Unilateral inguinofemoral lymphadenectomy in patients with early-stage vulvar squamous cell carcinoma and a unilateral metastatic sentinel lymph node is safe.

OBJECTIVE: Optimal management of the contralateral groin in patients with early-stage vulvar squamous cell carcinoma (VSCC) and a metastatic unilateral inguinal sentinel lymph node (SN) is unclear. We analyzed patients who participated in GROINSS-V I or II to determine whether treatment of the contralateral groin can safely be omitted in patients with a unilateral metastatic SN. METHODS: We selected the patients with a unilateral metastatic SN from the GROINSS-V I and II databases. We determined the incidence of contralateral additional non-SN metastases in patients with unilateral SN-metastasis who underwent bilateral inguinofemoral lymphadenectomy (IFL). In those who underwent only ipsilateral groin treatment or no further treatment, we determined the incidence of contralateral groin recurrences during follow-up. RESULTS: Of 1912 patients with early-stage VSCC, 366 had a unilateral metastatic SN. Subsequently, 244 had an IFL or no treatment of the contralateral groin. In seven patients (7/244; 2.9% [95% CI: 1.4%-5.8%]) disease was diagnosed in the contralateral groin: five had contralateral non-SN metastasis at IFL and two developed an isolated contralateral groin recurrence after no further treatment. Five of them had a primary tumor ≥30 mm. Bilateral radiotherapy was administered in 122 patients, of whom one (1/122; 0.8% [95% CI: 0.1%-4.5%]) had a contralateral groin recurrence. CONCLUSION: The risk of contralateral lymph node metastases in patients with early-stage VSCC and a unilateral metastatic SN is low. It appears safe to limit groin treatment to unilateral IFL or inguinofemoral radiotherapy in these cases.

Prospective evaluation of IOTA logistic regression models LR1 and LR2 in comparison with subjective pattern recognition for diagnosis of ovarian cancer in an outpatient setting.

OBJECTIVE: To determine whether International Ovarian Tumor Analysis (IOTA) logistic regression models LR1 and LR2 developed for the preoperative diagnosis of ovarian cancer could also be used to differentiate between benign and malignant adnexal tumors in the population of women attending gynecology outpatient clinics. METHODS: This was a single-center prospective observational study of consecutive women attending our gynecological diagnostic outpatient unit, recruited between May 2009 and January 2012. All the women were first examined by a Level-II ultrasound operator. In those diagnosed with adnexal tumors, the IOTA-LR1/2 protocol was used to evaluate the masses. The LR1 and LR2 models were then used to assess the risk of malignancy. Subsequently, the women were also examined by a Level-III examiner, who used pattern recognition to differentiate between benign and malignant tumors. Women with an ultrasound diagnosis of malignancy were offered surgery, while asymptomatic women with presumed benign lesions were offered conservative management with a minimum follow-up of 12 months. The initial diagnosis was compared with two reference standards: histological findings and/or a comparative assessment of tumor morphology on follow-up ultrasound scans. All women for whom the tumor classification on follow-up changed from benign to malignant were offered surgery. RESULTS: In the final analysis, 489 women who had either or both of the reference standards were included. Their mean age was 50 years (range, 16-91 years) and 45% were postmenopausal. Of the included women, 342/489 (69.9%) had surgery and 147/489 (30.1%) were managed conservatively. The malignancy rate was 137/489 (28.0%). Overall, sensitivities of LR1 and LR2 for the diagnosis of malignancy were 97.1% (95% CI, 92.7-99.2%) and 94.9% (95% CI, 89.8-97.9%) and specificities were 77.3% (95% CI, 72.5-81.5%) and 76.7% (95% CI, 71.9-81.0%), respectively (P > 0.05). In comparison with pattern recognition (sensitivity 94.2% (95% CI, 88.8-97.4%), specificity 96.3% (95% CI, 93.8-98.0%)), the specificities of the IOTA models were significantly lower (P < 0.0001). A significantly higher number of women would have been offered surgery for suspected cancer if the women had been assessed using the IOTA models instead of pattern recognition (213/489 (43.6%) vs 142/489 (29.0%); P < 0.001). CONCLUSIONS: The IOTA models maintained their high sensitivity when used in an outpatient setting. Specificity was relatively low, which indicates that a significant proportion of the women would have been offered unnecessary surgery for suspected ovarian cancer. These findings show that the IOTA models could be used as a first-stage test to diagnose ovarian cancer in an outpatient setting, but a different second-stage test is required to minimize the number of false-positive findings. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

The natural history of endometrial polyps.

STUDY QUESTION: What is the natural history of endometrial polyps in women who are managed expectantly? SUMMARY ANSWER: The growth rates of expectantly managed polyps vary considerably and cannot be accurately predicted. WHAT IS KNOWN ALREADY: The majority of polyps detected on ultrasound are treated surgically, and therefore little is known about their natural history. Some polyps have been reported to regress spontaneously without the need for treatment; however, the factors predictive of regression are unknown. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study conducted at the Department of Gynaecology, University College London Hospitals. We searched our ultrasound clinic database between July 1997 and September 2015, to identify women aged 18 years or older with endometrial polyps that were managed expectantly for ≥6 months. All women attended for a minimum of two ultrasound scans. PARTICIPANTS/MATERIALS, SETTING, METHODS: A single expert operator performed all ultrasound scans. Those with <6-month follow-up and those who were taking hormonal contraception, HRT or tamoxifen were excluded from the study. The mean diameter of each polyp was calculated from the measurements in three perpendicular planes. The polyp growth rate was expressed as annual percentage change in the mean diameter. Non-parametric tests and the Fisher's exact test were used to compare differences in polyp mean diameters and growth rates between women of different demographic characteristics. To correct for multiple significance testing, we used the Bonferroni method, giving the level of probability at which findings were considered significant as P < 0.0029 (as 17 tests were undertaken). MAIN RESULTS AND THE ROLE OF CHANCE: We included 112 women with endometrial polyps, which were expectantly managed over a median period of 22.5 months (range, 6-136). The annual endometrial polyp growth rate varied with a median of 1.0% (interquartile range, -6.5 to 14.3). There was no association between women's demographic characteristics or polyps' morphology and their growth rates. Eleven out of 75 (15% (95% CI, 6.9%-23.1%)) women who initially did not have abnormal uterine bleeding subsequently developed abnormal bleeding during the follow-up period. Polyp growth rate was not associated with the subsequent development of abnormal uterine bleeding (P = 0.397). Seven out of 112 (6.3% (95% CI, 1.8%-10.8%)) women had complete regression of their polyps without treatment during a median follow-up period of 28 months (range, 9-56). Spontaneous regression appeared to occur more frequently in premenopausal women (P = 0.016) and in those who presented with abnormal uterine bleeding at diagnosis (P = 0.004); however, the differences did not reach statistical significance after correction for multiple comparisons. LIMITATIONS, REASONS FOR CAUTION: This study was retrospective and therefore may be prone to selection and information biases. The lack of histological confirmation on all ultrasound diagnoses may also be considered as a limitation. WIDER IMPLICATIONS OF THE FINDINGS: Women should be advised that the growth pattern of an individual polyp cannot be accurately predicted; however, a small proportion of polyps do regress spontaneously. There was no correlation between polyps' growth rate and the subsequent development of abnormal uterine bleeding. In view of that, routine monitoring of asymptomatic polyps by ultrasound is not helpful and encouraging women to report clinical symptoms is more useful in deciding whether treatment is required. In contrast to previous studies, we found that polyps may regress more frequently in premenopausal women and in those who presented with abnormal uterine bleeding; a larger sample size would give us greater power to detect a difference in these subgroups of women. STUDY FUNDING/COMPETING INTERESTS: No study funding was received and no competing interests are present. TRIAL REGISTRATION NUMBER: N/A.

Time to diagnosis of Type I or II invasive epithelial ovarian cancers: a multicentre observational study using patient questionnaire and primary care records.

OBJECTIVE: To compare time to diagnosis of the typically slow-growing Type I (low-grade serous, low-grade endometrioid, mucinous, clear cell) and the more aggressive Type II (high-grade serous, high-grade endometrioid, undifferentiated, carcinosarcoma) invasive epithelial ovarian cancer (iEOC). DESIGN: Multicentre observational study. SETTING: Ten UK gynaecological oncology centres. POPULATION: Women diagnosed with primary EOC between 2006 and 2008. METHODS: Symptom data were collected before diagnosis using patient questionnaire and primary-care records. We estimated patient interval (first symptom to presentation) using questionnaire data and diagnostic interval (presentation to diagnosis) using primary-care records. We considered the impact of first symptom, referral and stage on intervals for Type I and Type II iEOC. MAIN OUTCOME MEASURES: Patient and diagnostic intervals. RESULTS: In all, 78% of 60 Type I and 21% of 134 Type II iEOC were early-stage. Intervals were comparable and independent of stage [e.g. median patient interval for Type I: early-stage 0.3 months (interquartile range 0.3-3.0) versus late-stage 0.3 months (interquartile range 0.3-4.5), P = 0.8]. Twenty-seven percent of women with Type I and Type II had diagnostic intervals of at least 9 months. First symptom (questionnaire) was also similar, except for the infrequent abnormal bleeding (Type I 15% versus Type II 4%, P = 0.01). More women with Type I disease (57% versus 41%, P = 0.04) had been referred for suspected gynaecological cancer. Median time from referral to diagnosis was 1.4 months for women with iEOC referred via a 2-week cancer referral to any specialty compared with 2.6 months (interquartile range 2.0-3.7) for women who were referred routinely to gynaecology. CONCLUSION: Overall, shorter diagnostic delays were seen when a cancer was suspected, even if the primary tumour site was not recognised to be ovarian. Despite differences in carcinogenesis and stage for Type I and Type II iEOC, time to diagnosis and symptoms were similar. Referral patterns were different, implying subtle symptom differences. If symptom-based interventions are to impact on ovarian cancer survival, it is likely to be through reduced volume rather than stage-shift. Further research on histological subtypes is needed. TWEETABLE ABSTRACT: No difference in time to diagnosis for Type I versus Type II invasive epithelial ovarian cancers.

Cks overexpression enhances chemotherapeutic efficacy by overriding DNA damage checkpoints.

Cdc kinase subunit (Cks) proteins Cks1 and Cks2 are adaptor-like proteins that bind many cyclin-dependent kinases. A wealth of clinical data has shown that Cks proteins are overexpressed in many types of human cancers and this often correlates with increased tumor aggressiveness. Previously, we showed that Cks overexpression abrogates the intra-S-phase checkpoint, a major barrier to oncogene-mediated transformation. Interestingly, the intra-S-phase checkpoint is crucial for the cellular response to replication stress, a major pathway of apoptosis induction by many chemotherapeutic agents. Here, we demonstrate cancer cells that overexpress Cks1 or Cks2 override the intra-S-phase checkpoint in the presence of replication stress-inducing chemotherapies such as 5-Fluorouracil (5-FU) and methotrexate (MTX) leading to enhanced sensitivity in vitro and in vivo. Furthermore, enforced expression of Cks1 in an MTX-resistant breast cancer cell line was found to restore drug sensitivity. Our results suggest that Cks proteins are important determinants of apoptosis induction of replication stress-inducing chemotherapies such as 5-FU.

Evidence for a time-dependent association between FOLR1 expression and survival from ovarian carcinoma: implications for clinical testing. An Ovarian Tumour Tissue Analysis consortium study.

BACKGROUND: Folate receptor 1 (FOLR1) is expressed in the majority of ovarian carcinomas (OvCa), making it an attractive target for therapy. However, clinical trials testing anti-FOLR1 therapies in OvCa show mixed results and require better understanding of the prognostic relevance of FOLR1 expression. We conducted a large study evaluating FOLR1 expression with survival in different histological types of OvCa. METHODS: Tissue microarrays composed of tumour samples from 2801 patients in the Ovarian Tumour Tissue Analysis (OTTA) consortium were assessed for FOLR1 expression by centralised immunohistochemistry. We estimated associations for overall (OS) and progression-free (PFS) survival using adjusted Cox regression models. High-grade serous ovarian carcinomas (HGSC) from The Cancer Genome Atlas (TCGA) were evaluated independently for association between FOLR1 mRNA upregulation and survival. RESULTS: FOLR1 expression ranged from 76% in HGSC to 11% in mucinous carcinomas in OTTA. For HGSC, the association between FOLR1 expression and OS changed significantly during the years following diagnosis in OTTA (Pinteraction=0.01, N=1422) and TCGA (Pinteraction=0.01, N=485). In OTTA, particularly for FIGO stage I/II tumours, patients with FOLR1-positive HGSC showed increased OS during the first 2 years only (hazard ratio=0.44, 95% confidence interval=0.20-0.96) and patients with FOLR1-positive clear cell carcinomas (CCC) showed decreased PFS independent of follow-up time (HR=1.89, 95% CI=1.10-3.25, N=259). In TCGA, FOLR1 mRNA upregulation in HGSC was also associated with increased OS during the first 2 years following diagnosis irrespective of tumour stage (HR: 0.48, 95% CI: 0.25-0.94). CONCLUSIONS: FOLR1-positive HGSC tumours were associated with an increased OS in the first 2 years following diagnosis. Patients with FOLR1-negative, poor prognosis HGSC would be unlikely to benefit from anti-FOLR1 therapies. In contrast, a decreased PFS interval was observed for FOLR1-positive CCC. The clinical efficacy of FOLR1-targeted interventions should therefore be evaluated according to histology, stage and time following diagnosis.

Use of IOTA simple rules for diagnosis of ovarian cancer: meta-analysis.

OBJECTIVES: To present data on prospective evaluation of the International Ovarian Tumor Analysis (IOTA) 'simple-rules' tool for the diagnosis of ovarian cancer and to perform a meta-analysis of studies that utilized the same diagnostic method. METHODS: In the present study a level-II ultrasound operator systematically assessed the tumors of women with an ultrasound diagnosis of adnexal tumor(s) according to the IOTA simple-rules protocol to determine the risk of the tumor being malignant. The results of simple rules were compared with the 'pattern recognition' method and with histological findings. This validation study was included in the subsequent meta-analysis, for which we searched MEDLINE, EMBASE and Cochrane from the publication of the first study in 2008. The terms used were 'simple rules', 'simple rules ovarian', 'ovar tumor' and 'ultrasound'. Quality assessment was performed using the modified Quality Assessment of the Diagnostic Accuracy of Studies (QUADAS-2) checklist. Random effects meta-analysis was used to calculate pooled estimates of sensitivity and specificity for the simple-rules tool, and meta-regression was used to investigate heterogeneity across the studies. RESULTS: Three hundred and three women were included in the validation study with 168 (55.4%) benign, 19 (6.3%) borderline and 116 (38.3%) malignant tumors on histological examination. The rules were applicable in 237 (78.2%) of the tumors and for these tumors, sensitivity was 96.2% (95% CI, 90.5-99.0%) and specificity was 88.6% (95% CI, 82.0-93.5%). Six of the 88 discovered studies were included in the meta-analysis along with the current validation study, which resulted in inclusion of a total of 3568 patients. When the meta-analysis was performed the pooled sensitivity (when the rules were applicable) was 93% (95% CI, 90-96%) (I(2) = 32.1%) and the pooled specificity was 95% (95% CI, 93-97%) (I(2) = 78.1%). Heterogeneity was observed across the studies. Sensitivity was higher and specificity lower in the study populations in which the prevalence of malignant tumors was greatest. CONCLUSION: The simple rules protocol could be used in 76-89% of tumors and is an accurate test for the diagnosis of ovarian cancer. Assessment by an ultrasound expert is required when the protocol cannot be applied.

Histological confirmation of breast cancer registration and self-reporting in England and Wales: a cohort study within the UK Collaborative Trial of Ovarian Cancer Screening.

BACKGROUND: In research studies, accurate information of cancer diagnosis is crucial. In women with breast cancer (BC), we compare cancer registration (CR) in England/Wales and self-reporting with independent confirmation. METHODS: In the UK Collaborative Trial of Ovarian Cancer Screening, notification of BC diagnosed between randomisation and 31 December 2009 was obtained through (1) CR (17 October 2011) and (2) self-reporting using postal-questionnaire. Breast cancer was confirmed using a detailed questionnaire (BC questionnaire BCQ) completed by the treating clinician (gold standard). Apparent sensitivity and positive-predictive value of CR/self-reporting vs BCQ were calculated. RESULTS: Of 1065 women with possible BC notification, diagnosis was confirmed in 932 (87.5%). A total of 3.1% (28 out of 918) of BC CR and 12.4% (128 out of 1032) of women with self-reported BC only had in-situ carcinoma on BCQ. Another 4.6% (43 out of 932) of BCQ-confirmed cancer did not have a BC registration, and 3.6% (34 out of 932) did not self-report BC. Apparent sensitivity of CR and self-reporting vs BCQ were 95.4 and 96.4%, respectively. Positive-predictive value of self-reporting (87.1%) was significantly lower than that of CR (96.8%). Women aged<65 were more likely to over report in-situ carcinoma as BC. Overall, 73 (6.8%) women would have been misclassified/missed if CR, and 167 (15.6%) if self-reporting data alone was used. CONCLUSION: This study confirms the reliability of BC registration in England/Wales and highlights the fact that 1 in 10 women self-reporting BC might only have in-situ breast carcinoma.

Elevated mRNA expression of CHAC1 splicing variants is associated with poor outcome for breast and ovarian cancer patients.

BACKGROUND: The role of CHAC1 (cation transport regulator-like protein 1), a recently identified component of the unfolded protein response (UPR) pathway, in gynaecological cancers has not yet been characterised. Now, this work illustrates CHAC1 mRNA expression and associated clinical outcome in breast and ovarian cancer. METHODS: The prognostic value of CHAC1 and its two transcript variants was investigated in 116 breast and 133 ovarian tissues using quantitative real-time reverse-transcriptase PCR. Subsequently, we conducted functional studies using short-interfering RNA-mediated knockdown and plasmid-mediated overexpression of CHAC1 in breast and ovarian cancer cells. RESULTS: Poorly differentiated tumours exhibited higher CHAC1 mRNA expression (breast cancer: P=0.004; ovarian cancer: P=0.024). Hormone receptor-negative breast tumours and advanced-staged ovarian cancers demonstrated elevated CHAC1 mRNA expression levels (P<0.001 and P=0.026, respectively). The multivariate survival analysis showed a prognostic value of both transcript variants in breast cancer (transcript variant 1: RR(death) 6.7 (2.4-18.9); P<0.001), RR(relapse) 6.7 (2.1-21.3); P=0.001); (transcript variant 2: RR(death) 4.9 (2.0-12.4); P<0.001), RR(relapse) 8.0 (2.4-26.8); P<0.001). Ovarian cancer patients aged younger than 62.6 years with high CHAC1 mRNA expression showed poorer relapse-free- and overall-survival (P=0.030 and P=0.012, respectively). In functional studies CHAC1 knockdown suppressed cell migration, whereas ectopic overexpression opposed these effects. CONCLUSION: High CHAC1 mRNA expression could be an independent indicator for elevated risk of cancer recurrence in breast and ovarian cancer.

Hypomethylation along with increased H19 expression in placentas from pregnancies complicated with fetal growth restriction.

The expression of imprinted genes is regulated by epigenetic modifications, such as DNA methylation. Many imprinted genes are expressed in the placenta and affect nutrient transfer capacity of the placental exchange barrier. The H19 gene is abundantly expressed by the human placenta and is implicated in the pathogenesis of congenital growth disorders such as Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes. The aim of this study was to investigate the role of DNA methylation on H19 transcription and imprinting, in the pathophysiology of fetal growth restriction (FGR). Thirty one and 17 placentas from FGR-complicated and normal pregnancies were collected, respectively. We studied gene transcription, genotyping and methylation analysis of the AluI H19 on exon 5 polymorphism. Placental expression levels of H19 were significantly increased in the FGR group. The H19 mRNA levels were similar between normal placental samples that demonstrated loss and maintenance of imprinting. Placentas from growth-restricted pregnancies had lower methylation levels compared to normals, in the H19 promoter region. We have demonstrated an increased H19 transcription in the FGR group of placentas. The hypomethylation of the H19 promoters is compatible with the aberrant expression. The association of these two findings is reported for the first time in placental tissues, however, its significance remains unknown. Whether the results of this study represent an adaptation of the placenta to hypoperfusion, or they are part of FGR pathophysiology has to be further investigated.

Tubal ligation and the risk of ovarian cancer: review and meta-analysis.

BACKGROUND: The reduction of ovarian cancer (OC) risk in women with a history of tubal ligation (TL) has been reported repeatedly, mostly on small populations. We have aimed to provide a critical overview of the studies available to date and to conduct a meta-analysis. METHODS: There were 40 relevant studies identified. The studies were divided into two groups for strict and extended meta-analysis, respectively. Subgroup analysis was performed for age, time dependency since TL, histological types of OC and BReast CAncer (BRCA) mutation. RESULTS: Meta-analysis of 13 strictly selected studies showed a reduced risk of epithelial OC by 34%. The protective effect of TL was confirmed even in a subgroup of women 10-14 years after the procedure. The risk reduction was confirmed for the endometrioid (RR = 0.40) and serous (RR = 0.73) cancers but not for mucinous. CONCLUSIONS: The review of relevant articles, as well as the meta-analysis of selected studies, yields consistent data on a significant reduction of OC risk in women who had undergone TL. The results of this meta-analysis should provide an impulse for further research on the etiology of ovarian epithelial cancers, focusing particularly on the importance of retrograde transport of endometrial cells.

Serum oestrogen receptor alpha and beta bioactivity are independently associated with breast cancer: a proof of principle study.

BACKGROUND: Oestrogens play a crucial role in breast carcinogenesis. Earlier studies have analysed the serum levels of endogenous hormones measured by conventional assays. In this study, we analysed the capacity of serum from breast cancer cases and controls to transactivate the oestrogen receptor alpha (ER-alpha) and beta (ER-beta). METHODS: We used a receptor oestrogen-responsive element (ERE) -- the green fluorescent protein (GFP)-reporter test system in Saccharomyces cerevisiae. Oestrogen receptor-alpha or ER-beta bioactivity was determined in serum from 182 randomly chosen postmenopausal women with breast cancer and from 188 age-matched controls. RESULTS: High serum ER-alpha and ER-beta bioactivity were independently associated with the presence of breast cancer. Women whose levels of serum ER-alpha and ER-beta bioactivity were in the highest quintile among controls had a 7.57-(95% confidence interval (CI): 2.46-23.32; P=0.0004) and a 10.14 (95% CI: 3.19-32.23; P<0.0001)-fold risk for general and oestrogen receptor-positive breast cancer, respectively. CONCLUSION: The use of serum ER-alpha and ER-beta bioactivity assays as clinical tools in the management of breast cancer warrants further research. Future studies will dictate whether surrogate markers of ER-alpha and ER-beta bioactivity will provide a means to monitor the efficacy of anti-endocrine, adjuvant and chemopreventive strategies.

Gene methylation data--a new challenge for bioinformaticians?

OBJECTIVES: Changes in the status of DNA methylation, known as epigenetic alterations, are among the most common molecular alterations in human neoplasia. For the first time, we reported on the analysis of fecal DNA from patients with CRC to determine the feasibility, sensitivity and specificity of this approach. We want to present basic information about DNA methylation analysis in the context of bioinformatics, the study design and several statistical experiences with gene methylation data. Additionally we outline chances and new research questions in the field of DNA methylation. METHODS: We present current approaches to DNA methylation analysis based on one reference study. Its study design and the statistical analysis is reflected in the context of biomarker development. Finally we outline perspectives and research questions for statisticians and bioinformaticians. RESULTS: Identification of at least three genes as potential DNA methylation-based tumor marker genes (SFRP2, SFRP5, PGR). CONCLUSIONS: DNA methylation analysis is a rising topic in molecular genetics. Gene methylation will push the extension of biobanks to include new types of genetic data. Study design and statistical methods for the detection of methylation biomarkers must be improved. For the purpose of establishing methylation analysis as a new diagnostic/prognostic tool the adaptation of several approaches has become a challenging field of research activity.

[Tumorectomy in conscious patient with suspected pregnancy associated breast cancer under cervical epidural anesthesia]. / Tumorektomie am wachen Patienten in zervikaler Epiduralanästhesie bei schwangerschaftsassoziiertem Mammakarzinom.

In this report we present a 35 year old pregnant woman (no significant disease in patient history, non smoker, primipara, gestational week 15) who had to undergo elective tumorectomy due to suspected pregnancy associated breast cancer. General anesthesia during pregnancy can potentially be harmful for the fetus (hypoxia, acidosis, premature delivery, teratogenicity). We decided to anesthetize the patient with a cervical segmental epidural block.

MeCP2 and MBD2 expression in human neoplastic and non-neoplastic breast tissue and its association with oestrogen receptor status.

This study analysed mRNA expression of two members of the methyl-CpG-binding protein family - MeCP2 and MBD2 - in human non-neoplastic (n=11) and neoplastic (n=57) breast tissue specimens using a quantitative real-time PCR method. We observed higher expression levels of MeCP2 mRNA in neoplastic tissues than in non-neoplastic tissues (P=0.001), whereas no significant differences for MBD2 were detected. When studying the relations between the most important clinicopathologic features of breast cancer and the mRNA expression level of both MBDs, we found that oestrogen receptor (OR)-positive breast cancer specimens contained higher levels of MeCP2 mRNA than did OR-negative cancers (P=0.005). Furthermore, we observed statistically significantly higher levels of MeCP2 in non-neoplastic tissues expressing high levels of OR as compared to those expressing low levels (P=0.017). Finally, using a linear regression model, we identified a statistically significant association between OR expression and MeCP2 mRNA expression in neoplastic and non-neoplastic breast tissue specimens (P=0.003). In conclusion, we were able to demonstrate for the first time that there exists a strong association between OR status and MeCP2 mRNA expression. Furthermore, we speculate that MeCP2, regulated by OR, plays a key role in the differentiation processes in human breast tissues.

Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas.

Gene therapeutic approaches currently favor adenoviral vectors over alternatively available vector systems. Ovarian cancer represents an attractive model for an intraperitoneal adenovirus-based gene therapy, which is now under intensive clinical investigation. Adenovirus-mediated gene transfer depends on adequate virus uptake and thus on the presence of sufficient amounts of high-affinity coxsackie-adenovirus receptor (CAR) and alphavbeta3- and alphavbeta5 integrins on target cells. This fact has been ignored in most ongoing clinical trials. This investigation, therefore, determined expression of CAR by immunohistochemistry in 37 ovarian carcinomas and compared it with that of alphavbeta3 and alphavbeta5 integrins. In all samples, except one undifferentiated carcinoma, CAR was immunohistochemically demonstrable. Grade 1 tumors exhibited stronger CAR immunostaining as compared with higher-grade cancers (P < 0.03). Integrins alphavbeta3 and alphavbeta5 were detectable in 62% and 65% of carcinomas, respectively, and staining for both classes correlated positively (P < 0.005). Cancers classified as undifferentiated completely lacked alphavbeta3 expression. Furthermore, in undifferentiated and grade 3 carcinomas the three molecules studied exhibited marked distributional heterogeneity with regard to focal positivity and negativity within the same tumor. Either the absence of CAR, alphavbeta3 and alphavbeta5 or the pronounced heterogeneity in their expression might seriously compromise the efficiency of adenovirus-based gene therapy in ovarian cancer.

Epigenetic downregulation of the retinoic acid receptor-beta2 gene in breast cancer.

A growing body of evidence supports the hypothesis that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene which induces apoptosis and that the chemopreventive and therapeutic effects of retinoids are due to induction of RAR-beta2. During breast cancer progression, RAR-beta2 is reduced or even lost. It is known from studies of other tumor-suppressor genes that methylation of the 5'-region is the cause of loss of expression. Several groups demonstrated that this is also true for the RAR-beta2 in breast cancer by treating breast cancer cell lines with a demethylating agent and examining expression of the RAR-beta2 gene in response to a challenge with retinoic acid. Studies using sodium bisulfite genomic sequencing as well as methylation specific PCR showed that a number of breast cancer cell lines as well as breast cancer tissue showed signs of methylation. The RAR-beta2 gene was unmethylated in non-neoplastic breast tissue as well as in other normal tissues. A combination of retinoic acid with demethylating agents as well as with histone deacetylase inhibitors acts synergistically to inhibit growth. This review presents data that suggest that treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality. Both the time of commencement of chemoprevention and the choice of substances that are able either to prevent de novo methylation or to reverse methylation-caused gene silencing may be important considerations.

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

BACKGROUND: Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. METHODS: Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. RESULTS: Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim. CONCLUSIONS: Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.

Inhibition of store-operated calcium entry contributes to the anti-proliferative effect of non-steroidal anti-inflammatory drugs in human colon cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and angiogenesis in colorectal cancer. We examined a possible involvement of store-operated calcium (SOC) entry in human colon carcinoma cells (HRT-18), which require calcium for proliferation. Acetyl-salicylic-acid (ASA), mefenamic acid (MEF) and sulindac sulfide (SUS) inhibited cell proliferation with the following order of potency: SUS > MEF >> ASA. SUS but not MEF and ASA induced apoptosis following low-dose treatment. Furthermore, SUS and MEF significantly altered the cell cycle distribution. The ability of NSAIDs to inhibit SOC entry was assessed by measuring the intracellular calcium concentration ([Ca2+]i) in response to calcium store depletion using the endoplasmic calcium ATPase inhibitor thapsigargin. SUS and MEF, but not ASA significantly inhibited SOC entry. A causal link between SOC entry inhibition and anti-proliferative activity was tested using the inorganic SOC entry inhibitor La3+ and the specific organic inhibitor N-1-n-octyl-3,5-bis-(4-pyridyl)triazole (DPT). Both La3+ and DPT inhibited cell proliferation and SOC entry. Analogous to MEF, the anti-proliferative effect of DPT was mediated by cell cycle arrest and not by induction of apoptosis. These data indicate a role of SOC entry for cell proliferation in cancer cells and suggest a novel anti-proliferative NSAID mechanism in addition to its known influence on lipid metabolism.