Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing

Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients’ plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.     

Improving early breast cancer detection: focus on methylation

The need for additional breast cancer screening tools is indisputably high, as one may conclude from the high rate of interval malignancies in women undergoing regular screening. DNA promoter methylation frequently occurs during breast carcinogenesis and is an early event in this process. Moreover, a field defect for methylation has been described and methylation values can reliably be assessed in limited amounts of DNA. Simultaneous detection of methylation of a panel of genes in breast fluids and/or blood derivatives could be both sufficiently specific and sensitive to be of additive value to current imaging-based screening methods. This review describes the recent developments in methylation detection in breast fluids, serum and plasma that paved the way for large prospective studies. These studies will provide us with the definite answer as to what will be the additive value of defining the methylation status of specific genes to current imaging-based screening methods.     

Detection of cancer-specific epigenomic changes in biofluids: Powerful tools in biomarker discovery and application

The genetic and epigenetic material originating from tumour that can be found in body fluids of individuals with cancer harbours tumour-specific alterations and represents an attractive target for biomarker discovery. Epigenetic changes (DNA methylation, histone modifications and non-coding RNAs) are present ubiquitously in virtually all types of human malignancies and may appear in early cancer development, and thus they provide particularly attractive markers with broad applications in diagnostics. In addition, because changes in the epigenome may constitute a signature of specific exposure to certain risk factors, they have the potential to serve as highly specific biomarkers for risk assessment. While reliable detection of cancer-specific epigenetic changes has proven to be technically challenging, a substantial progress has been made in developing the methodologies that allow an efficient and sensitive detection of epigenomic changes using the material originating from body fluids. In this review we discuss the application of epigenomics as a tool for biomarker research, with the focus on the analysis of DNA methylation in biofluids.     

Detection and relevance of epigenetic markers on ctDNA: recent advances and future outlook

Liquid biopsy, a minimally invasive approach, is a highly powerful clinical tool for the real‐time follow‐up of cancer and overcomes many limitations of tissue biopsies. Epigenetic alterations have a high potential to provide a valuable source of innovative biomarkers for cancer, owing to their stability, frequency, and noninvasive accessibility in bodily fluids. Numerous DNA methylation markers are now tested in circulating tumor DNA (ctDNA) as potential biomarkers, in various types of cancer. DNA methylation in combination with liquid biopsy is very powerful in identifying circulating epigenetic biomarkers of clinical importance. Blood‐based epigenetic biomarkers have a high potential for early detection of cancer since DNA methylation in plasma can be detected early during cancer pathogenesis. In this review, we summarize the latest findings on DNA methylation markers in ctDNA for early detection, prognosis, minimal residual disease, risk of relapse, treatment selection, and resistance, for breast, prostate, lung, and colorectal cancer.     

Prognostic and predictive roles for circulating biomarkers in gastrointestinal cancer

Circulating tumor cells (CTCs) and circulating free DNA (cfDNA) have been studied as promising prognostic and predictive tumor-derived biomarkers in the bloodstream of patients with gastrointestinal malignancies because they may be an alternative noninvasive tool to tumor tissue biopsies. Quantification and molecular characterization of CTCs and cfDNA may provide additional insights into cancer biology, potentially revealing novel targets to individualize cancer care. The present article aims to review the biology and current methods to assess CTCs and cfDNA, and the efforts to establish both tumor-derived biomarkers as prognostic and predictive factors in esophageal, gastric and colorectal cancer.     

A plasma metabolite panel as biomarkers for early primary breast cancer detection

In recent years, metabolites have attracted substantial attention as promising novel biomarkers of various diseases. However, breast cancer plasma metabolite studies are still in their infancy. Here, we investigated the potential of metabolites to serve as minimally invasive, early detection markers of primary breast cancer. We profiled metabolites extracted from the plasma of primary breast cancer patients and healthy controls using tandem mass spectrometry (UHPLC-MS/MS and FIA-MS/MS). Two metabolites were found to be upregulated, while 16 metabolites were downregulated in primary breast cancer patients compared to healthy controls in both the training and validation cohorts. A panel of seven metabolites was selected by LASSO regression analysis. This panel could differentiate primary breast cancer patients from healthy controls, with an AUC of 0.87 (95% CI: 0.81 ~ 0.92) in the training cohort and an AUC of 0.80 (95% CI: 0.71 ~ 0.87) in the validation cohort. These significantly differentiated metabolites are mainly involved in the amino acid metabolism and breast cancer cell growth pathways. In conclusion, using a metabolomics approach, we identified metabolites that have potential value for development of a multimarker blood-based test to complement and improve early breast cancer detection. The panel identified herein might be part of a prescreening tool, especially for younger women or for closely observing women with certain risks, to facilitate decision making regarding which individuals should undergo further diagnostic tests. In the future, the combination of metabolites and other blood-based molecular marker sets, such as DNA methylation, microRNA, and cell-free DNA mutation markers, will be an attractive option.     

Antibody microarray analysis of the serum proteome in primary breast cancer patients

Noninvasive biomarkers are urgently needed for detecting breast cancer as early as possible since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. There are currently no such biomarkers in clinical use as a diagnostic tool. Proteomic analysis of protein expression patterns in body fluids has potential for use in identifying biomarkers of breast cancer. The aim of this study was to compare protein expression levels in the sera of primary breast cancer patients and healthy controls. An antibody microarray tool with 23 antibodies immobilized on nitrocellulose slides was used to determine the levels of acute phase proteins, interleukins, and complement factors in the sera of 101 study participants (49 women with primary breast cancer and 52 healthy age-matched controls). Statistical analysis of reaction intensities identified 6 proteins that showed significantly (p < 0.05) different levels in breast cancer patients vs. healthy subjects. The neural network distinguished cancer patients from controls with a sensitivity of 69% and a specificity of 76%. Thus, antibody microarray analysis could be used as a tool for the development of improved diagnostics and biomarker discovery for breast cancer patients. Further validation of the results and de novo screening of new biomarkers could facilitate the early diagnosis of breast cancer.     

Diagnostic and prognostic role of cell‐free DNA testing for colorectal cancer patients

Circulating cell‐free DNA (cfDNA) was found in increased amounts in cancer patients and tumor‐associated molecular alteration can be detected in cancer patient's samples. For this reason, the cfDNA analysis is actually considered as a new concept of liquid biopsy. We evaluated the presence and integrity of plasma cfDNA by ALU‐based qPCR and the methylation profile of OSMR and SFRP1 genes promoter in a large cohort of colorectal cancer (CRC) patients (n = 114) in comparison to healthy subjects (n = 56) and patients with adenomatous lesions (n = 22). Moreover, we studied the prognosis value focusing on histopathological staging and survival. The cfDNA concentration and the integrity index were increased in CRC patients. The ALU83 and ALU244 fragment dosage showed a moderate discriminant capacity between CRC patients and controls and CRC and adenoma patients. Especially, cfDNA was significantly higher in CRC patients at advanced histopathological stage. In addition, the increased cfDNA level was associated with poor prognosis. A comparison of methylation profile in matched tissue and plasma on 25 CRC patients was performed and only three mismatched cases were observed. A lower methylation quantification was observed in cfDNA than tissue DNA. The cfDNA methylation frequency was statistically different in controls, adenoma and CRC patients and this frequency increased with the histopathological stage of tumor. The adenoma and CRC patients methylated cfDNA showed a higher quantity of ALU83 and ALU244. An integrated approach, combining the detection of ALU fragments and cancer type‐specific epigenetic alteration, can improve diagnostic efficiency and better define the prognostic value for CRC disease.     

Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening

Introduction

For early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.

Methods

We examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.

Results

Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.

Conclusions

Tumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.     

Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation-specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell-free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer-specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer-free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.     

Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification

BackgroundTargeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis.Patients and methodsWe developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients’ outcomes.ResultsMedian methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016).ConclusionsComprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.     

Assessment of cell-free DNA (cfDNA) concentrations in the perioperative period can predict risk of recurrence in patients with non-metastatic breast cancer

BackgroundCirculating cell-free DNA (cfDNA) is a potential non-invasive biomarker of disease status in patients with cancer, and provides important diagnostic and prognostic information in breast cancer. The goal of this study was to quantify cfDNA concentrations during the perioperative period and investigate its potential utility to detect recurrence outcomes in patients with breast cancer.MethodsSixty-two (n = 62) patients with non-metastatic breast cancer, undergoing curative-intent surgery were screened for inclusion. Blood samples were collected from these patients: pre-operatively (Preop) and post-operatively (PO) at either of the following PO time points; PO week 1-2, PO week 3-4 and PO weeks 5-12 following surgery. cfDNA was extracted and quantified using nanodrop spectrophotometer.ResultsIn a cohort of 62 patients (age, median (IQR), 51.5(45.0-65.0) years), with a median follow-up of 90 months (interquartile range (IQR),60-120 months), significant association was observed between cfDNA concentrations and risk of recurrence in patients with breast cancer. The group of patients who had disease recurrence during follow-up had significantly higher cfDNA concentrations (cutoff:400 ng/ml) compared to the group of patients who remain disease-free (Preop and PO period: p < 0.0001). The median Recurrence Free Survival (RFS) between the Disease Recurrence (DR) and the Disease Free (DF) groups of patients with breast cancer were 12(20-28.5) months and 72.00 (96-120) months; p < 0.0001). Univariate and multivariate cox regression analysis indicated that postoperative cfDNA concentration (Hazard ratio:5.0, 95% Confidence Interval:1.19-21.28, p = 0.028) was an independent negative prognostic factor for RFS in patients with non-metastatic breast cancer.ConclusionOur study demonstrated that high postoperative cfDNA is associated with increased risk of future recurrence in patients with non-metastatic breast cancer. Further, prospective studies are warranted to validate its clinical utility in breast cancer.     

Epigenetic biomarkers in lung cancer

Lung cancer mortality is strongly associated with the predominant diagnosis of late stage lesions that hampers effective therapy. Molecular biomarkers for early lung cancer detection is an unmet public health need and the lung cancer research community worldwide is putting a lot of effort to utilise major lung cancer population programmes in order to develop such molecular tools. The study of cancer epigenetics in the last decade has radically altered our views in cancer pathogenesis, providing new insights in biomarker development for risk assessment, early detection and therapeutic stratification. DNA methylation and miRNAs have rapidly emerged as potential biomarkers in body fluids showing promise to assist the clinical management of lung cancer. These new developments are exemplified in this review, demonstrating the huge potential of clinical cancer epigenetics, but also critically discussing the necessary validation steps to bring epigenetic biomarkers towards clinical implementation and the weaknesses of current biomarker studies.     

Prognostic role of PIK3CA mutations of cell‐free DNA in early‐stage triple negative breast cancer

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early‐stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse‐free survival (RFS; P = 0.0072) and breast cancer‐specific survival (BCSS; P = 0.016), according to the log‐lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early‐stage TNBC patients and it was associated with PI3K pathway‐dependent AR phosphorylation.     

DNA Methylation and Colorectal Cancer

Colorectal cancer (CRC) is one of the major cancers in the world and the second death-causing cancer in the USA. CRC development involves genetic and epigenetic alterations. Changes in DNA methylation status are believed to be involved at different stages of CRC. Promoter silencing via DNA methylation and hypomethylation of oncogenes alters gene expression and can be used as a tool for the early detection of colonic lesions. DNA methylation use as a diagnostic and prognostic marker has been described for many cancers including CRC. CpG island methylator phenotype (CIMP) is one of the underlying CRC mechanisms. This review aims to define methylation signatures in CRC. The analysis of DNA methylation profile in combination with the pathological diagnosis would be useful in predicting CRC tumor evolution and their prognostic behavior.     

Methylation profiling of normal individuals reveals mosaic promoter methylation of cancer-associated genes

Epigenetic silencing by promoter methylation of genes associated with cancer initiation and progression is a hallmark of tumour cells. As a consequence, testing for DNA methylation biomarkers in plasma or other body fluids shows great promise for detection of malignancies at early stages and/or for monitoring response to treatment. However, DNA from normal leukocytes may contribute to the DNA in plasma and will affect biomarker specificity if there is any methylation in the leukocytes. DNA from 48 samples of normal peripheral blood mononuclear cells was evaluated for the presence of methylation of a panel of DNA methylation biomarkers that have been implicated in cancer. SMART-MSP, a methylation specific PCR (MSP) methodology based on real time PCR amplification, high-resolution melting and strategic primer design, enabled quantitative detection of low levels of methylated DNA. Methylation was observed in all tested mononuclear cell DNA samples for the CDH1 and HIC1 promoters and in majority of DNA samples for the TWIST1 and DAPK1 promoters. APC and RARB promoter methylation, at a lower average level, was also detected in a substantial proportion of DNA samples. We found no BRCA1, CDKN2A, GSTP1 and RASSF1A promoter methylation in this sample set. Several individuals had higher levels of methylation at several loci suggestive of a methylator phenotype. In conclusion, methylation of many potential DNA methylation biomarkers can be detected in normal peripheral blood mononuclear cells, and is likely to affect their specificity for detecting low level disease. However, we found no evidence of promoter methylation for other genes indicating that panels of analytically sensitive and specific methylation biomarkers in body fluids can be obtained.     

The diagnostic and prognostic utility of prostate-specific antigen for diseases of the breast

Although prostate-specific antigen (PSA) is the most valuable tumor marker for the diagnosis and management of prostate carcinoma, it is widely accepted that PSA is not prostate specific. Numerous studies have shown that PSA is present in some female hormonally regulated tissues, principally the breast and its secretions. In this review, we summarize the findings of PSA in the breast, and focus on its potential for clinical applications in breast disease. PSA is produced by the majority of breast tumors and is a favorable indicator of prognosis in breast cancer. Low levels of PSA are released into the female circulation, and while the level of serum PSA is elevated in both benign and malignant breast disease, the molecular form of circulating PSA differs between women with and without breast cancer. These findings indicate that PSA may have potential diagnostic utility in breast cancer. PSA may also have a clinical application in benign breast disease, as both the level and molecular form of PSA differ between Type I and II breast cysts. High levels of PSA have been reported in nipple aspirate fluid (NAF) and recent studies have shown that the concentration of PSA in NAF is inversely related to breast cancer risk, indicating that NAF PSA may represent a clinical tool for breast cancer risk assessment. Thus, PSA represents a marker with numerous potential clinical applications as a diagnostic and/or prognostic tool in breast disease.     

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.