Measures of Abdominal Adiposity and Risk of Stroke: A Dose-Response Meta-analysis of Prospective Studies.

OBJECTIVE: Waist circumference, waist-to-hip ratio and waist-to-height ratio, which are the indicators or measures of abdominal adiposity, have long been hypothesized to increase the risk of stroke; yet evidence accumulated till date is not conclusive. Here, we conducted a dose-response meta-analysis to summarize evidences of the association between these measures of abdominal adiposity and the risk of stroke. METHODS: PubMed and Web of Science databases were searched from inception to May 2015. Two investigators independently conducted the study selection and data extraction. Dose-response relationships were assessed by the generalized least squares trend estimation, while the summary effect estimates were evaluated by the use of fixed- or random-effect models. Subgroup and sensitivity analyses were performed to assess the potential sources of heterogeneity and the robustness of the pooled estimation. Publication bias of the literature was evaluated using Begg's and Egger's test. RESULTS: Altogether 15 prospective cohort studies were identified in this study. The summary of relative risks (95% confidence intervals) of stroke for the highest versus the lowest categories was 1.28 (1.18-1.40) for waist circumference, 1.32 (1.21-1.44) for waist-to-hip ratio, and 1.49 (1.24-1.78) for waist-to-height ratio. For a 10-cm increase in waist circumference, the relative risk of stroke increased by 10%; for a 0.1-unit increase in waist-to-hip ratio, the relative risk increased by 16%; and for a 0.05-unit increase in waist-to-height ratio, the relative risk increased by 13%. There was evidence of a nonlinear association between waist-to-hip ratio and stroke risk, Pnonlinearity=0.028. CONCLUSION: Findings from our meta-analysis indicated that waist circumference, waist-to-hip ratio, and waist-to-height ratio were positively associated with the risk of stroke, particularly ischemic stroke.

Efficacy and safety of canagliflozin in subjects with type 2 diabetes: systematic review and meta-analysis.

PURPOSE: To assess the efficacy and safety of the novel sodium glucose co-transporter 2 (SGLT2) inhibitor-canagliflozin for type 2 diabetes (T2DM). METHODS: A search of Medline (1946-January 2014), Embase (1950-January 2014), and The Cochrane Library for randomized controlled trials of canagliflozin compared to placebo or active comparator in T2DM was performed. Clinical Trials website and unpublished U.S. Food and Drug Administration data were also searched. RESULTS: Ten trials including 6,701 patients were analyzed. Compared with placebo, canagliflozin produced absolute reductions in glycated hemoglobin A1c levels when used as monotherapy (weighted mean difference (WMD) -1.08%, 95% confidence interval (CI) [-1.25 to -0.90], p < 0.00001) or add-on treatment (WMD -0.73%, 95%CI [-0.84 to -0.61], p < 0.00001). When compared with other active comparators, canagliflozin significantly reduced HbA1c by -0.21% (WMD, 95%CI [-0.33 to -0.08], p = 0.001). Canagliflozin led to greater body weight loss (vs. placebo, WMD -2.81 kg, 95%CI [-3.26 to -2.37]; vs. active comparators, WMD -3.49 kg, 95%CI [-4.86 to -2.12]). Hypoglycemia with canagliflozin was similar to placebo or sitagliptin, and was lower than glimepiride (risk ratio (RR) 0.15, 95%CI [0.10 to 0.22]). Genital tract infections were more common with canagliflozin (vs. placebo, RR 3.76, 95%CI [2.23 to 6.35]; vs. active comparators, RR 4.95, 95%CI [3.25 to 7.52]). Similar incidences of urinary tract infections were noted with canagliflozin compared with control groups. CONCLUSION: Canagliflozin led to improvements in reducing glycated hemoglobin A1c levels and body weight with low risk of hypoglycemia in patients with T2DM. Common adverse effects including genital tract infections and osmotic diuresis-related AEs were identified and reviewed. Risks of cardiovascular events are even less certain, and more data on long-term effects are needed.

The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer: potential biomarkers.

MicroRNAs (miRNAs) are a class of small noncoding RNAs whose expression changes are associated with cancer development and invasion. We hypothesized that miR-10b and miR-373, which are increased in lymphatic metastatic tissues, could be directly assayed in the plasma and used to detect the lymph node status of breast cancer patients. Between November 2009 and January 2012, 35 breast ductal carcinoma patients with lymph node metastasis (N patients), 25 ductal carcinoma patients without lymph node metastasis (N(0) patients), and ten healthy female donors were enrolled in the study. Circulating miR-10b and miR-373 were determined in preoperative plasma samples by reverse transcription quantitative real-time PCR assay. In preliminary tests, the plasma levels of circulating miR-10b and miR-373 were found to be significantly higher in ten breast cancer patients with lymph node metastasis compared to ten N(0) patients and ten normal donors (P < 0.01). On validation analysis, the median value level of miR-10b in the 35 N patients was 4.44-fold (P < 0.01) increased, and miR-373 was 4.38-fold (P < 0.01) increased in comparison to the 25 N(0) patients. MiR-10b was used for differentiation of N patients from N(0) patients; the odds ratio was 2.19, and the value of the area under the receiver-operating curve (AUC) was 0.80, with sensitivity of 71 % and specificity of 72 %. For miR-373, the odds ratio was 2.62, and the AUC was 0.84, with sensitivity of 68 % and specificity of 89 %. A combination of the two circulating miRNAs further enhanced the sensitivity to 72 % and the specificity to 94.3 %. Our data suggest that circulating miRNA-10b and miRNA-373 are potential biomarkers for detecting the lymph node status of breast cancer.

Clinical characteristics, allergic response to autologous semen, and desensitization in patients with postorgasmic illness syndrome.

Introduction: Postorgasmic illness syndrome (POIS) is rare and includes a cluster of physical and cognitive symptoms that occur after ejaculation. The pathogenesis and effective treatments remain unclear. Aim: This study aimed to characterize the symptomatology of POIS, study the allergic response of autologous semen in patients and controls, and evaluate the effects of desensitization therapy. Methods: The clinical characteristics of 24 Chinese patients with POIS were analyzed. Skin prick tests, intracutaneous tests, and specific IgE detection were performed with autologous semen. Five patients were desensitized via subcutaneous injections of autologous semen. Outcomes: Evaluated outcomes included the clinical features of POIS; scores of the Self-rating Anxiety Scale (SAS), Self-rating Depression Scale (SDS), and visual analog scale (VAS) of symptoms; skin reactions; desensitization with diluted autologous seminal fluid; and the IgE reactivity patterns of immunoblotting and enzyme-linked immunosorbent assay in vitro. Results: The most common symptom cluster was the general cluster, and the most prevalent symptoms were extreme fatigue and inattention. A total of 66.67% (14/21) of the patients had no symptoms or milder symptoms after nocturnal emission than after intercourse or masturbation. Of the patients, 87.5% (21/24) had psychiatric symptoms and 53.85% (7/13) had abnormal sex hormone levels. The SAS and SDS scores of the high and low VAS groups were significantly higher than those of the control group. Pearson analysis showed that the correlation coefficient between the SAS and VAS was 0.607 (P < .01) and that between the SDS and VAS was 0.490 (P < .05). The patients and healthy donors all had positive intracutaneous test results with their own semen, negative skin prick test results, and no IgE specific to autologous semen. Most patients (4/5) did not achieve ideal therapeutic effects with desensitization. Clinical Implications: Allergy is not the main pathogenesis of POIS, and desensitization with autologous semen is not effective for most patients. Strengths and Limitations: This project included the largest number of patients with POIS in China and assessed the allergic response to autologous semen and the effect of desensitization therapy. There is no objective method for evaluating the efficacy of desensitization with autologous semen. Conclusions: IgE-mediated semen allergy is not the main pathogenesis of POIS, and there is a positive chance that POIS is related to psychological factors. Most patients do not respond to desensitization with autologous semen, and POIS treatment should be individualized, especially in cases with uncertain causes.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

Effects of salinomycin on human ovarian cancer cell line OV2008 are associated with modulating p38 MAPK.

This study investigated the anticancer effect and mechanism of salinomycin, a selective inhibitor of cancer stem cell, on human ovarian cancer cell line OV2008 in vitro and in vivo. The growth inhibitory effect of salinomycin on ovarian cancer cell line OV2008 was determined by measuring cell viability using the resazurin reduction assay. Apoptotic nuclear morphology was visualized by 4,6-diamino-2-phenylindole staining technique. The percentages of apoptotic cells and cell cycle parameters were detected by flow cytometry. The activity of p38 mitogen-activated protein kinase (p38 MAPK) was analyzed by Bio-Plex phosphoprotein assay. In vivo activity of salinomycin was assayed through tumor growth. Salinomycin caused concentration- (0.01-200 µM) and time-dependent (24-72 h) growth inhibitory effects in OV2008. Cell nuclear morphology observations showed that salinomycin-treated OV2008 cells displayed typical apoptotic characteristics. Salinomycin significantly increased the percentages of apoptotic cells in OV2008, showing a concentration- and time-dependent manner. There was no cell cycle arrest in the G1/G0, S, and G2/M phases between salinomycin-treated cells and control cells. Salinomycin also enhanced the phosphorylation of p38 MAPK. Moreover, salinomycin significantly inhibited the growth of the ovarian xenograft tumors. Salinomycin exhibited significant growth inhibition and induction of apoptosis in the human ovarian cancer cell line OV2008. The data suggested that salinomycin-induced apoptosis in OV2008 might be associated with activating p38 MAPK and merits further investigations.

Methylation signature of lymph node metastases in breast cancer patients.

BACKGROUND: Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers. METHODS: The quantitative methylation analysis was performed using the SEQUENOM's EpiTYPER™ assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis. CONCLUSIONS: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis.

Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma.

BACKGROUND: Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank's plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input. METHODS: Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA. RESULTS: Instead of 400-800 µL (routine input in the laboratory), 50 µLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA. CONCLUSIONS: The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

Epigenetic therapy for breast cancer.

Both genetic and epigenetic alterations can control the progression of cancer. Genetic alterations are impossible to reverse, while epigenetic alterations are reversible. This advantage suggests that epigenetic modifications should be preferred in therapy applications. DNA methyltransferases and histone deacetylases have become the primary targets for studies in epigenetic therapy. Some DNA methylation inhibitors and histone deacetylation inhibitors are approved by the US Food and Drug Administration as anti-cancer drugs. Therefore, the uses of epigenetic targets are believed to have great potential as a lasting favorable approach in treating breast cancer.

MALDI-TOF mass array analysis of RASSF1A and SERPINB5 methylation patterns in human placenta and plasma.

Differences in DNA methylation patterns between placenta and blood cells of pregnant women have been suggested as potential biomarkers for noninvasive prenatal diagnostic strategies, including for common obstetrical complications, such as preeclampsia. New findings in epigenetic origins of fetal or placental disorders may improve our ability for optimal management of these conditions. Using a novel high-throughput mass spectrometry on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass array, we compared the quantitative methylation changes of RASSF1 and SERPINB5 (also known as MASPIN) genes in placenta and plasma samples. We analyzed the methylation status of a total of 3569 CpG dinucleotides on these two genes in 83 different samples: 50 plasma samples (20 from pregnant women and 30 from nonpregnant women) and 33 placenta tissue samples (25 from normal pregnancies and eight from preeclamptic pregnancies). The aim of this study was to assess the utility of epigenetic changes as biomarkers for noninvasive prenatal diagnostic procedures. Using a two-way hierarchical cluster analysis, significantly different methylation levels of the RASSF1 gene were found between placenta (normal and preeclamptic) and plasma samples of pregnant women. Although the SERPINB5 gene was hypomethylated in placenta DNA more than in plasma DNA, it did not demonstrate significant differences between studied groups. The MALDI-TOF mass spectrometry analysis of placenta and plasma DNA methylation patterns may serve as a tool for the study of gender-independent biomarkers in noninvasive prenatal diagnosis.

Correlation of telomere length shortening with promoter methylation profile of p16/Rb and p53/p21 pathways in breast cancer.

Unregulated cell growth, a major hallmark of cancer, is coupled with telomere shortening. Measurement of telomere length could provide important information on cell replication and proliferation state in cancer tissues. Telomere shortening and its potential correlation with downregulation of cell-cycle regulatory elements were studied by the examination of relative telomere length and methylation status of the TP53, P21 and P16 promoters in tissues from breast cancer patients. Telomere length was measured in 104 samples (52 tumors and paired adjacent normal breast tissues) by quantitative PCR. Methylation profile of selected genes was analyzed in all samples using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results demonstrated a significant shortening of tumor telomere regions compared with paired adjacent normal tissues (P<0.001). Similarly, telomere lengths were significantly shorter in advanced stage cases and in those with higher histological grades (P<0.05). Telomere shortening in cancer tissues was correlated with a different level of hypermethylation in the TP53, P21 and P16 promoters (r=-0.33, P=0.001; r=-0.70, P<0.0001 and r=-0.71, P<0.0001, respectively). The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, telomere shortening and promoter hypermethylation of related genes both might serve as breast cancer biomarkers.

Epigenetics of ovarian cancer: from the lab to the clinic.

OBJECTIVE: Ovarian cancer remains the most lethal gynaecological cancer. Various molecular changes have been identified and have shown promise for their diagnostic, prognostic and curative capacity but still need further validation. Among different mechanisms, the present article reviews the importance of epigenetic changes in ovarian cancer. METHODS: Recent literature relevant to epigenetics of ovarian cancer has been reviewed. RESULTS: Greater insight into the epigenetic phenomena DNA methylation, histone modification and posttranscriptional gene downregulation by microRNAs is provided. In addition, the contribution of epigenetic control of gene expression to ovarian oncology is analysed and its potential in the clinic is considered. CONCLUSIONS: Although the epigenetics of ovarian cancer is still in its beginnings, it holds promising potential in early stage ovarian cancer detection, evaluation of prognosis/drug resistance and targeted cancer treatment.

Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors.

BACKGROUND: With the aim to simplify cancer management, cancer research lately dedicated itself more and more to discover and develop non-invasive biomarkers. In this connection, circulating cell-free DNA (ccf DNA) seems to be a promising candidate. Altered levels of ccf nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been found in several cancer types and might have a diagnostic value. METHODS: Using multiplex real-time PCR we investigated the levels of ccf nDNA and mtDNA in plasma samples from patients with malignant and benign breast tumors, and from healthy controls. To evaluate the applicability of plasma ccf nDNA and mtDNA as a biomarker for distinguishing between the three study-groups we performed ROC (Receiver Operating Characteristic) curve analysis. We also compared the levels of both species in the cancer group with clinicopathological parameters. RESULTS: While the levels of ccf nDNA in the cancer group were significantly higher in comparison with the benign tumor group (P < 0.001) and the healthy control group (P < 0.001), the level of ccf mtDNA was found to be significantly lower in the two tumor-groups (benign: P < 0.001; malignant: P = 0.022). The level of ccf nDNA was also associated with tumor-size (<2 cm vs. >2 cm<5 cm; 2250 vs. 6658; Mann-Whitney-U-Test: P = 0.034). Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (cut-off: 1866 GE/ml; sensitivity: 81%; specificity: 69%; P < 0.001) and between the tumor group and the healthy controls using ccf mtDNA as marker (cut-off: 463282 GE/ml; sensitivity: 53%; specificity: 87%; P < 0.001). CONCLUSION: Our data suggests that nuclear and mitochondrial ccf DNA have potential as biomarkers in breast tumor management. However, ccf nDNA shows greater promise regarding sensitivity and specificity.

High-throughput hacking of the methylation patterns in breast cancer by in vitro transcription and thymidine-specific cleavage mass array on MALDI-TOF silico-chip.

Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design.

Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer.

BACKGROUND: Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters. METHODS: Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein. RESULTS: The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed. CONCLUSION: Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens.

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs). STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing. RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci. CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.

Circulating cell-free DNA as a potential biomarker for minimal and mild endometriosis.

It has recently been reported that high concentrations of circulating cell-free (ccf) nucleic acids in plasma and serum could be used as biomarkers for non-invasive monitoring a wide variety of malignant and benign proliferations and inflammatory conditions. Endometriosis is one of the most common benign gynaecological proliferations with inflammatory activation in premenopausal women. Real-time multiplex polymerase chain reaction was used for synchronized quantification of the glyceraldehyde-3-phosphate dehydrogenase gene sequence in nuclear DNA (nDNA) and the ATP synthase-8 gene sequence in mitochondrial DNA (mtDNA). DNA was extracted from 500 microl serum and plasma of 19 cases with endometriosis to measure the total amount of ccf nDNA and ccf mtDNA. The concentration of ccf nDNA in plasma was significantly higher in the endometriosis group than in the control group (P = 0.046). The cut-off value selected by a receiver operating characteristic curve could provide a sensitivity of 70% and a specificity of 87% to discriminate between the minimal or mild cases and normal controls. The finding of significantly increased concentrations of ccf nDNA in plasma of patients with endometriosis suggests that ccf nDNA might be a potential biomarker for developing non-invasive diagnostic test in endometriosis.

Current understanding of mitochondrial DNA in breast cancer.

The recent surge in mitochondrial research has been driven by the identification of mitochondria-associated diseases and the role of mitochondria in apoptosis and aging. Mitochondrial DNA (mtDNA) has been proposed to be involved in carcinogenesis because of its high susceptibility to mutations and limited repair mechanisms in comparison to nuclear DNA. As mtDNA lacks introns, it has been suggested that most mutations will occur in coding sequences. The subsequent accumulation of mutations may lead to tumor formation. By virtue of their clonal nature, high copy number and high frequent mutations may provide a powerful molecular biomarker for the detection of cancer. It has been suggested that the extent of mtDNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In this review article, we aim to provide a brief summary of our current understanding of mitochondrial genetics and biology, review the mtDNA alterations reported in breast cancer, and offer some perspectives as to the emergence of mtDNA mutations, including their functional consequences in cancer development, diagnostic criteria, and therapeutic implications.

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry.

PURPOSE: To examine the potential high throughput capability and efficiency of an automated DNA extraction system in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status. METHODS: A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray mass spectrometric system. RESULTS: We determined that as little as 15 pg of RHD-positive genomic DNA could be detected in a background of 585 pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and specificity of 96.1 and 96.1%, respectively. CONCLUSION: Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using real-time PCR.