KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>KRAS </it>gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR)-targeted therapies are being investigated for use in ovarian carcinoma. As <it>KRAS </it>mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of <it>KRAS </it>mutations in ovarian carcinoma.</p> <p>Methods</p> <p>The presence of <it>KRAS </it>mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE) tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas) and 22 benign tissue samples from a total of 394 patients were examined. <it>KRAS </it>mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival.</p> <p>Results</p> <p><it>KRAS </it>mutations were found in 60 (15%) samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92%) samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to <it>KRAS </it>mutation status.</p> <p>Conclusion</p> <p><it>KRAS </it>mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The <it>KRAS </it>mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC), it may be important for prediction of response to EGFR-targeted therapies.</p

RPE65 of Retinal Pigment Epithelium, A Putative Receptor Molecule for Plasma Retinol-Binding Protein, is Expressed in Human Keratinocytes

Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid–retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes

The impact of p16(ink4a) positivity in invasive vulvar cancer on disease-free and disease-specific survival, a retrospective study

Purpose To evaluate HPV and p16(ink4a) status as prognostic factors in patients with invasive vulvar cancer. Methods Retrospective analysis of disease-free (DFS) and disease-specific survival (DSS) of patients with invasive vulvar cancer at a single tertiary care center. Histology, HPV and p16(ink4a) status were evaluated in the context of a global multicenter trial. Logistic regression models were performed to identify the impact of p16(ink4a) positivity. Results 135 patients were included in the analysis. 32 (23.7%) showed a p16(ink4a) expression of over 25%. Disease-free and disease-specific survival was longer in p16(ink4a) positive patients (23 vs. 10 months, p = 0.004, respectively, 29 vs. 21 months, p = 0.016). In multivariate analysis, p16(ink4a) positivity was an independent parameter for DFS (p = 0.025, HR: 2.120 (1.100-4.085)), but not for DSS (p = 0.926, HR: 1.029 (0.558-1.901), in contrast to age and tumor stage. Conclusions Age and tumor stage negatively affect survival. However, disease-free survival is significantly longer in patients with p16(ink4a) positive invasive vulvar cancer

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

<p>Abstract</p> <p>Background</p> <p>The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.</p> <p>Methods</p> <p>Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan^®Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, <it>hMAM </it>and <it>EpCAM </it>gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.</p> <p>Results</p> <p>Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. <it>EpCAM </it>gene expression was detected in 19% and 5% of patients, respectively, whereas <it>hMAM </it>gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.</p> <p>Conclusions</p> <p>The panel of six genes was found superior to <it>EpCAM </it>and <it>hMAM </it>for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.</p

Protein tyrosine phosphatases expression during development of mouse superior colliculus

Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis

First steps toward harmonized human biomonitoring in Europe : demonstration project to perform human biomonitoring on a European scale

'Reproduced with permission from Environmental Health Perspectives'Background: For Europe as a whole, data on internal exposure to environmental chemicals do not yet exist. Characterization of the internal individual chemical environment is expected to enhance understanding of the environmental threats to health. Objectives: We developed and applied a harmonized protocol to collect comparable human biomonitoring data all over Europe. Methods: In 17 European countries, we measured mercury in hair and cotinine, phthalate metabolites, and cadmium in urine of 1,844 children (5–11 years of age) and their mothers. Specimens were collected over a 5-month period in 2011–2012. We obtained information on personal characteristics, environment, and lifestyle. We used the resulting database to compare concentrations of exposure biomarkers within Europe, to identify determinants of exposure, and to compare exposure biomarkers with healthbased guidelines. Results: Biomarker concentrations showed a wide variability in the European population. However, levels in children and mothers were highly correlated. Most biomarker concentrations were below the health-based guidance values. Conclusions: We have taken the first steps to assess personal chemical exposures in Europe as a whole. Key success factors were the harmonized protocol development, intensive training and capacity building for field work, chemical analysis and communication, as well as stringent quality control programs for chemical and data analysis. Our project demonstrates the feasibility of a Europe-wide human biomonitoring framework to support the decision-making process of environmental measures to protect public health.The research leading to these results received funding for the COPHES project (COnsortium to Perform Human biomonitoring on a European Scale) from the European Community’s Seventh Framework Programme [FP7/2007–2013] under grant agreement 244237. DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) was co-funded (50%:50%) by the European Commission LIFE+ Programme (LIFE09/ENV/BE/000410) and the partners. For information on both projects as well as on the national co-funding institutions, see http://www.eu-hbm.info/. The sponsors had no role in the study design, data collection, data analysis, data interpretation or writing of the report

Exposure determinants of cadmium in European mothers and their children

© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother–child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24–52 years of age), and 1689 children (5–12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011–2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1–28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18–86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1–13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9–12 years had 7% (95% CI 1–13%) higher UCd (p=0.04) than children 5–8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.Financially supported by the 7th EU framework programe(DGResearch – No. 244237-COPHES),LIFE+ 2009(DG Environment – LIFE09ENV/BE000410-DEMOCOPHES),with addi- tional co-funding from DEMOCOPHES partners

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570