURATION: In vitro cultures of primary endometriotic stromal cells from 6 patients subjected to laparoscopy for benign pathologies with histologically confirmed endometriosis; and immortalized endometrial stromal (St-T1b) and endometriotic epithelial cells (12Z) were utilized to assess the effects of UA and UB on endometriotic cell properties. Results were validated in three-dimensional (3D) in vitro co-culture spheroids of 12Z and primary endometriotic stroma cells of one patient, and organoids from 3 independent donors with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects on cell growth were measured by non-radioactive colorimetric assay to measure cellular metabolic activity as an indicator of cell viability (MTT assay) and flow cytometric cell cycle assay on primary cultures, St-T1b, and 12Z. Apoptosis analyses, the impact on in vitro adhesion, migration, and invasion were evaluated in the cell lines. Moreover, Real-Time Quantitative Reverse Transcription polymerase chain reaction (RT-qPCR) assays were performed on primary cultures, St- T1b and 12Z to evaluate a plausible mechanistic contribution by factors related to proteolysis (matrix metalloproteinase 2, 3 and 9 -MMP2, MMP3, MMP9-, and tissue inhibitor of metalloproteinases -TIMP-1-), cytoskeletal regulators (Ras-related C3 botulinum toxin substrate 1 -RAC1-, Rho-associated coiled-coil containing protein kinase 2 -ROCK2-), and cell adhesion molecules (Syndecan 1 -SDC1-, Integrin alpha V-ITGAV-). Finally, the urolithins effects were evaluated on spheroids and organoids by formation, viability, and drug screen assays. MAIN RESULTS AND THE ROLE OF CHANCE: 40 µM UA and 20 µM UB produced a significant decrease in cell proliferation in the primary endometriotic cell cultures (P < 0.001 and P < 0.01, respectively) and in the St-T1b cell line (P < 0.001 and P < 0.05, respectively). In St-T1b, UA exhibited a mean half-maximum inhibitory concentration (IC50) of 39.88 µM, while UB exhibited a mean IC50 of 79.92 µM. Both 40 µM UA and 20 µM UB produced an increase in cells in the S phase of the cell cycle (P < 0.01 and P < 0.05, respectively). The same concentration of UA also increased the percentage of apoptotic ST-t1b cells (P < 0.05), while both urolithins decreased cell migration after 24 h (P < 0.001 both). Only the addition of 5 µM UB decreased the number of St-T1b adherent cells. TIMP-1 expression was upregulated in response to treating the cells with 40 µM UA (P < 0.05). Regarding the 12Z endometriotic cell line, only 40 µM UA decreased proliferation (P < 0.01); while both 40 µM UA and 20 µM UB produced an increase in cells in the G2/M phase (P < 0.05 and P < 0.01, respectively). In this cell line, UA exhibited a mean IC50 of 40.46 µM, while UB exhibited a mean IC50 of 54.79 µM. UB decreased cell migration (P < 0.05), and decreased the number of adherent cells (P < 0.05). Both 40 µM UA and 20 µM UB significantly decreased the cellular invasion of these cells; and several genes were altered when treating the cells with 40 µM UA and 10 µM UB. The expression of MMP2 was downregulated by UA (P < 0.001), and expression of MMP3 (UA P < 0.001 and UB P < 0.05) and MMP9 (P < 0.05, both) were downregulated by both urolithins. Moreover, UA significantly downregulated ROCK2 (P < 0.05), whereas UB treatment was associated with RAC1 downregulation (P < 0.05). Finally, the matrix adhesion receptors and signaling (co)receptors SDC1 and ITGAV were downregulated upon treatment with either UA or UB (P < 0.01 and P < 0.05, respectively in both cases). Regarding the effects of urolithins on 3D models, we have seen that they significantly decrease the viability of endometriosis spheroids (80 µM UA and UB: P < 0.05 both) as well as affecting their area (40 µM UA: P < 0.05, and 80 µM UA: P < 0.01) and integrity (40 µM UA and UB: P < 0.05, 80 µM UA and UB: P < 0.01). On the other hand, UA and UB significantly inhibited organoid development/outgrowth (40 and 80 µM UA: P < 0.0001 both; 40 µM UB: P < ns-0.05-0.001, and 80 µM UB: P < 0.01-0.001-0.001), and all organoid lines show urolithins sensitivity resulting in decreasing viability (UA exhibited a mean IC50 of 33.93 µM, while UB exhibited a mean IC50 of 52.60 µM). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed on in vitro endometriosis models. WIDER IMPLICATIONS OF THE FINDINGS: These in vitro results provide new insights into the pathogenetic pathways affected by these compounds and mark their use as a potential new therapeutic strategy for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.

Diagnosis of endometriosis in the 21st century.

Endometriosis is a common disease but, due to the wide spectrum of symptoms, diagnosis can be delayed 8-12 years. Laparoscopy is nowadays the gold standard for diagnosis. A less invasive method could shorten the time to diagnosis. The aim of this review is to systematically summarize the literature regarding possible less invasive methods for the diagnosis of endometriosis. Electronic databases, including MEDLINE/PubMed, Cochrane, and Google Scholar, were searched to identify relevant studies; 53 publications contributed to this review. Low invasive tests including imaging, genetic tests, biomarkers, or miRNAs could be the key for establishing a less invasive diagnosis of endometriosis. The findings generally support that different methods can differently contribute to the diagnosis, also depending on the type of endometriosis. For example, transvaginal ultrasound has a sensitivity of 93% and a specificity of 96% in the diagnosis of endometrioma, while superficial/peritoneal endometriosis cannot be detected with imaging processes. Although several non-invasive tests including imaging, genetic tests, biomarkers, or miRNAs show promising diagnostic potential, further research is required before they can be recommended in routine clinical care. The combination of low invasive tests may be the solution to a reliable low invasive diagnosis of endometriosis.

Seminal plasma (SP) induces a rapid transforming growth factor beta 1 (TGFß1)-independent up-regulation of epithelial-mesenchymal transdifferentiation (EMT) and myofibroblastic metaplasia-markers in endometriotic (EM) and endometrial cells.

PURPOSE: To study if short-term exposure (2 h and 6 h) of endometrial/endometriotic tissues and cells to 10% seminal plasma (SP) can induce EMT/metaplasia. METHODS: Basic research experimental study was carried out in a University hospital-based fertility center. Semen samples, peritoneal fluid (PF) from endometriosis patients, endometrial biopsy from premenopausal women, immortalized endometriotic epithelial cell line (12Z), and immortalized endometrial stromal cell line (St-T1b) were studied. Rapid stain identification test (RSID), TGFß1 immunofluorescence of washed sperms, TGFß1-ELISA of SP and PF, in vitro study (2 h and 6 h incubation) and real-time PCR of endometrial tissue and cell lines to analyze gene expression of EMT/metaplasia markers and mediators were done. RESULTS: SP is still detectable in washed semen. TGFß1 was expressed on the plasma membrane of the sperms and was significantly more concentrated in SP (88.17 ng/ml) than PF. 10% SP induced an up-regulation of alpha smooth muscle actin expression in endometrial tissue (p = 0.008) and in 12Z cells (p = 0.05), mostly TGFß1-independent. TWIST expression was persistently significantly down-regulated while Snail1 and 2 were up-regulated, though insignificant. CONCLUSION: Our results provide novel evidence to support that even in semen washed twice, SP is still detectable. The changes in EMT/metaplasia markers and mediators give a new insight into a possible effect of SP on the pathogenesis of endometriosis.

Importance of transvaginal ultrasound applying elastography for identifying deep infiltrating endometriosis - a feasibility study.

PURPOSE: To evaluate the presence of a lesion indicative of endometriosis with transvaginal elastography. MATERIALS AND METHODS: Transvaginal ultrasound and clinical examination were carried out in 48 women with clinical symptoms indicative of endometriosis. In 31 cases strain values were measured at two regions of interest (ROIs) in the Douglas's cul-de-sac during a cycle of compression and decompression with a vaginal probe. RESULTS: A significant difference was found for the ratio of the ROI measuring points in the Douglas' cul-de-sacs of women with a palpable nodule in examination compared to women without a palpable nodule (p = 0.002). CONCLUSION: The ratio of strain values between two ROIs in the Douglas' s cul-de-sac is associated with the presence of an endometriotic lesion. In the future, these findings could allow for a more detailed pre-surgical evaluation and possibly serve as a novel diagnostic tool for predicting deep infiltrating endometriosis.

ESHRE guideline: management of women with endometriosis.

STUDY QUESTION: What is the optimal management of women with endometriosis based on the best available evidence in the literature? SUMMARY ANSWER: Using the structured methodology of the Manual for ESHRE Guideline Development, 83 recommendations were formulated that answered the 22 key questions on optimal management of women with endometriosis. WHAT IS KNOWN ALREADY: The European Society of Human Reproduction and Embryology (ESHRE) guideline for the diagnosis and treatment of endometriosis (2005) has been a reference point for best clinical care in endometriosis for years, but this guideline was in need of updating. STUDY DESIGN, SIZE, DURATION: This guideline was produced by a group of experts in the field using the methodology of the Manual for ESHRE Guideline Development, including a thorough systematic search of the literature, quality assessment of the included papers up to January 2012 and consensus within the guideline group on all recommendations. To ensure input from women with endometriosis, a patient representative was part of the guideline development group. In addition, patient and additional clinical input was collected during the scoping and review phase of the guideline. PARTICIPANTS/MATERIALS, SETTING, METHODS: NA. MAIN RESULTS AND THE ROLE OF CHANCE: The guideline provides 83 recommendations on diagnosis of endometriosis and on the treatment of endometriosis-associated pain and infertility, on the management of women in whom the disease is found incidentally (without pain or infertility), on prevention of recurrence of disease and/or painful symptoms, on treatment of menopausal symptoms in patients with a history of endometriosis and on the possible association of endometriosis and malignancy. LIMITATIONS, REASONS FOR CAUTION: We identified several areas in care of women with endometriosis for which robust evidence is lacking. These areas were addressed by formulating good practice points (GPP), based on the expert opinion of the guideline group members. WIDER IMPLICATIONS OF THE FINDINGS: Since 32 out of the 83 recommendations for the management of women with endometriosis could not be based on high level evidence and therefore were GPP, the guideline group formulated research recommendations to guide future research with the aim of increasing the body of evidence. STUDY FUNDING/COMPETING INTEREST(S): The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the implementation of the guideline. The guideline group members did not receive payment. All guideline group members disclosed any relevant conflicts of interest (see Conflicts of interest). TRIAL REGISTRATION NUMBER: NA.

The prognostic impact of age in patients with triple-negative breast cancer.

The purpose of this study was to assess the prognostic impact of age in patients with triple-negative breast cancer (TNBC). 1,732 patients with primary TNBC were analyzed. Five age cohorts (≤30, 31-40, 41-50, 51-60, and >60 years) at diagnosis were correlated with clinical/pathological parameters. Univariate and multivariate analyses were used to examine the effect of age on disease-free (DFS), distant disease-free (DDFS), and overall survival (OS). In patients with TNBC, increasing age at diagnosis was inversely correlated with tumor grade (P < 0.0001); likelihood of being non-Caucasian (P = 0.0001); likelihood of getting chemotherapy (P < 0.0001); and positively correlated with DFS (P = 0.0003); DDFS (P < 0.0001); and OS (P < 0.0001). The median DFS for patients 31-40 and older than 60 years was 4 years [95 % confidence interval (95 % CI) 2-5] and 8 years (95 % CI 5-14, respectively, P = 0.0003). The DDFS and OS were also statistically significantly shorter for younger patients. In multivariate analysis, tumor size, nodal stage, tumor grade, and age remained significant independent prognostic variables. Clinical characteristics of TNBC differ by age group, patients ≤40 years have poorer survival despite more aggressive systemic therapy.

Optimizing TESE-ICSI by laser-assisted selection of immotile spermatozoa and polarization microscopy for selection of oocytes.

For most azoospermic men testicular sperm extraction (TESE) is the only treatment, however it presents challenges for the ART laboratory, as the retrieval of motile spermatozoa is difficult. In the absence of sperm movement no unequivocal distinction can be made between either dead or immotile, but vital spermatozoa. However, a single laser shot directed to the tip of the tail allows recognition of viability because the flagellum coils at the area of impact. To rank the quality and the maturity of oocytes, polarization microscopy can be used. The zona score and the visualization of the meiotic spindle correlate with implantation and pregnancy rates. We compared 65 TESE-ICSI cycles of the years 2007 and 2008 (Group 1, G1) with 58 TESE-ICSI cycles of the years 2009 and 2010 (Group 2, G2). Testicular spermatozoa were injected according to motility and morphology into selected oocytes. In G1 both, oocyte and spermatozoa were rated using light microscopy only, whereas in G2 the laser was used for sperm selection and the oocytes were rated by light and polarization microscopy. In G2 we enhanced our fertilization rate (FR) significantly in comparison to G1 (G1 42.1% vs. G2 52.7%, p < 0.001). The fertilization rate with immotile, but vital spermatozoa improved significantly when applying laser-based selection (p = 0.006). The laser selection of immotile spermatozoa and the use of polarization microscopy can enhance the FR of TESE-ICSI. No negative effect of the laser was seen on birth rates. The FR with immotile, but vital spermatozoa clearly benefits from laser selection and is a non-hazardous and safe method for the selection of viable but immotile sperm. To our knowledge this is the first report using new technology creating novel endpoints for the analysis of spermatozoa and oocytes in TESE-ICSI.

Chemotherapy-Induced Amenorrhea - An Update.

Because of the heterogeneity in the definition of chemotherapy-induced amenorrhea (CIA) there are distinct differences in the literature with regard to its incidence as well as its dependence on various influencing factors. The occurrence of CIA varies greatly depending on the applied chemotherapy. The pathogenesis of CIA is especially based on a reduction of ovarian reserves. Various sonographic and biochemical factors can be used to exclude or confirm CIA. This is particularly important when an endocrine therapy with tamoxifen is not possible and the use of aromatase inhibitors is under consideration. CIA and especially the frequently thereby resulting early menopause can lead to pronounced restrictions in the quality of life of the affected patients, not least due to the resulting infertility. On the other hand, various studies have shown that CIA may have a positive prognostic significance. Thus, the identification of measures to prevent CIA (for example, through the use of GnRH analogues) is of particular importance.

Role of the CAG repeat polymorphism of the androgen receptor gene in polycystic ovary syndrome (PCOS).

BACKGROUND: Polycystic ovary syndrome (PCOS) is a frequent heterogenic disorder with a familial background. Androgenic effects, determining the clinical features of the syndrome, are mediated by the androgen receptor (AR), whose activity is modulated by a genetic polymorphism. We investigated the role of the CAG repeat polymorphism of the androgen receptor in PCOS. METHODS: In the infertility unit of a university clinic, 72 PCOS patients were compared with 179 ovulatory controls undergoing a standardized diagnostic work-up. The number of CAG repeats was determined by PCR, labelling with IR-800 and PAGE. X-chromosome inactivation was assessed by a methylation-sensitive assay. RESULTS: Compared to controls, PCOS patients displayed a shorter mean CAG repeat length, encoding for higher AR activity (P=0.001). CAG repeat length correlated inversely with oligomenorrhea, a central androgen dependent feature of the syndrome (P=0.005). In a binomial regression analysis including BMI, LH and free testosterone, CAG repeat length was identified as an independent risk factor for PCOS (P=0.002). CONCLUSIONS: The CAG repeat polymorphism could constitute one of the genetic factors modulating the syndrome's phenotype, contributing to its clinical heterogeneity and associated metabolic consequences.

mRNA-expression of ERα, ERß, and PR in clonal stem cell cultures obtained from human endometrial biopsies.

Background. Proliferation and differentiation of the endometrium are regulated by estrogen and progesterone. The enormous regenerative capacity of the endometrium is thought to be based on the activity of adult stem cells. However, information on endocrine regulatory mechanisms in human endometrial stem cells is scarce. In the present study, we investigated the expression of ERα, ERß, and PR in clonal cultures of human endometrial stem cells derived from transcervical biopsies. Methods. Endometrial tissue of 11 patients was obtained by transcervical biopsy. Stromal cell suspensions were plated at clonal density and incubated for 15 days. Expression of ERα, ERß and PR was determined by qPCR prior to and after one cloning round, and normalized to 18 S rRNA expression. Results. Expression of ERα and ERß was downregulated by 64% and 89%, respectively (P = 0.002 and P < 0.001). In contrast, PR was not significantly downregulated, due to a more heterogenous expression pattern. Conclusions. Culture of human endometrial stroma cells results in a downregulation of ERα and ERß, while expression of PR remained unchanged in our patient collective. These results support the hypothesis that stem cells may not be subject to direct stimulation by sex steroids, but rather by paracrine mechanisms within the stem cell niche.

Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant.

Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved-thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2 ± 4.5% (fresh) to 13.6 ± 1.8 (DMSO), 9.5 ± 1.7 (PrOH), or 6.8 ± 1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2 ± 2.5%) and primary follicles (28.1 ± 5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2 ± 3 and 5.4 ± 2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.

miR-145-dependent targeting of junctional adhesion molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness.

Micro RNAs are small non-coding RNAs, which regulate fundamental cellular and developmental processes at the transcriptional and translational level. In breast cancer, miR-145 expression is downregulated compared with healthy control tissue. As several predicted targets of miR-145 potentially regulate cell motility, we aimed at investigating a potential role for miR-145 in breast cancer cell motility and invasiveness. Assisted by Affymetrix array technology, we demonstrate that overexpression of miR-145 in MDA-MB-231, MCF-7, MDA-MB-468 and SK-BR-3 breast cancer cells and in Ishikawa endometrial carcinoma cells leads to a downregulation of the cell-cell adhesion protein JAM-A and of the actin bundling protein fascin. Moreover, podocalyxin and Serpin E1 mRNA levels were downregulated, and gamma-actin, transgelin and MYL9 were upregulated upon miR-145 overexpression. These miR-145-dependent expression changes drastically decreased cancer cell motility, as revealed by time-lapse video microscopy, scratch wound closure assays and matrigel invasion assays. Immunofluorescence microscopy demonstrated restructuring of the actin cytoskeleton and a change in cell morphology by miR-145 overexpression, resulting in a more cortical actin distribution, and reduced actin stress fiber and filopodia formation. Nuclear rotation was observed in 10% of the pre-miR-145 transfected MDA-MB-231 cells, accompanied by a reduction of perinuclear actin. Luciferase activation assays confirmed direct miR-145-dependent regulation of the 3'UTR of JAM-A, whereas siRNA-mediated knockdown of JAM-A expression resulted in decreased motility and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. Our data identify JAM-A and fascin as novel targets of miR-145, firmly establishing a role for miR-145 in modulating breast cancer cell motility. Our data provide a rationale for future miR-145-targeted approaches of antimetastatic cancer therapy.

Prognostic impact of discordance between triple-receptor measurements in primary and recurrent breast cancer.

BACKGROUND: We evaluated discordance in expression measurements for estrogen receptor (ER), progesterone receptor (PR), and HER2 between primary and recurrent tumors in patients with recurrent breast cancer and its effect on prognosis. METHODS: A total of 789 patients with recurrent breast cancer were studied. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH. Repeat markers for ER, PR, and HER2 were available in 28.9%, 27.6%, and 70.0%, respectively. Primary and recurrent tumors were classified as triple receptor-negative breast cancer (TNBC) or receptor-positive breast cancer (RPBC, i.e. expressing at least one receptor). Discordance was correlated with clinical/pathological parameters. RESULTS: Discordance for ER, PR, and HER2 was 18.4%, 40.3%, and 13.6%, respectively. Patients with concordant RPBC had significantly better post-recurrence survival (PRS) than discordant cases; patients with discordant receptor status had similarly unfavorable survival as patients with concordant TNBC. IHC scores for ER and PR showed weak concordance between primary and recurrent tumors. Concordance of HER2-FISH scores was higher. CONCLUSIONS: Concordance of quantitative hormone receptor measurements between primary and recurrent tumors is modest consistent with suboptimal reproducibility of measurement methods, particularly for IHC. Discordant cases have poor survival probably due to inappropriate use of targeted therapies. However, biological change in clinical phenotype cannot be completely excluded.

Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.

Accuracy of classification of breast ultrasound findings based on criteria used for BI-RADS.

OBJECTIVE: To assess the accuracy of categorization of breast ultrasound findings based on scoring for malignancy using the sonographic breast imaging-reporting and data system (BI-RADS). METHODS: Breast ultrasound was performed in 2462 patients between 2001 and 2004 at our unit. Sonographic findings were scored using analog criteria as in BI-RADS for breast ultrasound (mass shape, margin, orientation, posterior acoustic features, lesion boundary, echo pattern). Each lesion was described using these features and classified into categories 1 to 5 according to the BI-RADS for breast ultrasound. Categorization and biopsy results were compared. RESULTS: In twenty-two (0.9%) patients breast ultrasound could not be evaluated because of extreme density of tissue. Normal breast ultrasound belonging to Category 1 was found in 871 (35.4%) patients. Simple cysts classified as Category 2 were observed in 712 (28.9%) women. In 491 (19.9%) patients, apparently benign solid masses (Category 3) were found. Suspicious masses were observed in 225 (9.1%) women and masses highly suggestive of malignancy were found in 141 (5.7%) patients (Categories 4 and 5, respectively). Histological examinations were available from 84 (17.1%) masses that had been classified by BI-RADS as Category 3, in 97 (43.1%) from Category 4 and 106 (75.2%) from Category 5. Accordingly, the rate of malignant findings was 1.2% (n = 1) in Category 3, 17% (n = 16) in Category 4 and 94% (n = 100) in Category 5. CONCLUSION: Scoring breast ultrasound findings for malignancy based on criteria used for BI-RADS breast ultrasound has a high accuracy, comparable to that obtained by BI-RADS for mammography.