Ramin Radpour

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

BackgroundWith 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.MethodsUsing 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.ResultsWhile 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).ConclusionOur 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.

Hypermethylation of Tumor Suppressor Genes Involved in Critical Regulatory Pathways for Developing a Blood-Based Test in Breast Cancer

Background Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients. Methods and Findings To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples. Conclusions Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.

Levels of circulating cell-free nuclear and mitochondrial DNA in benign and malignant ovarian tumors

OBJECTIVE: To analyze the levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in patients with benign and malignant ovarian tumors using a gold-standard assay and to investigate whether quantitative alterations of the circulating cell-free species have values in the management of the patients. METHODS: One hundred four patients were recruited for this study. We developed a quantitative, multiplex polymerase chain reaction to measure the levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in serum and plasma of patients with epithelial ovarian cancer, benign epithelial ovarian tumors, or endometriosis. The levels of the circulating cell-free DNA were compared with those of a healthy, age-matched control group. RESULTS: The patients with epithelial ovarian cancer had significantly higher amounts of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in plasma compared with the healthy control group (mean of nuclear DNA 10,723/2,591 and mean of mitochondrial DNA 4,918,978/2,294,264, P=.009 and 0.022, respectively) and with the other group with benign ovarian diseases (mean of nuclear DNA 10,723/2,965 and mean of mitochondrial DNA 4,918,978/1,597,551, P=.027 and 0.002, respectively). However, no relationship between levels of the circulating cell-free DNA and the pathological parameters as well as CA 125 measurement in patients with epithelial ovarian cancer was found. A significant difference between the epithelial ovarian cancer and endometriosis group was found in circulating cell-free mitochondrial DNA but not in circulating cell-free nuclear DNA (mean of mitochondrial DNA 4,918,978/2,273,988 and mean of nuclear DNA 10,723/3,291, P=.013 and 0.105, respectively). CONCLUSION: Elevated levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in epithelial ovarian cancer may have diagnostic value. Our finding suggests that the circulating molecules might be potential biomarkers in the disease. LEVEL OF EVIDENCE: III

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

BackgroundAlterations 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.MethodsPeripheral 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.ResultsThe 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.ConclusionEarly 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.

Genetic Investigations of CFTR Mutations in Congenital Absence of Vas Deferens, Uterus, and Vagina as a Cause of Infertility

A qualitative diagnosis of infertility requires attention to male and female physical abnormalities including endocrine anomalies and genetic conditions that interfere with reproduction. Many genes are likely to be involved in the complex process of reproduction. Congenital bilateral absence of the vas deferens (CBAVD) is a genital form of cystic fibrosis (CF) that is responsible for 2%-6% of male infertility. The incidence of CF varies in different populations; therefore, the incidence of CBAVD will also vary in different populations. The spectrum and distribution of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations differ between CBAVD and CF patients and are comparable to control individuals. Combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR protein. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). Females with CF are found to be less fertile than normal healthy women. Because of techniques such as intracytoplasmic sperm injection (ICSI), CBAVD patients are now able to father children. Such couples, however, have an increased risk of having a child with cystic fibrosis, and therefore genetic testing and counseling should be provided. Around 10% of obstructive azoospermia is congenital and due to mutations in the CF gene. This review highlights the relationship of mutations in the CFTR gene with CBAVD and CAUV.

Methylation signature of lymph node metastases in breast cancer patients

BackgroundInvasion 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.MethodsThe 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).ResultsThe 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.ConclusionsThe 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.

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.

Integrated Epigenetics of Human Breast Cancer: Synoptic Investigation of Targeted Genes, MicroRNAs and Proteins upon Demethylation Treatment

Background The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2′-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. Methods and Findings Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. Conclusions In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.

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. (Mol Cancer Res 2008;6(11):1702–9)

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.