A comparison of four technologies for detecting p53 aggregates in ovarian cancer.

The tumor suppressor protein p53 is mutated in half of all cancers and has been described to form amyloid-like structures, commonly known from key proteins in neurodegenerative diseases. Still, the clinical relevance of p53 aggregates remains largely unknown, which may be due to the lack of sensitive and specific detection methods. The aim of the present study was to compare the suitability of four different methodologies to specifically detect p53 aggregates: co-immunofluorescence (co-IF), proximity ligation assay (PLA), co-immunoprecipitation (co-IP), and the p53-Seprion-ELISA in cancer cell lines and epithelial ovarian cancer tissue samples. In 7 out of 10 (70%) cell lines, all applied techniques showed concordance. For the analysis of the tissue samples co-IF, co-IP, and p53-Seprion-ELISA were compared, resulting in 100% concordance in 23 out of 30 (76.7%) tissue samples. However, Co-IF lacked specificity as there were samples, which did not show p53 staining but abundant staining of amyloid proteins, highlighting that this method demonstrates that proteins share the same subcellular space, but does not specifically detect p53 aggregates. Overall, the PLA and the p53-Seprion-ELISA are the only two methods that allow the quantitative measurement of p53 aggregates. On the one hand, the PLA represents the ideal method for p53 aggregate detection in FFPE tissue, which is the gold-standard preservation method of clinical samples. On the other hand, when fresh-frozen tissue is available the p53-Seprion-ELISA should be preferred because of the shorter turnaround time and the possibility for high-throughput analysis. These methods may add to the understanding of amyloid-like p53 in cancer and could help stratify patients in future clinical trials targeting p53 aggregation.

Perinatal and 2-year neurodevelopmental outcome in late preterm fetal compromise: the TRUFFLE 2 randomised trial protocol.

INTRODUCTION: Following the detection of fetal growth restriction, there is no consensus about the criteria that should trigger delivery in the late preterm period. The consequences of inappropriate early or late delivery are potentially important yet practice varies widely around the world, with abnormal findings from fetal heart rate monitoring invariably leading to delivery. Indices derived from fetal cerebral Doppler examination may guide such decisions although there are few studies in this area. We propose a randomised, controlled trial to establish the optimum method of timing delivery between 32 weeks and 36 weeks 6 days of gestation. We hypothesise that delivery on evidence of cerebral blood flow redistribution reduces a composite of perinatal poor outcome, death and short-term hypoxia-related morbidity, with no worsening of neurodevelopmental outcome at 2 years. METHODS AND ANALYSIS: Women with non-anomalous singleton pregnancies 32+0 to 36+6 weeks of gestation in whom the estimated fetal weight or abdominal circumference is <10th percentile or has decreased by 50 percentiles since 18-32 weeks will be included for observational data collection. Participants will be randomised if cerebral blood flow redistribution is identified, based on umbilical to middle cerebral artery pulsatility index ratio values. Computerised cardiotocography (cCTG) must show normal fetal heart rate short term variation (≥4.5 msec) and absence of decelerations at randomisation. Randomisation will be 1:1 to immediate delivery or delayed delivery (based on cCTG abnormalities or other worsening fetal condition). The primary outcome is poor condition at birth and/or fetal or neonatal death and/or major neonatal morbidity, the secondary non-inferiority outcome is 2-year infant general health and neurodevelopmental outcome based on the Parent Report of Children's Abilities-Revised questionnaire. ETHICS AND DISSEMINATION: The Study Coordination Centre has obtained approval from London-Riverside Research Ethics Committee (REC) and Health Regulatory Authority (HRA). Publication will be in line with NIHR Open Access policy. TRIAL REGISTRATION NUMBER: Main sponsor: Imperial College London, Reference: 19QC5491. Funders: NIHR HTA, Reference: 127 976. Study coordination centre: Imperial College Healthcare NHS Trust, Du Cane Road, London, W12 0HS with Centre for Trials Research, College of Biomedical & Life Sciences, Cardiff University. IRAS Project ID: 266 400. REC reference: 20/LO/0031. ISRCTN registry: 76 016 200.

Influence of Pregnancy-Related Conditions on Human Epididymis Protein 4 Serum Levels in Comparison to CA 125 - a Prospective Cohort Trial.

Introduction HE4 and CA 125, two established biomarkers for assessing adnexal masses in non-pregnant women, are hardly investigated in pregnancy, especially in pregnancy-associated conditions. The aim was to evaluate HE4 and CA 125 levels in the course of pregnancy and to assess the impact of pregnancy disorders, contractions and rupture of membranes on HE4 and CA 125 serum levels in order to use these parameters for evaluation of adnexal masses in pregnancy. Patients and Methods Blood samples (n = 238) of 201 women seen at the Medical University of Innsbruck, Austria, were prospectively obtained during pregnancy and postpartum. Serum concentrations of HE4 and CA 125 were analyzed. ROMA index was calculated by the premenopausal formula. Results HE4 serum levels were highest in the third trimester. Contractions (p < 0.001), rupture of membranes (p = 0.005) and pregnancy-associated diseases (p = 0.003) were associated with higher HE4 levels. As much as 97.5% of HE4 measurements remained below the recommended cut-off for premenopausal women (70 pmol/l). CA 125 levels were not altered by pregnancy-associated conditions. Generally, CA 125 exhibited a wider serum level variability, exceeding the established cut-off of 35 U/ml in 16.4%. Conclusions HE4 serum levels are influenced by several pregnancy-related conditions leading to significantly higher levels in these cases. Despite differing medians according to trimester, the 95th percentile cut-offs and almost all maximum values during the entire course of pregnancy were below the established cut-off for premenopausal women. It was also superior to the performance of ROMA index. Therefore, HE4 can be used as a valuable negative predictive marker for the assessment of adnexal masses during pregnancy.

Screening for Open Spina Bifida in a Routine Clinical Setting at the First-Trimester Scan: A Prospective Multicentre Cohort Study.

OBJECTIVE: The aim of this study was to investigate the performance of screening for open spina bifida (OSB) integrated into the routine first-trimester screening. METHOD: This is a prospective multicentre study of 4,755 women undergoing first-trimester ultrasound scans over a 4-year period. Measurements of the brainstem (BS) diameter and brainstem-to-occipital-bone (BSOB) distance were performed. The cisterna magna (CM) was measured in the tilted axial view. RESULTS: Pregnancy outcome data were available for 4,658 fetuses included in this study. There were 5 fetuses with OSB, and in all of them, the BS/BSOB ratio and the CM measurements were abnormal. The sensitivity and specificity of a BS/BSOB ratio >1 were 100%. The sensitivity of a CM width <5th centile was 100%, and the specificity was 95.1%. In 4.6% of cases, the BS/BSOB ratio was between the 95th percentile and 1. In 87.1% of these cases, the CM was normal, and 12.9% had a CM below the 5th percentile. CONCLUSION: Screening for OSB is feasible in routine first-trimester scans. The BS/BSOB ratio shows a very good sensitivity and specificity. In cases with near-normal values for the BS/BSOB ratio, the CM width might be helpful for further assessment.

Impact of clinical factors and surgical outcome on long-term survival in high-grade serous ovarian cancer: a multicenter analysis.

INTRODUCTION: Long-term survivors of ovarian cancer are a unique group of patients in whom prognostic factors for long-term survival have been poorly described. Such factors may provide information for a more personalized therapeutic approach. The objective of this study is to determine further characteristics of long-term survivors with high-grade serous ovarian cancer. METHODS: Long-term survivors were defined as patients living longer than 8 years after first diagnosis and were recruited within seven high volume centers across Europe from November 1988 to November 2008. The control group included patients with high-grade serous ovarian cancer with less than 5 years' survival identified from the systematic 'Tumorbank ovarian cancer' database. A subanalysis of Charité patients only was performed separately for in-depth analysis of tumor dissemination. Propensity score matching with nearest-neighbor caliper width was used to match long-term survivors and the control group regarding age, FIGO stage, and residual tumor. RESULTS: A total of 276 patients with high-grade serous ovarian cancer were included, divided into 131 long-term survivors and 145 control group patients. After propensity score matching and multivariable adjustment, platinum sensitivity (p=0.002) was an independent favorable prognostic factor whereas recurrence (p<0.001) and ascites (p=0.021) were independent detrimental predictors for long-term survival. Significantly more long-term survivors tested positive for mutation in the BRCA1 gene than the BRCA2 gene (p=0.016). Intraoperatively, these patients had less tumor involvement of the upper abdomen at initial surgery (p=0.024). Complexity of surgery and surgical techniques were similar in both cohorts. CONCLUSION: Platinum sensitivity constitutes a favorable factor for long-term survival whereas tumor involvement of the upper abdomen, ascites, and recurrence have a negative impact. Based on clinical estimation, long-term survival is associated with combinations of clinical, surgical, and molecular factors.

AB0 blood groups and rhesus factor expression as prognostic parameters in patients with epithelial ovarian cancer - a retrospective multi-centre study.

BACKGROUND: AB0 blood groups and Rhesus factor expression have been associated with carcinogenesis, response to treatment and tumor progression in several malignancies. The aim of the present study was to test the hypothesis that AB0 blood groups and Rhesus factor expression are associated with clinical outcome in patients with epithelial ovarian cancer (EOC). METHODS: AB0 blood groups and Rhesus factor expression were evaluated in a retrospective multicenter study including 518 patients with EOC. Their association with patients' survival was assessed using univariate and multivariable analyses. RESULTS: Neither AB0 blood groups nor Rhesus factor expression were associated with clinico-pathological parameters, recurrence-free, cancer-specific, or overall survival. In a subgroup of patients with high-grade serous adenocarcinoma, however, blood groups B and AB were associated with a better 5-year cancer-specific survival rate compared to blood groups A and 0 (60.3 ± 8.6% vs. 43.8 ± 3.6%, p = 0.04). Yet, this was not significant in multivariable analysis. CONCLUSIONS: AB0 blood groups and Rhesus factor expression are both neither associated with features of biologically aggressive disease nor clinical outcome in patients with EOC. Further investigation of the role of the blood group B antigen on cancer-specific survival in the subgroup of high-grade serous should be considered.

Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - a study of the OVCAD consortium.

Cyclin E, coded by the genes CCNE1 and CCNE2, is the main regulator for transition from G1 to S phase determining cell division. CCNE1 and CCNE2 are known oncogenes in many cancer entities. Especially CCNE1 has frequently been associated with gene amplifications in various malignancies, emphasising its role as a putative oncogene. We determined gene expression and copy number of CCNE1 and CCNE2 by quantitative polymerase chain reaction (PCR) from 172 International Federation of Obstetrics and Gynecology (FIGO) II/III/IV stage serous epithelial ovarian cancer (EOC) tissues and analysed its impact on outcome. Furthermore, whole transcriptome gene expression changes correlating with CCNE1 expression were determined by microarray technology, interpreted by Signalling Pathway Impact Analysis (SPIA), Tool for Inferring Network of Genes (TINGe), and illustrated by hive plots. Protein-protein interaction (PPI) networks were also used for the interpretation. Interestingly, and contradictory to most reports and intuitive expectations, high CCNE1 expression correlated with better overall survival (p=0.005) if corrected for usual clinicopathologic parameters and a molecular subclassification. Using different grading systems or only high graded tumours had no impact on this correlation. Copy number of CCNE1 was increased in 25% of cases which correlated highly significantly with expression but showed no impact on outcome. CCNE2 had no impact on outcomes at all. Whole genome transcriptome analysis revealed 1872 differentially expressed genes correlated to CCNE1 expression, which were significantly enriched with genes from five pathways (e.g. cell cycle and viral carcinogenesis pathway were up-regulated and the Fanconi anaemia pathway was down-regulated). High CCNE1 gene expression is a significant and independent predictor for prolonged overall survival in FIGO III/IV EOC patients. This upside down impact of CCNE1 on survival probably reflects the special characteristic of EOC with tumour dissemination in the near anaerobic peritoneal cavity as the predominant cause of death, compared to other cancer entities where distant metastasis are predominantly lethal.

Determination of tumor-infiltrating CD8+ lymphocytes in human ovarian cancer.

Tumor-infiltrating immune cells and their prognostic value have been analyzed in various malignancies. Although tissue microarray (TMA) has been used in some of these studies, it is still questionable whether this technique can represent the results of infiltrating CD8+ cells obtained from whole-tissue sections (WTS). The aim of this study was to assess and compare the density of tumor-infiltrating CD8+ cells in ovarian cancer using TMA and WTS. CD8+ lymphocytes were immunohistochemically stained on WTS and TMA cores from 37 ovarian cancer patients and quantified using the image analysis software HistoQuest. Four different areas were selected on the WTS, namely (i) tumor; (ii) stroma; (iii) mixed; and (iv) dense, whereby dense represented areas of most abundant CD8+ cells. On the TMA, (i) the whole TMA cores and (ii) areas containing only epithelial tumor tissue were analyzed. The Pearson correlation and principal component analysis was used to estimate the correlation of results from different techniques. CD8+ lymphocytes showed highly correlated measurements between tumor, mixed, and dense areas. Moderate correlations were found between each of these 3 measurements and stroma. CD8+ cell counts from WTS showed moderate correlation with TMA cell counts. Consistently, principal component analysis showed 3 clusters (i) tumor, dense, mixed; (ii) stroma; and (iii) TMA areas. Taken together, when the prognostic impact of tumor-infiltrating CD8+ cells in ovarian cancer is investigated with TMA technique, a moderate correlation with WTS results has to be considered.

The N-terminally truncated p53 isoform Δ40p53 influences prognosis in mucinous ovarian cancer.

OBJECTIVE: The tumor suppressor p53 generates the N-terminally truncated isoforms Δ40p53 and Δ133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer. METHODS: Δ40p53, Δ133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase-quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, Δ40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status. RESULTS: In endometrioid cancer specimens, Δ133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated Δ40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high Δ40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094-0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193-1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53ß and Δ40p53α in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters. CONCLUSIONS: We show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of Δ40p53 in patients with mucinous ovarian cancer.

MDM2 SNP309 modifies the prognostic significance of the p53 mutational status in patients with ovarian cancer.

A single nucleotide polymorphism (SNP309) of MDM2 causes elevated MDM2 levels and an attenuation of p53 function. The aim of the present study was to examine the clinical relevance of the MDM2 SNP309 in ovarian cancer.MDM2 SNP309 genotype was analyzed in 198 patients with primary ovarian cancer. MDM2 expression was investigated using immunohistochemistry. A functional yeast-based assay and subsequent sequencing were performed to determine p53 mutational status. Of the patients, 44.4% (88 of 198) exhibited the common variant (T/T), 40.9% (81 of 198) the heterozygous variant (T/G) and 14.7% (29 of 198) the homozygous variant (G/G) MDM2 SNP309 genotype. MDM2 SNP309 was not associated with p53 mutational status, MDM2 expression, clinicopathological parameters or prognosis. In patients with the T allele (T/T and T/G genotype), p53 wild type carcinomas were associated with significantly improved recurrence-free (p<0.001) and overall survival (p<0.001) as compared to p53 mutant carcinomas. In contrast, p53 mutational status did not possess prognostic relevance in G/G carriers. A possible functional impairment of the p53 pathway caused by the G/G genotype of the MDM2 SNP309 may modify the association between p53 mutational status and prognosis in ovarian cancer.

Clinical relevance of TAp73 and ΔNp73 protein expression in ovarian cancer: a series of 83 cases and review of the literature.

The p73 gene gives rise to the full-length transactivation competent TAp73 and the N-terminally truncated isoform ΔNp73, which inhibits TAp73 and wild-type p53. The clinical relevance of TAp73 and ΔNp73 protein expression has not yet been evaluated in ovarian cancer. TAp73 and ΔNp73 expression was examined using immunohistochemistry and reverse transcription-polymerase chain reaction in 83 and 64 ovarian cancer specimens, respectively. A yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status. TAp73 and ΔNp73 protein expression was found in 73 of 83 (88%) and 48 of 83 (57.8%) ovarian cancer samples, respectively. The majority of cases showed immunostaining in both the nucleus and cytoplasm of tumor cells. TAp73 and ΔNp73 protein expression correlated with messenger RNA quantification in 25 of 64 (39.1%) and 37 of 64 (57.8%) cancer specimens, respectively. TAp73 and ΔNp73 protein expression was not associated with the p53 mutational status, clinicopathologic parameters, and prognosis of the examined ovarian cancer cases. Although TAp73 and ΔNp73 protein expression did not possess prognostic significance for ovarian cancer in this study, a potential clinical role of p73 isoforms cannot be definitively excluded due to limitations of immunohistochemistry.

Clinical relevance of dominant-negative p73 isoforms for responsiveness to chemotherapy and survival in ovarian cancer: evidence for a crucial p53-p73 cross-talk in vivo.

PURPOSE: We aimed to determine the clinical role of the p53 family members p53 and p73 in the responsiveness to platinum-based chemotherapy and survival in ovarian cancer, considering their cross-talk and the p53 polymorphism at codon 72. EXPERIMENTAL DESIGN: A detailed analysis of p53 and p73 in a series of 122 ovarian cancers was done. We used a functional yeast-based assay to determine the p53 mutational status. Red yeast colonies, indicating mutant p53, were subsequently sequenced to determine the specific p53 alteration. p53 mutations were divided into two groups according to their previous characterization in the literature: those that efficiently inhibit transcriptionally active TAp73 function and those that do not. A p53 polymorphism at codon 72 was determined in corresponding normal tissue or blood of ovarian cancer patients. Isoform-specific p73 expression analysis using real-time reverse transcription-PCR has previously been done in the majority of ovarian cancers included in this study. In a retrospective chart review, responsiveness to chemotherapy was assessed, and survival data with long follow-up times were collected. RESULTS: Eighty of 122 (65.6%) of ovarian cancers harbored p53 mutations. p53 mutational status was an important determinant of responsiveness to platinum-based chemotherapy in all patients with a residual tumor of <2 cm in diameter after initial surgery (wild-type versus mutant, P = 0.029). In addition, p53 mutational status was a strong prognosticator for recurrence-free and overall survival (P < 0.0001 and P = 0.003, respectively) in univariate analyses. High expression levels of dominant-negative p73 isoforms (DeltaNp73 and DeltaN'p73) significantly correlated with chemotherapeutic failure (P = 0.048) and with worse recurrence-free and overall survival in patients with p53 mutant cancers (P = 0.048 and P = 0.005, respectively). Eight p53 mutations, present in 19 cases, were found that efficiently inhibit TAp73 (i.e., 175H, 220C, 245S, 245D, 248W, 248Q, 266E, and 273H). Patients with p53 mutations that efficiently inhibit TAp73 function had a significantly shorter overall survival than patients with p53 mutations of unknown effect on TAp73 (P = 0.044). The p53 polymorphism at codon 72 had no influence on responsiveness to chemotherapy or survival. CONCLUSION: We provide the first clinical evidence that dominant-negative p73 isoforms contribute to drug resistance in vivo, underscoring the importance of a p53-p73 cross-talk. NH2-terminally truncated p73 isoforms were of significant clinical effect by providing an additional unfavorable factor for response to platinum-based chemotherapy and survival in p53 mutant ovarian cancers.