Eleftherios P. Samartzis

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast

Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade‐ and oestrogen receptor (ER)‐matched invasive carcinomas of no special type (IC‐NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray‐based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC‐NSTs, and recurrent mutations affecting mitogen‐activated protein kinase family genes and NBPF10. RNA‐sequencing analysis identified 17 high‐confidence fusion genes, eight of which were validated and two of which were in‐frame. No recurrent fusions were identified in an independent series of MPCs and IC‐NSTs. Forced expression of in‐frame fusion genes (SLC2A1–FAF1 and BCAS4–AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out‐of‐frame rearrangements was found in one MPC and in 13% of HER2‐positive breast cancers, identified through a re‐analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild‐type CDK12 in a CDK12‐null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities.

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.

Loss of ARID1A /BAF250a-expression in endometriosis: a biomarker for risk of carcinogenic transformation?

Mutations of the tumor-suppressor gene ARID1A result in the loss of protein expression of the BRG-associated factor 250a (BAF250a), a large subunit of transcription-regulating Human SWI/SNF complexes, which have an important role in the control of cell proliferation and tumor suppression. ARID1A mutations are particularly frequent in endometriosis-associated ovarian clear cell and endometrioid carcinomas, and were recently described as a possible key mechanism and early step in the transformation of endometriosis into cancer. Here, we examined the immunohistochemical expression pattern of BAF250a in a tissue microarray including 74 endometriosis and 30 endometrium samples. Ovarian cancer samples (n=136) served as a control. Epithelial BAF250a expression was assessable in 90/104 (87%) and stromal BAF250a expression in 95/104 (91%) of the endometriosis, and endometrium cases due to lack of adequate tissue in some spots. Complete lack of BAF250a expression was observed in three endometriomas (n=3/20, 15%) and one deep-infiltrating endometriosis sample (n=1/22, 5%), but in none of the peritoneal endometriosis (n=0/16) and eutopic endometrium samples (n=0/30). A comparison of the mean immunoreactivity scores revealed a significantly lower expression rate of BAF250a in endometriomas compared with normal endometrium (P<0.0005), as well as peritoneal (P=0.003) and deep-infiltrating endometriosis (P=0.02). Our data demonstrates that a complete loss of BAF250a expression is observable in some endometriotic lesions, especially in endometriomas. In addition, we report that a partial loss of BAF250a expression is occurring in the form of cell clusters indicating a clonal loss of BAF250a expression in these cells. The loss of expression of the tumor-suppressor protein BAF250a in some endometriomas possibly indicates a risk of malignant transformation in these cases, which could be of importance in the determination of individual treatment strategies. However, its role and value as a prognostic parameter in endometriosis needs to be further studied.

Effect of MRE11 Loss on PARP-Inhibitor Sensitivity in Endometrial Cancer In Vitro

Aim of the study To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment. Methods MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment. Results Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells. Conclusion Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

Protein expression of cancer testis antigens predicts tumor recurrence and treatment response to imatinib in gastrointestinal stromal tumors

Cancer testis antigens (CTAs) have been identified in various tumors as immunological tumor targets. In gastrointestinal stromal tumor (GIST), the prediction of malignant potential remains difficult but is crucial in the era of adjuvant imatinib treatment. Here, we analyzed the impact of CTAs on tumor recurrence and its role on the treatment response to imatinib. The expression of the most frequent CTAs MAGE‐A1, MAGE‐A3, MAGE‐A4, MAGE‐C1 and NY‐ESO‐1 was analyzed by immunohistochemistry. The duration between the initial operation and the tumor relapse was defined as recurrence free survival (RFS). All recurrent cases were treated with imatinib. The tumor response to imatinib was graded according to the modified CT response evaluation criteria. Patients with a CTA positive GIST (n = 23, 27%) had a significantly shorter RFS (p = 0.001) compared to negative cases (n = 63, 73%). The median RFS was 25 months in CTA positive patients and was not reached during the study period in CTA negative patients. According to the established staging criteria CTA positive tumors were predominantly high‐risk tumors (p = 0.001). The expression of MAGE‐A3 (p = 0.018) and NY‐ESO‐1 (p = 0.001) were associated with tumor progression under imatinib treatment. A tendency for worse tumor response to imatinib was observed in CTA positive tumors (p = 0.056). Our study confirms the expression of CTAs in GIST and their role as prognostic markers. It also draws attention to the potential impact of CTAs on the tumor response to imatinib.

The G protein-coupled estrogen receptor (GPER) is expressed in two different subcellular localizations reflecting distinct tumor properties in breast cancer

Introduction The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival. Methods The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy. Results A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012). Conclusion Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment.

Expression of the G protein-coupled estrogen receptor (GPER) in endometriosis: a tissue microarray study

BackgroundThe G protein-coupled estrogen receptor (GPER) is thought to be involved in non-genomic estrogen responses as well as processes such as cell proliferation and migration. In this study, we analyzed GPER expression patterns from endometriosis samples and normal endometrial tissue samples and compared these expression profiles to those of the classical sex hormone receptors.MethodsA tissue microarray, which included 74 samples from different types of endometriosis (27 ovarian, 19 peritoneal and 28 deep-infiltrating) and 30 samples from normal endometrial tissue, was used to compare the expression levels of the GPER, estrogen receptor (ER)-alpha, ER-beta and progesterone receptor (PR). The immunoreactive score (IRS) was calculated separately for epithelium and stroma as the product of the staining intensity and the percentage of positive cells. The expression levels of the hormonal receptors were dichotomized into low (IRS < 6) and high (IRS > =6) expression groups.ResultsThe mean epithelial IRS (+/−standard deviation, range) of cytoplasmic GPER expression was 1.2 (+/−1.7, 0–4) in normal endometrium and 5.1 (+/−3.5, 0–12) in endometriosis (p < 0.001), of nuclear GPER 6.4 (+/−2.6, 0–12) and 6.8 (+/−2.9, 2–12; p = 0.71), of ER-alpha 10.6 (+/−2.4, 3–12) and 9.8 (+/−3.0, 2–12; p = 0.26), of ER-beta 2.4 (+/−2.2; 0–8) and 5.6 (+/−2.6; 0–10; p < 0.001), and of PR 11.5 (+/−1.7; 3–12) and 8.1 (+/−4.5; 0–12; p < 0.001), respectively. The mean stromal IRS of nuclear GPER expression was 7.7 (+/−3.0; 2–12) in endometrium and 10.8 (+/−1.7; 6–12) in endometriosis (p < 0.001), of ER-alpha 8.7 (+/−3.1; 2–12) and 10.6 (+/−2.4; 2–12; p = 0.001), of ER-beta 1.8 (+/−2.0; 0–8) and 5.4 (+/−2.5; 0–10; p < 0.001), and of PR 11.7 (+/−0.9; 8–12) and 10.9 (+/−2.0; 3–12; p = 0.044), respectively. Cytoplasmic GPER expression was not detectable in the stroma of endometrium and endometriosis. The observed frequency of high epithelial cytoplasmic GPER expression levels was 50% (n = 30/60) in the endometriosis and none (0/30) in the normal endometrium samples (p < 0.001). High epithelial cytoplasmic GPER expression levels were more frequent in endometriomas (14/20, 70%; p = 0.01), as compared to peritoneal (9/18, 50%) or deep-infiltrating endometriotic lesions (7/22, 31.8%). The frequency of high stromal nuclear GPER expression levels was 100% (n = 74/74) in endometriosis and 76.7% (n = 23/30) in normal endometrium (p < 0.001). The frequency of high epithelial nuclear GPER expression levels did not differ between endometriosis and normal endometrium.ConclusionsThe present data indicate a unique GPER expression pattern in endometriosis, especially in endometriomas as compared to the normal endometrium. The overexpression of GPER in endometriotic lesions suggests a potential role for GPER in the hormonal regulation of endometriosis, which should be taken into consideration for future hormonal treatment strategies.

The Expression of Histone Deacetylase 1, But Not Other Class I Histone Deacetylases, Is Significantly Increased in Endometriosis

Class I histone deacetylases (HDACs-1-3) play an important role in steroid hormone–dependent gene expression and in modulating cell survival and proliferation. We analyzed their expression in a tissue microarray including 74 endometriosis samples and 30 normal endometrium controls. The mean HDAC-1 immunoreactivity score (IRS ± standard deviation) was 7.6 ± 2.5 in endometriosis and 5.3 ± 2.3 in normal endometrium (P < .001). In contrast, the IRSs of HDAC-2 and -3 were 11.7 ± 0.7 and 11.8 ± 1.1 in endometriosis and 11.6 ± 1.0 and 11.9 ± 0.4 in normal endometrium (P = .7 and P = .2), respectively. Significant correlations were found between HDAC-1 and estrogen (-alpha/-beta) and progesterone receptor expression. In conclusion, HDAC-1, but not HDAC-2/-3, was significantly increased in endometriosis and associated with steroid hormone receptor expression that may reflect interdependence. In context with the literature, specific inhibitors of HDAC-1 may have inhibitory activities similar to those of broad-spectrum HDAC inhibitors and may be clinically tolerated, which would increase their chance as an option in the treatment of endometriosis.

Six years after deregulation of emergency contraception in Switzerland: Has free access induced changes in the profile of clients attending an emergency pharmacy in Zürich?

ABSTRACT Objectives Emergency contraception (EC) has been freely accessible in Swiss pharmacies since November 2002. Today some groups are still concerned that free access might result in less use of efficient contraceptive methods, overuse and more risky sexual behaviour. Methods Profiles of EC users one and six years after deregulation were analysed with regard to age, contraceptive methods used, reasons for EC use, and last contact with a gynaecologist. Data were collected from a centrally located pharmacy. Written official assessment forms concerning 1500 women (750 in 2004 and 750 in 2009) were analysed. Results Free access to EC use had no impact on regular contraceptive behaviour. The percentage of pill and condom users was very high (85%). The percentage of EC-users without any contraception (17–18%) was the same over the years. In 2009, condom rupture was reported more frequently (p < 0.001). In addition significantly more women had used EC previously in their history. Conclusion Free access to EC has not resulted in less use of efficient contraceptive methods. In the context of falling abortion rates our results are reassuring. This also applies to adolescents, who mainly used EC as a back-up method and seldom in the context of unprotected intercourse.

Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study

BackgroundEpigenetic regulation is an important mechanism leading to cancer initiation and promotion. Histone acetylation by histone deacetylases (HDACs) represents an important part of it. The development of HDAC inhibitors has identified the utility of HDACs as a therapeutic target. Little is known about the epigenetic regulation of vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell cancer (VSCC). In this study, the expression of class I HDACs (HDAC 1, 2 and 3) was compared in a series of VIN and VSCC tissues.MethodsA tissue micro array (TMA) with specimens from 106 patients with high-grade VIN and 59 patients with vulvar cancer was constructed. The expression of HDACs 1, 2 and 3 were analyzed with immunohistochemistry (IHC). The nuclear expression pattern was evaluated in terms of intensity and percentage of stained nuclei and was compared between vulvar preinvasive lesions and vulvar cancer.ResultsHDAC 2 expression was significantly higher in VIN than in VSCC (p < 0.001, Fishers test). Also, 88.7% (n = 94/106) of VIN samples and only 54.5% (n = 31/57) of VSCC samples were scored at the maximum level. Conversely, HDAC 3 expression was significantly higher in VSCC (93%, 53/57) compared to VIN (73.6%, 78/106, p = 0.003), whereas only a small difference in the expression of HDAC 1 was found between these two entities of vulvar neoplasia.ConclusionsThese results suggest that epigenetic regulation plays a considerable role in the transformation of VIN to invasive vulvar neoplasia.