Specificity of Methylation Assays in Cancer Research: A Guideline for Designing Primers and Probes

DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers. A large number of cancer studies have been focused on identification of methylation changes as biomarkers (i.e., breast cancer). However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test. This highlights the critical need for specific primer and probe design to avoid false-positive detection of methylation profiling. The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step

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 P14ARF, 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 P14ARF 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 cance

Assessing the value of CAN-gene mutations using MALDI-TOF MS

Purpose: To identify cancer-linked genes, Sjöblom et al. and Wood et al. performed a genome-wide mutation screening in human breast and colorectal cancers. 140 CAN-genes were found in breast cancer, which in turn contained overall 334 mutations. These mutations could prove useful for diagnostic and therapeutic purposes. Methods: We used a MALDI-TOF MS 40-plex assay for testing 40 loci within 21 high-ranking breast cancer CAN-genes. To confirm mutations, we performed single-plex assays and sequencing. Results: In general, the mutation rate of the analyzed loci in our sample cohort was very low. No mutation from the 40 loci analyzed could be found in the 6 cell lines. In tissue samples, a single breast cancer tissue sample showed heterozygosity at locus c.5834G>A within the ZFYVE26 gene (Zinc finger FYVE domain-containing gene 26). Conclusions: Sjöblom et al./Wood et al. already showed that the vast majority of CAN-genes are mutated at very low frequency. Due to the fact that we only found one mutation in our cohort, we therefore assume that at the selected loci, mutations might be low-frequency events and therefore, more rarely detectable. However, further evaluation of the CAN-gene mutations in larger cohorts should be the aim of further studie

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

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

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

Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients

P53 pathway alterations in breast cancer for biomarker discovery

Breast cancer has the highest incidence rate amongst female cancers worldwide. Following the advent of mammography and the utilization of advanced treatment strategies breast cancer mortality rates have markedly declined. However, in order to treat the refractory and more aggressive forms of the disease, development of detection methods using novel biomarkers and more efficacious therapeutic protocols are still needed. P53 dysfunction and impaired p53 signalling has been reported in over 50% of human cancers. Impairment of p53 activity in various cancers has primarily been attributed to P53 gene mutations. In addition to gene mutations, however, alterations in the activity of upstream and/or downstream TP53 regulators (e.g. autoregulatory loops, proteins and miRNAs) also modulate the activity of the p53 pathway. The frequency of p53 mutations in breast cancer is lower than other cancers (approximately 20%). The impact of these additional p53 regulatory mechanisms on breast cancer pathology is poorly understood. To address this issue, the present study examined the following: I. Identify novel mechanisms of p53 pathway inactivation in breast cancer, independent of P53 gene mutations. II. Establish an association between aberrant DNA methylation of p53 regulatory pathways and downstream p53 activity. III. Evaluate differences in aberrant DNA methylation signatures between primary tumors and metastatic lesions and its contribution to pathology. In the first part of the study, we demonstrated that both P14ARF and PTEN (P <0.05 and P <0.01, respectively) promoters were hypermethylated in breast cancer patients. Hypermethylation of both promoters could potentially mediate p53 inactivation via the dysregulation of p53 auto-regulatory feedback loops. Indeed, proteomic characterization of breast cancer cell lines upon demethylation therapy revealed the overexpression of p53 regulatory proteins - Atic, Calr and Pcna. This indicates a putative role of DNA methylation on the regulation of p53 pathway activity. Furthermore, we identified a subset of differentially expressed miRNAs that targeted p53 pathway components. In this context, over-expression of miR-21 (P<0.05) is potentially important for the pathogenesis of wt-p53 breast cancer patients. Second, we were able to demonstrate a significant correlation between the hypermethylation of P16/Rb and P53/P21 promoters and the shortening of telomere length in breast cancer patients (r=-0.33, P=0.001; r=-0.70, P<0.0001 and r=-0.71, P<0.0001; respectively). Moreover, hypermethylation of P14ARF and PTEN was associated with increased mitochondrial DNA (mt-DNA) damages including: depletion of mt-DNA content (11.88-fold changes; P< 0.01) and enhancement of mutations in the D-loop region (36.36%). Lastly, we explored the methylation signature of 12 breast cancer candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14, P16, P21, PTEN and TIMP3). There was considerable heterogeneity in the methylation status of primary tumors and metastatic lesions. Higher methylation levels at promoter regions of APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 were identified in primary tumor tissues (P<0.05, P<0.05, P<0.01, P<0.0001, P<0.01, P<0.01, P<0.05, P<0.05, P<0.01; respectively), whereas in metastatic lymph node lesions only BMP6, BRCA1 and P16 were hypermethylated (P<0.05 and P<0.01, P<0.0001, P<0.05; respectively). In conclusion, the present study identified novel biomarkers for tumor specific and metastatic breast cancers. Aberrant methylation signature of APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14ARF, P16, P21, PTEN and TIMP3, as well as overexpression of miR-21, increased mt-DNA damages and shortened telomere length specifically identified tumor specific lesions. Moreover, hypermethylation of BMP6, BRCA1 and P16 gene promoters indicate their potential use as metastatic biomarkers. These novel biomarkers will be further examined for the effect of specific hypermethylated CpG sites on the transcriptional activity of each gene. Furthermore, establishing absolute percent methylation cutoffs for each breast tumor sub-type would enable their utilization in clinical laboratory settings

Cell-free DNA in the circulation as a potential cancer biomarker

In the course of the search for new biomarkers, circulating cell-free DNA (ccf-DNA) has become a popular target of interest. An elevated level of ccf-DNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since ccf-DNA in cancer patients often bears similar genetic and epigenetic features to the related tumor DNA, there is evidence that some of the ccf-DNA originates from tumoral tissue. This, and the fact that ccf-DNA can easily be isolated from the circulation and other body fluids of patients, makes it a promising candidate as a non-invasive biomarker of cancer. Yet ccf-DNA-based cancer tests have not come to fruitful clinical applications. This review evaluates the potential of ccf-DNA alterations as a biomarker for cancer management by addressing the question of how large the gap between ccf-DNA and the ideal cancer biomarker is

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

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