Methylated ctDNA Research Articles

The prognostic role of circulating tumour DNA detected prior to clinical diagnosis of colorectal cancer in the HUNT study

BackgroundToday, the prognostic tools available at the time of diagnosis in colorectal cancer (CRC) are limited. Better prognostic tools are a prerequisite for personalised treatment.This study aimed to investigate whether circulating tumour DNA (ctDNA) markers found in plasma before clinical diagnosis of CRC could contribute to the prediction of poor prognosis.MethodsThis observational cohort study included patients diagnosed with CRC stage I-III within 24 months following participation in the Trøndelag Health Study (n = 85). Known methylated ctDNA biomarkers of CRC were analysed by PCR in plasma. Outcomes were overall survival (OS), recurrence-free survival (RFS) and poor prognosis (PP). Candidate clinical and methylated ctDNA predictors of the outcomes were identified by Cox regression analyses.ResultsMethylated GRIA4 (HR 1.96 (1.06–3.63)), RARB (HR 9.48 (3.00–30.00)), SLC8A1 (HR 1.97 (1.03–3.77)), VIM (HR 2.95 (1.22–7.14)) and WNT5A (HR 5.83 (2.33–14.56)) were independent predictors of OS, methylated RARB (HR 9.67 (2.54–36.81)), SDC2 (HR 3.38 (1.07–10.66)), SLC8A1 (HR 2.93 (1.01–8.51)) and WNT5A (HR 6.95 (1.81–26.68)) were independent predictors of RFS and methylated RARB (HR 6.11 (1.69–22.18)), SDC2 (HR 2.79 (1.20–6.49)) and WNT5A (HR 5.57 (3.04–15.26)) were independent predictors of PP (p < 0.05).ConclusionsPrediagnostic ctDNA markers are promising contributors to predicting poor prognosis in CRC, potentially becoming one of the tools guiding more personalised treatment.

Clinical validation of Northstar response: A novel methylated ctDNA therapy response monitoring assay in patients with advanced gastrointestinal cancers.

e16000 Background: Circulating tumor DNA (ctDNA) offers a non-invasive method to comprehensively assess tumors. While most assays for therapy response monitoring rely on variant allele frequencies, methylated ctDNA has shown promise. However, accuracy has been limited by quantification precision. The CLIA-approved Northstar Response assay quantifies over 550 hyper-methylated loci and has demonstrated a 10-fold increase in detection signal over tissue-informed assays. Methods: We present an interim analysis from an ongoing prospective, observational study designed to assess the clinical validity of Northstar Response as a tool to monitor therapeutic response without prior tumor tissue. Our study aims to include 100 advanced GI cancer patients undergoing systemic therapies for advanced disease. Serial ctDNA measurements are collected at baseline, approximately every 30 days post-treatment initiation, and with imaging assessments. The primary endpoint is concordance between ctDNA and RECISTv1.1 criteria for disease response until progression or death. Results: Baseline ctDNA characteristics were examined from 44 patients, with 23 undergoing at least two longitudinal blood collections and subsequent imaging. The median age in this cohort was 65.5 years, with a male predominance (70.5%) and a majority identifying as White (79.5%). Cancers included esophageal (18.2%), hepatocellular (29.5%), biliary (15.9%) and colorectal (15.9%), among others. A majority (59.1%) had both primary and metastatic lesions. Baseline methylation scores ranged from 280 in biliary cancers to 100,000 in colorectal cancers. Those on second-line treatments had lower scores than first or third-line therapies. Methylation scores decreased in patients responding to treatment, indicating that these scores have the potential to serve as indicators of therapeutic efficacy. Conclusions: Baseline methylation scores differ across GI tumor types, disease burdens, and treatment lines, reflecting the complexity of using methylation as a biomarker. Northstar Response has shown initial promise in monitoring therapy response in GI cancers. More mature data, which will further elucidate the assay's clinical utility, will be presented at the meeting.

Progress of circulating tumor DNA methylation for gastric cancer screening and management

Circulating tumor DNA (ctDNA) is cell-free DNA released by tumors or circulating tumor cells, containing abundant tumor-specific information that can serve as biomarkers for cancer early screening, monitoring, prognosis, and prediction of treatment response. This is particularly attractive in the field of gastric cancer, where high-quality screening, monitoring, and prediction methods are currently lacking. Gastric cancer exhibits significant tumor heterogeneity, with large differences in genetic and epigenetic characteristics among different subgroups. Methylated ctDNA has high sensitivity and specificity, which can help clarify tumor genotyping and facilitate the formulation of precise diagnostic and therapeutic strategies. Furthermore, numerous studies have confirmed the unique advantages of methylated DNA in predicting treatment response, adjuvant therapy, and drug resistance assessment, which may be used in the future to enhance the efficacy of chemotherapy regimens and improve patient chemotherapeutic response, and even treat multidrug resistance. However, there are several challenges associated with methylated ctDNA, such as low sensitivity and specificity at single-target sites, limited association between some gastric cancer subtypes and ctDNA, off-target risks, and the lack of large-scale and high-quality clinical research evidence. This review mainly summarizes current research on the methylation status of ctDNA in gastric cancer and connects these findings to early screening, recurrence monitoring, and potential treatment opportunities for gastric cancer. With advances in technology and the deepening of interdisciplinary research, ctDNA detection will reveal more disease information and become an essential foundation for gastric cancer research and precision medicine treatment.

BCAT1, IKZF1 and SEPT9: methylated DNA biomarkers for detection of pan-gastrointestinal adenocarcinomas

Introduction Methylated circulating tumour DNA (ctDNA) blood tests for BCAT1/IKZF1 (COLVERA) and SEPT9 (Epi proColon) are used to detect colorectal cancer (CRC). However, there are no ctDNA assays approved for other gastrointestinal adenocarcinomas. We aimed to characterize BCAT1, IKZF1 and SEPT9 methylation in different gastrointestinal adenocarcinoma and non-gastrointestinal tumours to determine if these validated CRC biomarkers might be useful for pan-gastrointestinal adenocarcinoma detection. Methods Tissue DNA methylation data from colorectal (COAD, READ), gastroesophageal (ESCA, STAD), pancreatic (PAAD) and cholangiocarcinoma (CHOL) adenocarcinoma cohorts within The Cancer Genome Atlas were used for differential methylation analyses. Clinicodemographic predictors of BCAT1, IKZF1 and SEPT9 methylation, and the selectivity of hypermethylated BCAT1, IKZF1 and SEPT9 for colorectal adenocarcinomas in comparison to other cancers were each explored with beta regression. Results Hypermethylated BCAT1, IKZF1 and SEPT9 were each differentially methylated in colorectal and gastroesophageal adenocarcinomas. IKZF1 was differentially methylated in pancreatic adenocarcinoma. Hypermethylated DNA biomarkers BCAT1, IKZF1 and SEPT9 were largely stable across different stages of disease and were highly selective for gastrointestinal adenocarcinomas relative to other cancer types. Discussion Existing CRC methylated ctDNA blood tests for BCAT1/IKZF1 and SEPT9 might be usefully repurposed for use in other gastrointestinal adenocarcinomas and warrant further prospective ctDNA studies.

Clinical validation of Northstar Response, a novel methylated ctDNA therapy response monitoring assay in patients with advanced GI cancer undergoing active treatment.

756 Background: ctDNA is a non-invasive way to holistically assess the tumor. Several assays developed for therapy response monitoring rely on variant allele frequencies to detect scarce somatic variants in the blood. In contrast, methylated ctDNA measurement has shown promise as a treatment monitoring biomarker without requiring a tumor biopsy, yet accuracy is limited in ability to precisely quantify the amount of methylation present in the ctDNA. The CLIA-approved Northstar Response assay (BillionToOne, Inc.) quantifies on average 90 methylated loci, a 10-fold increase in signal over the average tissue-informed assays. Methods: This prospective, observational study assesses the clinical validity of Northstar Response, a quantitative methylated ctDNA therapeutic response monitoring assay that does not require prior tumor tissue. A cohort of 100 advanced cancer patients with a variety of GI malignancies submit serial ctDNA measurements in a blinded fashion. Eligibility includes advanced GI cancer patients starting a new line of systemic therapy (IO, targeted, chemo or combination) who are not pregnant or s/p organ or bone marrow transplantation. Blood samples are collected at baseline, at the time of imaging, and at least two intermediate time points (~30 days and 60 days post tx initiation). The primary endpoint is to measure the concordance and temporal relationship between disease progression measured by ctDNA compared to RECISTv1.1, clinical confirmation, or death. A Cox proportional hazards model will be used to quantify the association between the change in tumor methylation score (TMS; total methylated tumor molecules per 1000 assayed genomic equivalents) and clinical progression. The cohort will be split equally between a training and validation set. Results: Enrollment began in June 2023 and continues. To date, 28 patients with a variety of advanced GI cancers (table) have been enrolled with 37 samples processed. All samples have passed established QC metrics with average turn-around-time from receipt of sample to final report of 9.1 days (95% were &lt;15 days). TMS ranged from &lt;120 to &gt;160,000 across individual samples, indicating that the assay can detect a broad range of tumor load in blood. We detected a median of 69 methylated loci per sample (range 4-451). Out of 21 pts, 17 had at least one sample with TMS measured above the noise floor of 120. Conclusions: The Northstar Response assay can consistently detect ctDNA across a variety of advanced GI cancers. Enrollment continues with formal comparison to clinical progression planned. Additional data on the complete training set will be available at the meeting.[Table: see text]

Methylated ctDNA Quantification: Noninvasive Approach to Monitoring Hepatocellular Carcinoma Burden.

Noninvasive, precision monitoring of hepatocellular carcinoma (HCC) treatment efficacy would greatly facilitate personalized therapy and improve patient outcomes. We hypothesize that quantifying methylated circulating tumor DNA (ctDNA) can be used to effectively monitor HCC burden without the need for biopsy. Blood samples were collected from 25 patients, 21 with HCC and 4 with benign liver masses, at various timepoints throughout the course of treatment at a high-volume academic medical center. Quantification of methylated ctDNA molecules assessed CpG sites on more than 550 preselected cancer-specific amplicons. The tumor methylation score (TMS) was calculated by measuring the difference between the amount of methylation in the plasma and buffy coat with a normal cutoff value of 120 or less. Among 10 patients with surgical HCC (5 surgical resections and 5 liver transplants), TMS revealed a statistically significant, rapid postoperative decline in 9. One patient who had a persistently elevated TMS on postoperative day 1 was subsequently found to have had metastatic disease. Patients in the negative control cohort all had normal-range pre- and postoperative TMS. Preoperative TMS correlated moderately with tumor burden on pathology (Spearman r = 0.54) of surgical specimens. From 11 subjects undergoing systemic therapy or Y90 radioembolization, analysis of 16 time periods demonstrated that the change in TMS (ΔTMS) was better associated with tumor progression than the change in Δalpha-fetoprotein (area under the curve 0.800 and 0.783, respectively). A composite score combining ΔTMS and Δalpha-fetoprotein further improved performance for detecting tumor progression with an area under the curve of 0.892. These findings indicate that ctDNA methylation scores can effectively evaluate changes in tumor burden without the need for tumor biopsy.

Methylated Circulating Tumor DNA in Blood as a Tool for Diagnosing Lung Cancer: A Systematic Review and Meta-Analysis.

Lung cancer is the leading cause of cancer-related deaths, and early detection is crucial for improving patient outcomes. Current screening methods using computed tomography have limitations, prompting interest in non-invasive diagnostic tools such as methylated circulating tumor DNA (ctDNA). The PRISMA guidelines for systematic reviews were followed. The electronic databases MEDLINE, Embase, Web of Science, and Cochrane Library were systematically searched for articles. The search string contained three main topics: Lung cancer, blood, and methylated ctDNA. The extraction of data and quality assessment were carried out independently by the reviewers. In total, 33 studies were eligible for inclusion in this systematic review and meta-analysis. The most frequently studied genes were SHOX2, RASSF1A, and APC. The sensitivity and specificity of methylated ctDNA varied across studies, with a summary sensitivity estimate of 46.9% and a summary specificity estimate of 92.9%. The area under the hierarchical summary receiver operating characteristics curve was 0.81. The included studies were generally of acceptable quality, although they lacked information in certain areas. The risk of publication bias was not significant. Based on the findings, methylated ctDNA in blood shows potential as a rule-in tool for lung cancer diagnosis but requires further research, possibly in combination with other biomarkers.

Colorectal cancer detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study.

Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis. In this case-control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study. The most specific single markers were BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7-43.4) and the CRC specificity was 86% (95% CI 85.7-86.2). The findings were reproduced in an independent validation set of samples. Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme.

Methylated ctDNA as a biomarker for detecting previously undetectable clinically significant somatic mutations.

e15020 Background: Treatment selection liquid biopsies can be re-run upon clinical progression to identify the new clinically significant somatic mutations that might be contributing to resistance to therapy or that can help guide the selection of an alternative therapy. However, there are not currently any biomarkers or tests that suggest an increased likelihood for the detection of new, i.e. previously undetected, clinically significant somatic mutations in a subsequent treatment selection liquid biopsy assay. Methods: We explored whether a methylation-based liquid biopsy could indicate when new clinically significant alterations are more likely to be detected in a treatment selection liquid biopsy. As part of a retrospective study of metastatic lung cancer patients (N = 75 new treatment starts), both a treatment selection assay and a therapy monitoring methylation assay were performed on plasma collected pre-treatment for a subset of patients that underwent at least two separate treatment regimens. By quantifying the number of methylated ctDNA molecules at &gt; 500 genomic locations, the methylation assay assessed the total burden of methylated ctDNA (represented as tumor methylation score), as well as the percent of methylated molecules originating from newly methylated genomic loci, representing a change in the methylation profile. Results: When the methylation assay detected at least a 2x increase in the tumor methylation score, indicating a significant increase in the fraction of ctDNA present in the patient’s blood, new clinically significant somatic mutations (including actionable ERBB2 alterations) were found 83% of the time. This appeared to be more frequent than among patients without an increase in tumor methylation score, where new clinically significant somatic mutations were found 33% of the time. Importantly, when the increase in the methylation score was coupled with a significant change in the methylation profile, new clinically significant mutations were identified in 100% of patients. Conclusions: The preliminary data suggests that an overall increase in methylated ctDNA, in combination with a significant change in methylation profile, is correlated with detecting new, previously undetected clinically significant somatic mutations in a treatment selection assay. Leveraging this methylated ctDNA assay may inform clinicians as to when to run another treatment selection assay, thereby potentially identifying the appearance of clinically significant somatic mutations which could influence clinical decision making and affect patient clinical outcomes.

Abstract 5588: Methylated ctDNA dynamics correspond with clinical tumor load in metastatic lung cancer patients on therapy

Abstract Treatment monitoring assays are needed to accurately and rapidly assess the efficacy of cancer treatments. While imaging remains the gold standard for monitoring the efficacy of cancer treatment, the use of more sensitive tools, such as liquid biopsy, could be beneficial for the patient’s ultimate treatment outcome. Several liquid biopsy-based assays that measure circulating tumor DNA (ctDNA) have been developed to meet this need. However, approaches that rely on quantifying the variant allele fraction (VAF) of somatic variants may be inaccurate or inconsistent due to a scarcity of detected somatic variants in the ctDNA or may be logistically infeasible if they require a tumor biopsy a priori. Methylated ctDNA has shown promise as a biomarker for treatment monitoring without requiring a tumor biopsy, but current efforts are limited in their ability to precisely quantify the amount of methylation present in the ctDNA. We hypothesize that more precise quantification of methylated ctDNA could enable more accurate correspondence with clinical tumor load and cancer treatment outcomes. Here, we present a retrospective study characterizing how amounts of methylated ctDNA dynamically change through cancer therapy. We tested 75 patient-treatment events, with each event composed of patient samples from one pre-treatment and two post-treatment time points, all from patients with Stage III-IV metastatic lung cancer receiving targeted therapies or immunotherapy. We employed a novel methylation-based, tumor-naive liquid biopsy assay for treatment monitoring by counting the number of methylated molecules in cell-free DNA. We observed methylation amounts ranging from &amp;lt;50 to &amp;gt;50,000 total methylated tumor molecules per 1000 assayed genomic equivalents at individual time points, indicating that the assay can detect a broad range of tumor load in blood. In addition, we measured changes between time points as large as &amp;gt;96% decrease and &amp;gt;2800% increase in methylation. We found that methylation correlated with maximum variant allele fractions as measured by a treatment selection assay (r2 = 0.74). Moreover, in this tumor-naive assay, we identified a median of 58 informative methylated loci, significantly more than a median of ~1-5 informative SNVs identified in VAF-based tumor-naive assays, and ~4-16 informative SNVs identified in VAF-based tumor-informed assays. This ~6-10x increase in the number of informative loci enables much more precise quantification and underlies the assay’s robustness and precision across patients, time points, and therapies. Finally, these changes in normalized total tumor molecules correlated with radiographic responses on imaging, duration of response, and time on therapy. Overall, our findings support the utility of methylated ctDNA as a biomarker for monitoring tumor load in cancer patients. Citation Format: Patrick Ye, Brian Woodward, Robb Viens, Sydne Langpap, Katherine Shelburne, Wen Zhou, Joyce Zhu, Jan Wignall, Gary Palmer, David Tsao, Oguzhan Atay, Hatim Husain. Methylated ctDNA dynamics correspond with clinical tumor load in metastatic lung cancer patients on therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5588.

Abstract 2290: DNA methylation markers for sensitive detection of circulating tumor DNA in plasma from patients with gastroesophageal adenocarcinomas

Abstract Background: We have previously shown that detection of methylated ctDNA in the three genes c9orf50, KCNQ5, and CLIP4 is a highly sensitive method (named the TriMeth assay) to discriminate patients with colorectal cancer from healthy individuals (1). The publicly available Infinium HumanMethylation450K BreadChip DNA methylation array indicates that these three markers are also highly methylated in gastric and esophageal cancer. In this study, we investigate the ability of the TriMeth assay to detect ctDNA in patients with gastric and gastroesophageal junction (GEJ) adenocarcinomas. Methods: Purified DNA was extracted from tumor tissue and plasma from 63 patients with gastric/GEJ adenocarcinomas. Sodium bisulfite conversion was performed to distinguish methylated from unmethylated cytosines. Droplet digital PCR (ddPCR) with methylation-specific primers and probes was performed to detect hypermethylated sites in the three genes c9orf50, KCNQ5, and CLIP4. The TriMeth assay was considered positive if at least two out of three markers were methylated according to a predefined cut-off. Eleven plasma samples from healthy individuals were used as negative controls.Results: In tumor tissue, methylated copies were detected in all 29 surgical samples (100% sensitivity), with varying allele frequencies. In plasma, methylated copies were detected in 14 out of 18 samples (78% sensitivity) from patients with advanced disease, with varying sensitivity amongst the three markers (AUC for the three markers CLIP4, C9orf50, and KCNQ5 was 0.61, 0.86, and 0.94, respectively). In plasma from patients with resectable disease, methylated copies were detected in 10 of 17 samples (59% sensitivity), although only 7/17 (41% sensitivity) were positive according to the predefined cut-off. All 11 controls were negative for methylation signals (100% specificity). Conclusion and Further Perspectives: Using a pre-defined cut-off value, the TriMeth assay detected methylated DNA in all tumor samples. The sensitivity of the TriMeth assay in plasma was 78% in advanced disease and 41% in resectable disease. Further investigations of the TriMeth assay in patients with resectable gastric and GEJ adenocarcinomas are needed. We plan to validate the sensitivity of the TriMeth assay for treatment monitoring and detection of residual disease.

Abstract 3369: Utility of ctDNA-based targeted methylation MRD assay for hematological malignancies

Abstract Introduction: Minimal or measurable residual disease (MRD) is an emerging independent predictor of progression-free and overall survival in several hematologic diseases. Despite many different methods used in clinical trials, few assays have clinical utility as a surrogate endpoint in a limited number of hematologic malignancies. MRD assays for lymphomas are mostly performed in research settings due to lack of harmonized methods, heterogeneity of diseases, and need for a primary tumor sample. We explored the feasibility of a pan hematologic malignancy classifier (“pan heme classifier”) based on GRAIL’s methylation platform as a potential tumor-agnostic plasma-based MRD assay for hematologic malignancies. Methods: First, 428 plasma samples from various hematologic malignancies including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM) were blindly tested retrospectively using the pan heme classifier. The majority of samples (375/428; 88%) were from relapsed/refractory (R/R) disease. Cancer signal detection and signal origin prediction were explored at various detection thresholds. Next, sensitivity of the pan heme classifier was evaluated using 74 (22 and 14 unique patients in CLL and MCL, respectively) post-treatment samples with orthogonal methods (flow cytometry in CLL and clonoSEQ in MCL). Finally, dilution experiments were carried out in 4 DLBCL and 4 CLL patient samples. Results: Of 428 samples, 408 (95%) passed quality control. At a prespecified detection threshold, we observed high (91%) cancer detection rate with high cancer signal origin accuracies: 100% in CLL, &amp;gt;98% in MM, &amp;gt;95% in non-Hodgkin lymphoma (DLBCL, FL, MCL), and 87% in AML samples. As expected, targeting lower detection thresholds resulted in increased cancer-positive calls. Cancer was reproducibly detected in 48 of 54 (89%) cases where paired samples were taken prior to treatment (screening and cycle 1, day 1), demonstrating high biologic reproducibility. Analysis of posttreatment CLL and MCL samples with cell-based orthogonal MRD assays suggests the current assay limit of detection (LOD) is ~10-3-10-4 MVAF (a methylation-based tumor fraction estimate). These data suggest that cancer signal score is strongly correlated to the observed MVAF. Furthermore, serially diluted DLBCL and CLL patient plasma samples were spiked into healthy volunteer plasma samples, suggesting an LOD of 10-4 MVAF. Conclusions: These data demonstrate that the pan heme classifier can identify cancer signal from R/R patients across multiple hematologic malignancies. We are currently working to further optimize the assay’s specificity and sensitivity. These results support the development of a blood-based tumor agnostic methylated ctDNA MRD assay with potential utility in several hematologic indications. Citation Format: Veerendra Munugalavadla, Gary de Jesus, Aleksandra Markovets, Qinwen Liu, Oliver Venn, Rafael White, Giulia Fabbri, Paul Labrousse, Dan Stetson, Brian Dougherty, Darren Hodgson, Jill Walker, Anas Younes, Daniel Auclair. Utility of ctDNA-based targeted methylation MRD assay for hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3369.

Mutation-Agnostic Detection of Colorectal Cancer Using Liquid Biopsy-Based Methylation-Specific Signatures.

Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; P = .027).

Detection of circulating DNA methylated BCAT1 and IKZF1 in pancreatic adenocarcinoma.

597 Background: Pancreatic adenocarcinoma patients have poor survival outcomes and there are no non-invasive tests to aid diagnosis or therapy. Detection of circulating tumour DNA (ctDNA) methylated for BCAT1 and/or IKZF1 is over 60% sensitive for colorectal adenocarcinoma. Both pancreatic and colorectal adenocarcinomas are of endodermal origin, therefore, these methylated biomarkers might also have utility in detecting pancreatic cancer. The aim of this study was to investigate whether individuals with pancreatic adenocarcinoma have detectable methylated BCAT1 and IKZF1 DNA in circulation. Methods: In this pilot study, pre-treatment blood and clinicopathological findings were collected from 21 patients diagnosed with pancreatic adenocarcinoma. DNA isolated from plasma was bisulfite-converted and assayed for methylated BCAT1, IKZF1 and a non-methylated region in ACTB (for yield estimates). Samples with methylation in either gene was deemed positive. Chi -squared test was used to compare positivity between Stage I/II and Stage III/IV cases. The sum of percent (%) methylation ([average BCAT1]+[average IKZF1])/average ACTB) ± standard error was compared between stages using Kruskal-Wallis rank test. Results: 10/21 (47.6%) patients were positive for methylated BCAT1 and/or IKZF1. There was a trend of increasing positivity with advancing stage (Stage I/II 2/8 (25.0%) vs Stage III/IV 8/13 (61.5%, p=0.104)), and for higher % methylation with more advanced disease (Stage II 0.01%±0.004 vs Stage IV 5.0%±3.7, p=0.06). No other comorbidities or demographics were associated with positivity. Conclusions: Assay for methylated BCAT1 and IKZF1 ctDNA detects approximately two-thirds of late-stage pancreatic adenocarcinoma. Future studies are warranted to assess the clinical utility of these biomarkers for detection and monitoring of pancreatic cancer. Clinical trial information: 12616001138471.

Assessment of methylated BCAT1 and IKZF1 circulating tumor DNA as a potential diagnostic and prognostic biomarker in esophagogastic cancers.

348 Background: Esophagogastric cancers (EG cancers) are associated with high mortality and poor treatment outcomes due to advanced disease at diagnosis. Reliable non-invasive biomarkers are lacking for detection and prognostication. The aim of this study was to assess the clinical utility of methylated BCAT1 and IKZF1 circulating tumour DNA (ctDNA) for detection and prognosis of EG cancers. Methods: Blood samples were collected from patients diagnosed with either esophageal or gastric adenocarcinoma prior to commencing any treatment. DNA was extracted from plasma and analysed for methylation of BCAT1 and IKZF1, and ACTB as the reference gene, by multiplex qPCR. ctDNA positivity rates were assessed against cancer clinicopathological features using chi-squared tests and age-adjusted logistic regression with odds ratios (OR). Secondary outcome was overall survival after 12 months using cox-proportional hazard ratio (HR). Results: Ninety-seven patients (77/97 (79%) male; 70 (72%) with esophageal cancer and 27 (28%) with gastric cancer) were enrolled with median age 72 years (IQR = 61-79). Fifty-three (55%) were ctDNA positive. There was no difference between the ctDNA positivity rate of esophageal and gastric cancers (p &gt; 0.05). Positivity was significantly associated with node positive tumours (N0 = 6/23 (20.7%), N1 = 5/10 (50%), N2 = 11/17 (64.7%), N3 = 3/4 (75%); p = 0.01) and depth of invasion (T1 = 0/9 (0%), T2 = 5/12 (41.7%), T3 = 17/36 (47.2%), T4 = 5/6 (83.3%), p = 0.049). ctDNA positivity was independent of history of GORD or Barrett’s disease (p &gt; 0.05). There was a significant association between ctDNA and advanced stage disease (Stage I = 1/13 (7.7%), Stage II = 2/12 (16.7%), Stage III = 25/40 (62.5%), Stage IV = 25/32 (78.1%)), with stage IV cases having a 43-fold increased odds for a positive ctDNA result compared to stage I (OR = 43.1, 95%CI 4.7-392.0; p = 0.001). Patients testing positive for ctDNA had poorer overall survival outcomes compared to those testing negative (HR 4.23, 95% CI 1.84-9.68, p &lt; 0.001). Conclusions: Our study demonstrates a potential role for methylated BCAT1 and IKZF1 ctDNA as a biomarker for the detection of EG cancers. The detection of ctDNA is significantly associated with poorer survival outcomes, with further studies warranted to determine whether ctDNA can be an independent predictor of survival. The integration of this ctDNA assay into clinical practice could further improve risk stratification, which may guide treatment strategies.

Analysis of HOXA9 methylated ctDNA in ovarian cancer using sense-antisense measurement

DNA promoter methylation is an early event in tumorigenesis and holds promise as a valuable marker in ovarian cancer (OC). It can be measured using circulating tumor specific DNA (ctDNA) isolated from the bloodstream. Sensitivity, however, is a limiting factor of its diagnostic feasibility in OC. DNA methylation analyses are based on bisulfite conversion, resulting in two DNA strands that are no longer complementary. The current standard strategy would then target only one of the double stranded DNA strands, but the potential to increase the sensitivity by targeting both DNA strands is available. In this study, we aimed at evaluating the diagnostic potential of methylated HOXA9 ctDNA in OC by targeting both the DNA sense and antisense strand. Methylated HOXA9 was detected in the plasma of 47/79 (59.5%) patients with newly diagnosed OC using sense-antisense droplet digital PCR. Simultaneous sense-antisense measurement increased the sensitivity by 14.6% (51.9% to 59.5%) as compared to antisense only. In patients with FIGO stage I-II disease the sensitivity was increased by 25%. In conclusion, simultaneous measurement targeting both DNA strands can increase the sensitivity and the analytical approach appears valuable in the diagnostic setting of OC.

Use of circulating tumor DNA in colorectal cancer patients to assess tumor burden and response to therapy: An observational study.

3528 Background: Residual disease after treatment for colorectal cancer (CRC) poses a risk for recurrence but imaging and CEA are limited in their capacity to detect residual disease. A simple test is needed for assessing treatment response. This study determined whether levels of methylated BCAT1/IKZF1 DNA in blood correlate with tumor burden and whether post-treatment levels inform efficacy of different treatments for CRC. Methods: Patients with primary CRC had blood collected prior to treatment (n = 282, 59.9% males, median age 68.5y). Cell free DNA (cfDNA) was extracted from plasma and assayed for methylation in BCAT1 and IKZF1. Detection of methylation in either gene deemed a sample positive; levels were expressed as %methylation (average methylation/average cfDNA). Positive patients had additional samples collected post-treatment for early stage CRC (surgery, n = 31), advanced/metastatic CRC (surgery + adjuvant chemotherapy, n = 15), and rectal cancer (neoadjuvant therapy, surgery +/- chemotherapy, n = 6), or following mid-therapy suspension of treatment in advanced CRC (n = 24). Tumor size was expressed as the maximum diameter of the primary (assessed surgically or by MRI). Results: Pretreatment results increased with CRC stage. Positivity by stage was: I, 23.7% (14/59); II, 62.1% (54/87); III, 68.6% (70/102); IV, 85.3% (29/34). Level by stage: I, 0.0%; II, 0.06%; III, 0.07%; IV, 4.07%, p &lt; 0.001). Pretreatment levels correlated significantly with tumor size (r = 0.372, p &lt; 0.001). Post-treatment blood was collected a median 2.4mo (IQR 1.7-3.9) after therapy completion. Positivity decreased after completing treatment (Table), with 88.4% of cases (46/52) becoming ctDNA negative. All cases with complete treatment had a reduction in biomarker levels, whereas in those with incomplete therapy, 54.5% (12/22) remained positive and the pre- and post-treatment levels were not significantly different. Of those positive after treatment, 13 had a further blood sample: 8 had become ctDNA negative and all but one remained disease free. Five remained positive and all had further suspected or confirmed disease. Conclusions: Levels of methylated BCAT1 and IKZF1 DNA in blood correlated with tumor burden; levels became undetectable in the majority of patients following completion of planned curative intent treatment. The methylated ctDNA blood test aids monitoring of responses to therapy and identification of those cases with residual cancer who might benefit from ongoing therapy.[Table: see text]

Circulating tumor DNA and circumferential resection margin as key prognostic indicators for survival in rectal cancer.

3579 Background: Recurrence of colorectal cancer has been linked to the presence of epigenetic circulating tumour DNA (ctDNA) in patient plasma after surgery. The prognostic significance of ctDNA prior to treatment remains unknown. This study investigated the correlation between pre-treatment ctDNA and current radiological (MRI) prognostic markers in patients with rectal cancer. Methods: Forty-two patients with rectal cancer were enrolled, with all having staging MRI prior to treatment. Plasma was taken for ctDNA at diagnosis. The presence of either methylated branched-chain amino acid transaminase 1 (BCAT1) or Ikaros family zinc finger (IKZF1) in cell-free DNA from plasma was deemed a positive ctDNA result. Correlation of MRI prognostic indicators and ctDNA results was assessed with chi-square tests. Univariable and multivariable cox regression analyses were performed to determine variables associated with overall survival (OS). Results: Mean age was 64.4 years (SD 12.5) and majority were male (30/42, 71.4%). 11, 13, 9 &amp; 9 patients had stages I, II, III, IV respectively. Patients had a minimum follow-up of 36 months. Thirty-three (78.6%) patients received neoadjuvant chemoradiotherapy. 29 (69.0%) patients underwent surgical resection. 3-year survival rate was 64% in the overall group. 67% (n=28/42) of patients were positive for the methylated ctDNA at diagnosis. 11 out of 12 patients with a positive circumferential resection margin (CRM +) were ctDNA positive (p=0.03). Univariable analysis showed that prognostic indicators for OS were presence of extramural venous invasion (EMVI) (HR 3.0, 95% CI 1.1-8.4), CRM+ (HR 12.2, 95%CI 3.9-37.6), metastatic disease (HR 7.32, 95% CI 2.63-20.37) and ctDNA% methylation (HR 1.1, 95% CI 1.04-1.13) (Table 1). The presence of CRM+ and a positive ctDNA had a HR of 20.5 (95% CI 5.1-82.3). With multivariable analysis, including adjustment for age and EMVI, only CRM+/ctDNA+ variable was an independent predictor for poor survival (HR 20.2, 95% CI 4.5-90.9). Conclusions: In rectal cancer, almost all patients with CRM involvement have ctDNA, and these patients had the worst prognosis. Future studies with longitudinal ctDNA assessment pre and post treatment could potentially inform prognosis and help tailor patients’ treatment.[Table: see text]

EPCO-01. LUNG ADENOCARCINOMA BRAIN METASTASIS PREDICTION, PREVENTION, AND NON-INVASIVE DIAGNOSIS USING METHYLATION SIGNATURES WITHIN TISSUE AND CIRCULATING TUMOUR DNA

Abstract BACKGROUND One quarter of lung adenocarcinoma (LUAD) patients develop brain metastases (BM) and experience a poorer median survival of 12 months despite treatment. Clinical variables do not robustly predict who will develop BM and targeted preventative treatments are limited. Tumour DNA methylation signatures predict outcomes in other cancers and can be detected in circulating tumour DNA (ctDNA). This work predicts BM development from LUAD using methylation data, identifies novel potential treatment targets to prevent metastases, and detects LUAD-BM ctDNA non-invasively. METHODS DNA methylation profiling was undertaken on N=124 LUAD tumours. A gradient boosted regression model built on differentially methylated CpGs (DMCs) between tumours with and without BM in 70% of samples was validated in an independent 30% testing cohort. Nine paired BM samples were profiled and DMCs between their corresponding LUAD tissue were identified along with copy number (CN) alterations. A total of 47 LUAD-BM plasma samples underwent sequencing of immunoprecipitated methylated ctDNA and differentially methylated regions (DMRs) between LUAD-BM and intrinsic brain lesions were identified. RESULTS The methylation-based model significantly predicted time to brain metastasis development within the testing cohort independently from cancer stage in a multivariate analysis (HR=4.3, 95%CI 1.1–17, p=0.038). Genes/pathways involved in the process of brain metastasis were identified through assessment of 83K DMCs (FDR&amp;lt; 0.2, mean difference &amp;gt;|0.1|) between paired samples as well as the CN losses found in chromosome 12q/19 of BM samples. A total of 5.5K DMRs were identified that distinguish BM samples from gliomas or primary CNS lymphomas (FDR&amp;lt; 0.05, logFC &amp;gt;1). CONCLUSIONS DNA methylation signatures in lung adenocarcinomas predict brain metastasis development independently from prognostic clinical factors. Genes and pathways involved in metastasis were identified as novel potential therapeutic targets. Methylated circulating tumor DNA signatures differentiate lung brain metastases from other ring-enhancing brain lesions and may have potential for non-invasive diagnosis.

Epigenetic Analysis of Circulating Tumor DNA in Localized and Metastatic Prostate Cancer: Evaluation of Clinical Biomarker Potential.

Novel and minimally-invasive prostate cancer (PCa)-specific biomarkers are needed to improve diagnosis and risk stratification. Here, we investigated the biomarker potential in localized and de novo metastatic PCa (mPCa) of methylated circulating tumor DNA (ctDNA) in plasma. Using the Marmal-aid database and in-house datasets, we identified three top candidates specifically hypermethylated in PCa tissue: DOCK2, HAPLN3, and FBXO30 (specificity/sensitivity: 80%–100%/75–94%). These candidates were further analyzed in plasma samples from 36 healthy controls, 61 benign prostatic hyperplasia (BPH), 102 localized PCa, and 65 de novo mPCa patients using methylation-specific droplet digital PCR. Methylated ctDNA for DOCK2/HAPLN3/FBXO30 was generally not detected in healthy controls, BPH patients, nor in patients with localized PCa despite a positive signal in 98%–100% of matched radical prostatectomy tissue samples. However, ctDNA methylation of DOCK2, HAPLN3, and/or FBXO30 was detected in 61.5% (40/65) of de novo mPCa patients and markedly increased in high- compared to low-volume mPCa (89.3% (25/28) vs. 32.1% (10/31), p < 0.001). Moreover, detection of methylated ctDNA was associated with significantly shorter time to progression to metastatic castration resistant PCa, independent of tumor-volume. These results indicate that methylated ctDNA (DOCK2/HAPLN3/FBXO30) may be potentially useful for identification of hormone-naïve mPCa patients who could benefit from intensified treatment.