Zeinab Barekati

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.

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.

New Trends in Molecular Biomarker Discovery for Breast Cancer

Breast cancer is one of the most common and leading causes of cancer death in women. Early diagnosis, selection of appropriate therapeutic strategies, and efficient follow-up play an important role in reducing mortality. Recently, HER-2/neu in breast cancer has been routinely used to guide treatment of using Trastuzumab in less than 25-30% of patients. More new biomarkers will be still expected in the future to tailor treatments. However, there are still many obstacles in developing clinically useful biomarker tests for clinical practice. A lack of specificity of tumor markers and lack of sensitivity of testing systems have been noticed, which limit their clinical use. Finding biomarkers for breast cancer could allow physicians to identify individuals who are susceptible to certain types and stages of cancer to tailor preventive and therapeutic modalities based on the genotype and phenotype information. These biomarkers should be cancer specific, and sensitively detectable in a wide range of specimen(s) containing cancer-derived materials, including body fluids (plasma, serum, urine, saliva, etc.), tissues, and cell lines. This review highlights the new trends and approaches in breast cancer biomarker discovery, which could be potentially used for early diagnosis, development of new therapeutic approaches, and follow-up of patients.

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.

Targeted therapy in breast cancer: what's new?

Breast cancer is the most commonly diagnosed malignancy and one of the major causes of death among women. Breast cancer is also one of the most investigated diseases but whose biological features are still not well understood, several effective treating strategies having been explored in dealing with different types of advanced breast cancer, such as endocrine therapy and molecular targeted therapy. Trastuzumab is the first approved targeted anti-cancer agent to show an attractive response rate and outcomes in treating HER-2 positive metastatic breast cancer patients. However, primary or acquired trastuzumab resistance usually occurs some time into the use of trastuzumab and leads to treatment resistance or tumour progression. The promising results with trastuzumab targeted therapy encouraged further investigations in this area exploring several novel targeted agents aiming to overcome the resistance drawback of trastuzumab. In this review we discuss the major newly developed targeted agents in breast cancer treatment, including the novel anti-HER-2 monoclonal antibody pertuzumab or ertumaxomab, small molecular tyrosine inhibitor lapatinib, selective PARP1 inhibitor olaparib, mTOR inhibitor rapamycin analogues, and sheddase inhibitors. Many of these novel targeted drugs or molecules showed additional or complementary effects to trastuzumab therapy that need further and wider investigation.

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.

Effects of Amifostine in Combination With Cyclophosphamide on Female Reproductive System

This study investigated the cytoprotective effects of amifostine against the adverse effects of cyclophosphamide relying on ovarian cell death markers and the fertilization rate of the surviving follicles as a late outcome of the study. Combined pretreatment of amifostine with cyclophosphamide enabled partial recovery of antiapoptotic messenger RNA (mRNA) expression levels while a decrease in expression of BAX and Casp3 were identified. The pretreatment of amifostine to cyclophosphamide significantly reduced the proportion of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive primary and preantral follicles (P < .001 and P < .05, respectively). These findings were comparable with the results obtained from protein expression of cleaved caspase 3. The fertilization rate showed a significant capability of amifostine to improve fertilization potency of oocytes exposed to cyclophosphamide (P < .01). In conclusion, administration of amifostine prior to cyclophosphamide might serve as a promising protocol to improve female fertility.