Distinct Expression Levels and Patterns of Stem Cell Marker,Aldehyde Dehydrogenase Isoform 1 (ALDH1), in Human Epithelial Cancers

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn''t significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDH^br tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.     

ZNF385B and VEGFA Are Strongly Differentially Expressed in Serous Ovarian Carcinomas and Correlate with Survival

Background

The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods

Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results

Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = −130.5, p = 1.2×10⁻⁷) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10⁻⁶) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10⁻¹⁰ and FC = 46.2, p = 5.6×10⁻⁴, respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions

We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.     

Molecular pathology of ovarian cancer

Ovarian cancer is the leading cause of death among women with gynecologic malignancies. Epithelial tumors typically constitute 80-90% of ovarian malignancies and are classified primarily as serous, mucinous, endometrioid or clear cell. Current data indicate that each of these histologic subtypes is associated with distinct morphologic and molecular genetic alterations. We describe such genetic alterations with specific reference to histologic subtypes.     

Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.     

Statin Therapy Is Associated with Improved Survival in Patients with Non-Serous-Papillary Epithelial Ovarian Cancer: A Retrospective Cohort Analysis

Aim

To determine whether statin use is associated with improved epithelial ovarian cancer (OvCa) survival.

Methods

This is a single-institution retrospective cohort review of patients treated for OvCa between 1992 and 2013. Inclusion criteria were International Federation of Gynecology and Obstetrics (FIGO) stage I–IV OvCa. The primary exposures analyzed were hyperlipidemia and statin use. The primary outcomes were progression-free survival (PFS) and disease-specific survival (DSS).

Results

442 patients met inclusion criteria. The cohort was divided into three groups: patients with hyperlipidemia who used statins (n = 68), patients with hyperlipidemia who did not use statins (n = 28), and patients without hyperlipidemia (n = 346). OvCa outcomes were evaluated. When we analyzed the entire cohort, we found no significant differences in PFS or DSS among the groups. The median PFS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 21.7, 13.6, and 14.7 months, respectively (p = 0.69). Median DSS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 44.2, 75.7, and 41.5 months, respectively (p = 0.43). These findings did not change after controlling for confounders. However, a secondary analysis revealed that, among patients with non-serous-papillary subtypes of OvCa, statin use was associated with a decrease in hazards of both disease recurrence (adjusted HR = 0.23, p = 0.02) and disease-specific death (adjusted HR = 0.23, p = 0.04). To augment the findings in the retrospective cohort, the histology-specific effects of statins were also evaluated in vitro using proliferation assays. Here, statin treatment of cell lines resulted in a variable level of cytotoxicity.

Conclusion

Statin use among patients with non-serous-papillary OvCa was associated with improvement in both PFS and DSS.     

Sex Hormone-Binding Globulin (SHBG) Expression in Ovarian Carcinomas and Its Clinicopathological Associations

Sex hormone-binding globulin (SHBG) is known as a carrier protein. It is classically thought to be mainly synthesized in the liver and then secreted into the circulating system, where it binds to sex steroids with a high affinity and modulates the bio-availability of the hormones. Other organs known to produce SHBG include brain, uterus, testis, prostate, breast and ovary, and the local expressed SHBG may play an important role in tumor development. However, SHBG expression status and its clinicopathological significance in ovarian cancer cells are not reported yet. In our present study, we examined and found the variable SHBG expression in four ovarian cancer cell lines (OV-90, OVCAR-3, SKOV-3 and ES-2) by immunocytochemistry and Western blotting. We then extended our study to 248 ovarian carcinoma samples, which were collected at The Norwegian Radium Hospital, Oslo University Hospital with complete clinical information, and discovered that SHBG was variably expressed in these ovarian carcinomas. Higher level of SHBG expression was significantly associated with more aggressive histological subtype (p = 0.022), higher FIGO stage (p = 0.018) and higher histological grade (grade of differentiation, p = 0.020), although association between SHBG expression and OS/PFS was not observed. Our results demonstrate that ovarian cancer cells produce SHBG and higher SHBG expression in ovarian carcinoma is associated with unfavorable clinicopathological features.     

Risk of Ovarian Cancer and Inherited Variants in Relapse-Associated Genes

Background

We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk.

Methods and Findings

Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66–0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02–1.91).

Conclusions

Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.     

Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

Impact of Risk Factors on Different Interval Cancer Subtypes in a Population-Based Breast Cancer Screening Programme

Background

Interval cancers are primary breast cancers diagnosed in women after a negative screening test and before the next screening invitation. Our aim was to evaluate risk factors for interval cancer and their subtypes and to compare the risk factors identified with those associated with incident screen-detected cancers.

Methods

We analyzed data from 645,764 women participating in the Spanish breast cancer screening program from 2000–2006 and followed-up until 2009. A total of 5,309 screen-detected and 1,653 interval cancers were diagnosed. Among the latter, 1,012 could be classified on the basis of findings in screening and diagnostic mammograms, consisting of 489 true interval cancers (48.2%), 235 false-negatives (23.2%), 172 minimal-signs (17.2%) and 114 occult tumors (11.3%). Information on the screening protocol and women''s characteristics were obtained from the screening program registry. Cause-specific Cox regression models were used to estimate the hazard ratios (HR) of risks factors for interval cancer and incident screen-detected cancer. A multinomial regression model, using screen-detected tumors as a reference group, was used to assess the effect of breast density and other factors on the occurrence of interval cancer subtypes.

Results

A previous false-positive was the main risk factor for interval cancer (HR = 2.71, 95%CI: 2.28–3.23); this risk was higher for false-negatives (HR = 8.79, 95%CI: 6.24–12.40) than for true interval cancer (HR = 2.26, 95%CI: 1.59–3.21). A family history of breast cancer was associated with true intervals (HR = 2.11, 95%CI: 1.60–2.78), previous benign biopsy with a false-negatives (HR = 1.83, 95%CI: 1.23–2.71). High breast density was mainly associated with occult tumors (RRR = 4.92, 95%CI: 2.58–9.38), followed by true intervals (RRR = 1.67, 95%CI: 1.18–2.36) and false-negatives (RRR = 1.58, 95%CI: 1.00–2.49).

Conclusion

The role of women''s characteristics differs among interval cancer subtypes. This information could be useful to improve effectiveness of breast cancer screening programmes and to better classify subgroups of women with different risks of developing cancer.     

Sperm Recovery and IVF after Testicular Sperm Extraction (TESE): Effect of Male Diagnosis and Use of Off-Site Surgical Centers on Sperm Recovery and IVF

Objective

Determine whether testicular sperm extractions and pregnancy outcomes are influenced by male and female infertility diagnoses, location of surgical center and time to cryopreservation.

Patients

One hundred and thirty men undergoing testicular sperm extraction and 76 couples undergoing 123 in vitro fertilization cycles with testicular sperm.

Outcome Measures

Successful sperm recovery defined as 1–2 sperm/0.5 mL by diagnosis including obstructive azoospermia (n = 60), non-obstructive azoospermia (n = 39), cancer (n = 14), paralysis (n = 7) and other (n = 10). Obstructive azoospermia was analyzed as congenital absence of the vas deferens (n = 22), vasectomy or failed vasectomy reversal (n = 37) and “other”(n = 1). Sperm recovery was also evaluated by surgical site including infertility clinic (n = 54), hospital operating room (n = 67) and physician’s office (n = 11). Treatment cycles were evaluated for number of oocytes, fertilization, embryo quality, implantation rate and clinical/ongoing pregnancies as related to male diagnosis, female diagnosis, and use of fresh or cryopreserved testicular sperm.

Results

Testicular sperm recovery from azoospermic males with all diagnoses was high (70 to 100%) except non-obstructive azoospermia (31%) and was not influenced by distance from surgical center to laboratory. Following in vitro fertilization, rate of fertilization was significantly lower with non-obstructive azoospermia (43%, p = <0.0001) compared to other male diagnoses (66%, p = <0.0001, 59% p = 0.015). No differences were noted in clinical pregnancy rate by male diagnosis; however, the delivery rate per cycle was significantly higher with obstructive azoospermia (38% p = 0.0371) compared to diagnoses of cancer, paralysis or other (16.7%). Women diagnosed with diminished ovarian reserve had a reduced clinical pregnancy rate (7.4% p = 0.007) compared to those with other diagnoses (44%).

Conclusion

Testicular sperm extraction is a safe and effective option regardless of the etiology of the azoospermia. The type of surgical center and/or its distance from the laboratory was not related to success. Men with non-obstructive azoospermia have a lower chance of successful sperm retrieval and fertilization.     

The Local Effects of Ovarian Diathermy in an Ovine Model of Polycystic Ovary Syndrome

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30–100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.     

Renal Involvement in Non-Hodgkin Lymphoma: Proven by Renal Biopsy

Aims

To determine the spectrum of renal lesions in patients with kidney involvement in non-Hodgkin''s lymphoma (NHL) by renal biopsy.

Methods

The clinical features and histological findings at the time of the renal biopsy were assessed for each patient.

Results

We identified 20 patients with NHL and renal involvement, and the diagnosis of NHL was established following the kidney biopsy in 18 (90%) patients. The types of NHL include the following: chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 8), diffuse large B-cell lymphoma (n = 4), T/NK cell lymphoma (n = 3), lymphoplasmacytic lymphoma (n = 2), cutaneous T-cell lymphoma (n = 1), mucosa-associated lymphoid tissue lymphoma (n = 1) and mantle cell lymphoma (n = 1). All presented with proteinuria, and 15 patients had impaired renal function. The pathological findings included (1) membranoproliferative glomerulonephritis-like pattern in seven patients; (2) crescent glomerulonephritis in four; (3) minimal-change disease in three, and glomeruli without specific pathological abnormalities in three; (4) intraglomerular large B-cell lymphoma in one; (5) intracapillary monoclonal IgM deposits in one; (6) primary diffuse large B-cell lymphoma of the kidneys in one; and (7) lymphoma infiltration of the kidney in eight patients.

Conclusion

A wide spectrum of renal lesions can be observed in patients with NHL, and NHL may be first proven by renal biopsies for evaluation of kidney injury or proteinuria. Renal biopsy is necessary to establish the underlying cause of renal involvement in NHL.     

Deregulation of Microcephalin and ASPM Expression Are Correlated with Epithelial Ovarian Cancer Progression

Mutations in the MCPH1 (Microcephalin) and ASPM (abnormal spindle-like microcephaly associated) genes cause primary microcephaly. Both are centrosomal associated proteins involved in mitosis. Microcephalin plays an important role in DNA damage response and ASPM is required for correct division of proliferative neuro-epithelial cells of the developing brain. Reduced MCPH1 mRNA expression and ASPM mRNA over-expression have been implicated in the development of human carcinomas. Epithelial ovarian cancer (EOC) is characterised by highly aneuploid tumours. Previously we have reported low Microcephalin and high ASPM protein levels and associations with clinico-pathological parameters in malignant cells from ascitic fluids. To confirm these previous findings on a larger scale Microcephalin and ASPM expression levels and localisations were evaluated by immunohistochemistry in two cohorts; a training set of 25 samples and a validation set of 322 EOC tissue samples. Results were correlated to the associated histopathological data. In normal ovarian tissues the Microcephalin nuclear staining pattern was consistently strong. In the cancer tissues, we identified low nuclear Microcephalin expression in high grade and advanced stage tumours (p<0.0001 and p = 0.0438 respectively). ASPM had moderate to high nuclear and low to moderate cytoplasmic expression in normal tissue. Cytoplasmic ASPM expression decreased with tumour grade and stage in the serous subtype of EOC (p = 0.023 and p = 0.011 respectively). Cytoplasmic ASPM increased with tumour stage in the endometrioid subtype (p = 0.023). Increasing tumour invasiveness (T3) and lymph node involvement (N1) also correlated with a decrease in cytoplasmic ASPM in EOC (p = 0.02 and p = 0.04 respectively). We have validated previous findings of deregulated expression of Microcephalin and ASPM in EOC by confirming associations for low nuclear Microcephalin levels and high cytoplasmic ASPM levels in a larger scale tumour tissue study. Microcephalin and ASPM may prove useful biomarkers in EOC.