ctDNA Methylation Research Articles

Potential utility of methylation levels detected from circulating tumor DNA (ctDNA) in predicting molecular residual disease (MRD) in patients with resected non-small cell lung cancer (NSCLC).

e15535 Background: To improve the prognosis of resected lung cancer patients, growing efforts are being invested in finding the most optimal approach to detect MRD and predict relapse. In our study, we aimed to investigate the utility of ctDNA methylation profiling in detecting MRD from patients with resected early-stage NSCLC. Methods: Surgically-resected tumor tissues were obtained from 65 patients diagnosed with resectable stage IA-III NSCLC with various histological subtypes. Matched blood samples were also collected before surgery (baseline) and during regular follow-up after 2-8 weeks of surgery. Comprehensive somatic mutation and methylation level profile from circulating tumor DNA (ctDNA) were performed using unique molecular identifier-based targeted sequencing and targeted bisulfite sequencing, respectively. A tumor-informed MRD prediction model was constructed based on methylation levels obtained from the patient’s resected tumor tissue to calculate the corresponding methylation signal intensity from the matched plasma sample of the patient at baseline or other time points during follow-up, which is reflected as MRD score. Results: Of the 28 patients with baseline ctDNA methylation data, 28.6% (8/28) of the patients had elevated ctDNA methylation levels at first post-operative follow-up (F1) as compared to baseline, indicating the possibility of MRD. Meanwhile, of the 20 patients (71.4%, 20/28) who had reduced ctDNA methylation levels at F1, elevation of ctDNA methylation level was detected from 3 and 1 patients at second (F2) and third (F3) follow-up, respectively. Based on the MRD prediction model, 17.9% (5/28) of the patients had higher MRD scores at F1. Of the 23 patients with lower MRD scores at F1, 5, 1 and 1 patients had an elevation in MRD scores at F2, F3, and F4, respectively, which indicates a possible MRD. Disease relapse was radiologically confirmed after 10-16 months post-surgery in three patients with concomitant elevation of ctDNA methylation level and MRD score during follow-up between 2-9 months prior to radiologic relapse. Conclusions: Our results demonstrate that post-operative ctDNA methylation levels could be used to detect MRD in patients with resected NSCLC. Moreover, ctDNA methylation-based prediction model of MRD could serve as a potential model to predict relapse in early-stage as well as disease progression in advanced-stage NSCLC.

A noninvasive multi-analytic approach for lung cancer screening of patients with pulmonary nodules.

1550 Background: Low-dose computed tomography (LDCT) is an effective approach for lung cancer screening of high-risk patients with pulmonary nodules, however with varying false positive rates depending on the somewhat subjective judgement of the practice professional. Artificial intelligence derived from machine learning of comprehensive patient profiles, including multi-omics and clinical data, has the potential to provide more objective assessment of patient’s risk in order to aid clinician’s decision making. We have developed a multi-analyte algorithm-based assay (MAAA) that incorporates ctDNA mutation, ctDNA methylation, and protein biomarker profiles evaluated through non-invasive blood-based testing, as well as patient’s clinical information, to improve the diagnostic efficacy of lung cancer. Methods: 98 high-risk patients with pulmonary nodules were enrolled in two independent cohorts (68 for training/testing and 30 for independent validation). The malignancy of the pulmonary nodules were established through pathology of surgical-removed nodules. Prior to surgery, each patient was also subject to cell-free DNA-based sequencing for DNA mutation and DNA methylation profiling, as well as serum protein biomarker profiling. On the training/testing patient cohort, machine-learning-based predictive models were first built for malignancy status prediction based on each type of molecular or clinical features. A final ensemble model was then constructed to incorporate the measurements based on molecular and clinical markers to provide the ultimate recommendation on the malignancy of the pulmonary nodule. The performance of each individual model and the final ensemble model was benchmarked on the training/testing cohort, and also validated on the independent validation cohort. Results: On the 30-patient independent validation cohort, individual prediction models based on clinical information, protein marker, ctDNA mutation, and ctDNA methylation profiles achieved predictive AUC of 0.59, 0.48, 0.71, and 0.84, respectively. The final ensemble model achieved predictive AUC of 0.86, which has strongly indicated that an integrative, algorithm-based approach of multi-analytic molecular and clinical profiles greatly outperforms any single-analytic profiling. Conclusions: Multi-analyte algorithm-based approach can be utilized to assist in lung cancer screening for patients with pulmonary nodules. It has demostrated a high accuracy through independent validation, and has outperformed any single-analyte testing in our study.

P2.11-29 Diagnosis and Surveillance of Solitary Pulmonary Nodules with ctDNA Methylation Sequencing: Protocol for a Prospective Multicenter Study

P2.11-29 Diagnosis and Surveillance of Solitary Pulmonary Nodules with ctDNA Methylation Sequencing: Protocol for a Prospective Multicenter Study

Monitoring of circulating tumor DNA and its aberrant methylation in the surveillance of surgical lung Cancer patients: protocol for a prospective observational study

BackgroundDetection of circulating tumor DNA (ctDNA) is a promising method for postoperative surveillance of lung cancer. However, relatively low positive rate in early stage patients restricts its application. Aberrant methylation of ctDNA can be detected in blood samples, and may provide a more sensitive method. This study is designed to systematically evaluate and compare the detection of aberrant methylation and mutations in ctDNA among surgical non-small cell lung cancer (NSCLC) patients, aiming to investigate the feasibility of ctDNA detection as a means of lung cancer surveillance.MethodsThis is a prospective observational study. Consecutive surgical NSCLC patients will be recruited. Blood samples will be collected both before and after surgery (during the follow-up period), while matching tumor tissues and tumor-adjacent normal tissues will be collected during surgery. Quantitative analysis of aberrant methylation and mutations of ctDNA will be conducted in combination with a three-year follow-up data.DiscussionThis is the first registered prospective study designed to investigate the feasibility of ctDNA methylation detection as a means of postoperative lung cancer surveillance. We will systematically evaluate and compare the quantitative detection of ctDNA mutations and ctDNA methylation in surgical NSCLC patients, combining with the follow-up information. By integrating genetic and epigenetic information of ctDNA, more effective strategies for postoperative surveillance may be defined.Trial registrationThis study (MEDAL, MEthylation based Dynamic Analysis for Lung cancer) was registered on ClinicalTrials.gov on 08/05/2018 (NCT03634826; Pre-results).

Circulating ctDNA methylation quantification of two DNA methyl transferases in papillary thyroid carcinoma.

Both ctDNA and tissue genomic DNA of 57 PTC and 45 Goiter samples were isolated. After bisulfite modification, the methylation status was studied by Methylation-Sensitive High Resolution Melting (MS-HRM) assay technique. Four promoter regions of O6-methylguanine-DNA methyltransferase (MGMT) and three promoter regions of DNA methyltransferase 1 (DNMT1) were assessed. From seven candidate promoter regions of two methyltrasferase coding genes, the methylation status of ctDNA within MGMT (a), MGMT (c), MGMT (d), and DNMT1 (b) were meaningfully different between PTC cases and controls. However, the most significant differences were seen in circulating ctDNA MGMT (c) which was hypermethylated in 25 (43.9 %) of patients with PTC vs 2 (4. 4 %) of goiter samples. Between two selected DNA methyl transferase, the methylation of MGMT as the maintenance methyltransferase was significantly higher in PTC cases than goiter controls (P-value < .001). The resulting areas under the receiver operating characteristic (ROC) curve were 0.78 for MGMT (d) for PTC versus goiter samples that can represent the overall ability of MGMT (d) methylation status to discriminate between PTC and goiter patients. Among seven candidate regions of ctDNA the MGMT (c) and MGMT (d) showed higher sensitivity and specificity for PTC as a suitable candidates as biomarkers of PTC.

TP53, EGFR and PIK3CA gene variations observed as prominent biomarkers in breast and lung cancer by plasma cell-free DNA genomic testing

Use of plasma cell-free DNA genomic testing, also know as liquid biopsy, reveals information for early detection and monitoring of solid tumors. Our study reports the analysis of 113 lung and 18 breast cancer patients using commercially available platforms. Lung and breast cancer panel hotspot regions on the genes were investigated. There was a significant increase in isolation efficiency with very fresh blood samples of at least 15 millilitres which were processed in minutes. TP53 gene variations were detected in both types of tumors. Additionally, associations were found for EGFR variations in lung tumors and PIK3CA variations in breast tumors. Mutation assessment of these three genes are recommended as useful biomarkers for predictive studies, to follow up tumor growth and for personalized treatment. Mutations observed in this study warrant further investigation for follow up studies and may justify expression studies. However, in our subsequent studies, we intensify our tumor profiling strategy with other methods. However in terms of true personalized medicine,future plans would include repeating these studies with ctDNA size analysis and methylation analysis of the non-coding region in the individual tumors.

Applying circulating tumor DNA methylation in the diagnosis of lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Low dose computed tomography (LDCT) is commonly used for disease screening, with identified candidate cancerous regions further diagnosed using tissue biopsy. However, existing techniques are all invasive and unavoidably cause multiple complications. In contrast, liquid biopsy is a noninvasive, ideal surrogate for tissue biopsy that can identify circulating tumor DNA (ctDNA) containing tumorigenic signatures. It has been successfully implemented to assist treatment decisions and disease outcome prediction. ctDNA methylation, a type of lipid biopsy that profiles critical epigenetic alterations occurring during carcinogenesis, has gained increasing attention. Indeed, aberrant ctDNA methylation occurs at early stages in lung malignancy and therefore can be used as an alternative for the early diagnosis of lung cancer. In this review, we give a brief synopsis of the biological basis and detecting techniques of ctDNA methylation. We then summarize the latest progress in use of ctDNA methylation as a diagnosis biomarker. Lastly, we discuss the major issues that limit application of ctDNA methylation in the clinic, and propose possible solutions to enhance its usage.

Abstract P4-01-25: Blood-based DNA methylation as epigenetic biomarkers for non-invasive detection of breast cancer

Abstract Background: DNA methylation significantly contributes to all hallmarks of cancer. In particular, promoter hypermethylation has been identified as a potential marker for early detection of cancer, therapy monitoring, assessment of prognosis or prediction of therapy response. Here, we identified and validated breast cancer-specific methylation markers for diagnosis of the disease with high sensitivity and specificity. Methods: 151 bio-banked samples were obtained for a randomized blind study; 65 subjects diagnosed with breast cancer (Stage I to IV), 15 subjects with benign breast disease, 32 subjects diagnosed with other cancer types (11 colorectal, 9 liver and 12 lung cancer), and 39 healthy donor samples. Cell-free DNA was then extracted from the samples, bisulfite converted, and DNA methylation was quantified by using the IvyGene Platform. Finally, the raw data were collected and analyzed to evaluate the test performance. Results: A total of 53 of the 65 breast cancer samples were correctly identified for an overall calculated sensitivity of 89% with little difference between the sensitivity of detecting Stage I to Stage IV breast cancer (range 88% to 94%). The breast cancer subjects were correctly identified with 96% specificity. Additionally, 28 of 32 samples drawn from healthy donor subjects (specificity 95%) and all 15 samples drawn from subjects diagnosed with benign breast disease (specificity 100%) were also correctly identified. Of the samples drawn from subjects with cancer other than breast cancer, over 96% of lung cancer, colorectal cancer and liver cancer samples were correctly identified as not breast cancer. Conclusion: The results demonstrate the high diagnostic potential of ctDNA methylation markers in the blood for the detection of breast cancer. In addition, a quantitative analysis of ctDNA provides an opportunity for non-invasive detection of cancer and it's benefits can be anticipated to improve patient management and healthcare quality.

The value of methylated circulating tumor DNA as biomarkers in cancer diagnosis and prognosis

Circulating tumor DNA (ctDNA) in the blood of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This liquid biopsy allows for detection of copy number variations, tumor-specific mutations, epigenetic changes, and can be used to guide and improve treatment throughout real-time tracking the course of tumor disease. Aberrant methylation of specific gene regions can be a very consistent feature of cancer, in contrast to mutations, which makes ctDNA methylation amenable to the design of widely clinical applications in early diagnosis, monitoring disease status, predicting treatment response and predicting prognosis. Therefore, ctDNA methylation detection is considered as one of the most valuable methods for cancer diagnosis and risk assessment. Key words: Neoplasms; Circulating tumor DNA; DNA methylation; Prognosis

Liquid Biopsy as a Minimally Invasive Source of Thyroid Cancer Genetic and Epigenetic Alterations.

In the blood of cancer patients, some nucleic acid fragments and tumor cells can be found that make it possible to trace tumor changes through a simple blood test called “liquid biopsy”. The main components of liquid biopsy are fragments of DNA and RNA shed by tumors into the bloodstream and circulate freely (ctDNAs and ctRNAs). Tumor cells which are shed into the blood (circulating tumor cells or CTCs), and exosomes that have been investigated for non-invasive detection and monitoring several tumors including thyroid cancer. Genetic and epigenetic alterations of a thyroid tumor can be a driver for tumor genesis or essential for tumor progression and invasion. Liquid biopsy can be real-time representative of such genetic and epigenetic alterations to trace tumors. In thyroid tumors, the circulating BRAF mutation is now taken into account for both thyroid cancer diagnosis and determination of the most effective treatment strategy. Several recent studies have indicated the ctDNA methylation pattern of some iodine transporters and DNA methyltransferase as a diagnostic and prognostic biomarker in thyroid cancer as well. There has been a big hope that the recent advances of genome sequencing together with liquid biopsy can be a game changer in oncology.

Highly sensitive detection of DNA hypermethylation in melanoma cancer cells

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a promising biomarker for early cancer detection. This methylation status is reflected in the methylation pattern of ctDNA shed from the primary tumor; however, to realize the full clinical utility of ctDNA methylation detection via liquid biopsy for early cancer diagnosis, improvements in the sensitivity and multiplexability of existing technologies must be improved. Additionally, the assay must be cheap and easy to perform in a clinical setting. We report the integration of methylation specific PCR (MSP) to melt curve analysis on giant magnetoresistive (GMR) biosensors to greatly enhance the sensitivity of our DNA hybridization assay for methylation detection. Our GMR sensor is functionalized with synthetic DNA probes that target methylated or unmethylated CpG sites in the MSP amplicon, and measures the difference in melting temperature (Tm) between the two probes (ΔTm), giving an analytical limit of detection down to 0.1% methylated DNA in solution. Additionally, linear regression of ΔTm's for serial dilutions of methylated:unmethylated mixtures allows for quantification of methylation percentage, which could have diagnostic and prognostic utility. Lastly, we performed multiplexed MSP on two different genes, and show the ability of our GMR assay to resolve this mixture, despite their amplicons’ overlapping Tm's in standard EvaGreen melt analysis. The multiplexing ability of our assay and its enhanced sensitivity, without necessitating deep sequencing, represent important steps toward realizing an assay for the detection of methylated ctDNA in plasma for early cancer detection in a clinical setting.

555P - Correlation between ctDNA methylation and CEA in colorectal cancer

555P - Correlation between ctDNA methylation and CEA in colorectal cancer

The Use of Circulating Tumor DNA for Prognosis of Gastrointestinal Cancers.

Gastrointestinal cancers, including oesophageal, gastric and colorectal cancers (CRC) have high rates of disease recurrence despite curative resection. There are a number of recent studies that have investigated the use of circulating tumor DNA (ctDNA) for prognostic value in these cancers. We reviewed studies that had been published prior to March 2018 that assessed the prognostic values of ctDNA in patients with oesophageal and gastric cancers, gastrointestinal stromal tumors (GIST) and CRC. We identified 63 eligible clinical studies that focussed on recurrence and survival. Studies assessed investigated various ctDNA biomarkers in patients with different stages of cancer undergoing surgical resection, chemotherapy and no treatment. For oesophageal squamous cell carcinoma and oesophageal adenocarcinoma, methylation of certain genes such as APC and DAPK have been highlighted as promising biomarkers for prognostication, but these studies are limited and more comprehensive research is needed. Studies focusing on gastric cancer patients showed that methylation of ctDNA in SOX17 and APC were independently associated with poor survival. Two studies demonstrated an association between ctDNA and recurrence and survival in GIST patients, but more studies are needed for this type of gastrointestinal cancer. A large proportion of the literature was on CRC which identified both somatic mutations and DNA methylation biomarkers to determine prognosis. ctDNA biomarkers that identified somatic mutations were more effective if they were personalized based on mutations found in the primary tumor tissue, but ctDNA methylation studies identified various biomarkers that predicted increased risk of recurrence, poor disease free survival and overall survival. While the use of non-invasive ctDNA biomarkers for prognosis is promising, larger studies are needed to validate the clinical utility for optimizing treatment and surveillance strategies to reduce mortality from gastrointestinal cancers.

Abstract 3324: Diagnosis and prognosis of hepatocellular carcinoma with ctDNA methylation markers

Abstract Abstract An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumor DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive “liquid biopsy” for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation markers by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumor DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P&amp;lt;0.001) and was highly correlated with tumor burden, treatment response, and stage (P&amp;lt;0.001). Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P&amp;lt;0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC. Citation Format: Kang Zhang. Diagnosis and prognosis of hepatocellular carcinoma with ctDNA methylation markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3324.

Circulating CtDNA Methylation Quantification of Two DNA Methyl Transferases in Papillary Thyroid Carcinoma

Circulating CtDNA Methylation Quantification of Two DNA Methyl Transferases in Papillary Thyroid Carcinoma

Methylated circulating tumor DNA in blood: power in cancer prognosis and response.

Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This ‘liquid biopsy’ allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time ‘tracking’ in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients.

Tumor subtype-specific cancer-testis antigens as potential biomarkers and immunotherapeutic targets for cancers.

Cancer-testis (CT) antigens are potential targets for cancer immunotherapy because of their restricted expression in immune-privileged germ cells and various malignancies. Current application of CT-based immunotherapy has been focused on CT expression-rich tumors such as melanoma and lung cancers. In this study, we surveyed CT expression using The Cancer Genome Atlas (TCGA) datasets for ten common cancer types. We show that CT expression is specific and enriched within certain cancer molecular subtypes. For example, HORMAD1, CXorf61, ACTL8, and PRAME are highly enriched in the basal subtype of breast cancer; MAGE and CSAG are most frequently activated in the magnoid subtype of lung adenocarcinoma; and PRAME is highly upregulated in the ccB subtype of clear cell renal cell carcinoma. Analysis of CT gene expression and DNA methylation indicates that some CTs are regulated epigenetically, whereas others are controlled primarily by tissue- and subtype-specific transcription factors. Our results suggest that although for some CT expression is associated with patient outcome, not many are independent prognostic markers. Thus, CTs with shared expression pattern are heterogeneous molecules with distinct activation modes and functional properties in different cancers and cancer subtypes. These data suggest a cancer subtype-orientated application of CT antigen as biomarkers and immunotherapeutic targets.

Abstract 2663: Phase I study of azacitidine and cisplatin in patients with advanced head and neck or non-small cell lung cancer

Abstract Background: DNA hypomethylation agents such as azacitidine may be used for epigenetic sensitization of cisplatin treatment, and therefore leading to improvement of treatment outcome. We conducted a phase I study of azacitidine and cisplatin to determine safety, toxicity, and effect of global DNA methylation in peripheral blood mononuclear cells. Methods: Azacitidine was given as subcutaneous injection daily from day 1 to day 5, and cisplatin was given at 75 mg/m2 IV on day 8. The treatment was repeated every 28 days. Four patients received azacitidine 37 mg/m2 daily from day 1 to day 5 on dose level 1. Two patients were treated on dose level 2, and received azacitidine 60 mg/m2 daily from day 1 to day 5. Peripheral blood samples were collected before and after azacitidine treatment. DNA was extracted from peripheral blood mononuclear cells, and subjected to gas chromatography/mass spectrometry to measure the level of global DNA methylation, as reflected by the ratio of 5-methylcytosine to the sum of 5-methylcytosine and cytosine. Results: No patient had dose-limiting toxicities. Four patients completed at least 2 cycles of treatment, and were evaluated for clinical response by CT scan and DNA methylation effect. One partial response was noted in patient with metastatic tongue cancer after 2 cycles of treatment on dose level 1 of azacitidine. This patient received total 3 cycles of treatment and achieved free of progression for about 15 months on chemotherapy holiday. One patient with recurrent salivary gland cancer achieved stable disease after 2 cycles of treatment on dose level 2, and maintained in free of progression for more than 6 months after 4 cycles of treatment. There was inhibition of global DNA methylation after azacitidine treatment ranging from 12 to 36% in all 4 patients. Furthermore, the inhibition of global DNA methylation was associated with the increase of fetal hemoglobin in patients’ peripheral blood. Conclusion: Sequential administration of azacitidine followed by cisplatin is well tolerated, and demonstrates encouraging activity in patients with advanced head and neck cancer. This combination deserves further clinical investigation to study the correlation between clinical response and biological effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2663. doi:1538-7445.AM2012-2663

Novel molecular screening approaches in colorectal cancer

Traditional methods for colorectal cancer (CRC) screening have reduced rates of malignancy and death; however, low compliance and morbidities associated with invasive techniques have encouraged efforts for establishing equally effective, less invasive novel screening approaches. We review the current state of novel screening approaches in CRC to include CT colonography, fecal DNA, DNA methylation, micro-RNA, and protein and molecular markers.