Abstract B67: Genome-wide 5-hydroxymethylcytosine mapping in circulating cell-free DNA reveals prognostic implications in pancreatic ductal adenocarcinoma

AbstractThe 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancer marks H3K4me1 and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in the testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.

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Cell-free DNA promoter hypermethylation as a diagnostic marker for pancreatic ductal adenocarcinoma – An external validation study

Pancreatology ◽

10.1016/j.pan.2021.05.003 ◽

2021 ◽

Author(s):

Stine D. Henriksen ◽

Benjamin E. Stubbe ◽

Poul H. Madsen ◽

Julia S. Johansen ◽

Benny V. Jensen ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Validation Study ◽

Diagnostic Marker ◽

External Validation ◽

Promoter Hypermethylation ◽

Ductal Adenocarcinoma ◽

Cell Free Dna ◽

Free Dna ◽

External Validation Study

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Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA

Nature Communications ◽

10.1038/s41467-020-18965-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Gulfem D. Guler ◽

Yuhong Ning ◽

Chin-Jen Ku ◽

Tierney Phillips ◽

Erin McCarthy ◽

...

Keyword(s):

Pancreatic Cancer ◽

Early Stage ◽

Ductal Adenocarcinoma ◽

Cell Free Dna ◽

Early Stage Disease ◽

Non Invasive ◽

Cancer Pathogenesis ◽

Free Dna ◽

Stage Disease ◽

Circulating Cell Free Dna

Abstract Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92–0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

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Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma

Clinical Chemistry ◽

10.1373/clinchem.2017.283721 ◽

2018 ◽

Vol 64 (4) ◽

pp. 726-734 ◽

Cited By ~ 21

Author(s):

Min Kyeong Kim ◽

Sang Myung Woo ◽

Boram Park ◽

Kyong-Ah Yoon ◽

Yun-Hee Kim ◽

...

Keyword(s):

Kras Mutation ◽

Progression Free Survival ◽

Ductal Adenocarcinoma ◽

Multiplex Detection ◽

Kras Mutations ◽

Cell Free Dna ◽

Free Survival ◽

Free Dna ◽

Fractional Abundance ◽

Prognostic Implications

Abstract BACKGROUND Cell-free DNA (cfDNA) is known to provide potential biomarkers for predicting clinical outcome, but its value in pancreatic ductal adenocarcinoma (PDAC) has not been fully evaluated. The aim of this study was to evaluate the clinical applicability of quantitative analysis of multiplex KRAS mutations in cell-free DNA from patients with PDAC. METHODS A total of 106 patients with PDAC were enrolled in this prospective study. The concentration and fraction of KRAS mutations were determined through multiplex detection of KRAS mutations in plasma samples by use of a droplet digital PCR kit (Bio-Rad). RESULTS KRAS mutations were detected in 96.1% of tissue samples. Eighty patients (80.5%) harbored KRAS mutations in cfDNA, with a median KRAS mutation concentration of 0.165 copies/μL and a median fractional abundance of 0.415%. Multivariable analyses demonstrated that the KRAS mutation concentration [hazard ratio (HR), 2.08; 95% CI, 1.20–3.63] and KRAS fraction (HR, 1.73; 95% CI, 1.02–2.95) were significant factors for progression-free survival. KRAS mutation concentration (HR, 1.97; 95% CI, 1.05–3.67) also had prognostic implications for overall survival. Subgroup analyses showed that KRAS mutation concentration and fractional abundance significantly affected progression-free survival in resectable PDAC (P = 0.016). Moreover, when combined with the cancer biomarker CA19-9, the KRAS mutation concentration in cfDNA showed additive benefits for the prediction of overall survival. CONCLUSIONS This study demonstrates that multiplex detection of KRAS mutations in plasma cfDNA is clinically relevant, providing a potential candidate biomarker for prognosis of PDAC.

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Tumor cell-free DNA copy number instability compared to CA19-9 as an early predictor of response to systemic therapy in pancreatic ductal adenocarcinoma (PDAC).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e14524 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e14524-e14524

Author(s):

Madappa N. Kundranda ◽

Alexander Koenig ◽

Julia Beck ◽

Kirsten Bornemann-Kolatzki ◽

Jessica Coats ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Real Time ◽

Systemic Therapy ◽

Copy Number ◽

Ductal Adenocarcinoma ◽

Malignant Neoplasms ◽

Cell Free Dna ◽

Free Dna ◽

Time Assessment

e14524 Background: Humoral tumor markers are used clinically for real-time assessment of therapeutic efficacy. In pancreatic ductal adenocarcinoma (PDAC) the predominant marker is CA19-9, which is not expressed by 10 to 30% of patients depending on race. We compared plasma cell-free DNA (cfDNA) copy number based assay with changes in serum CA19-9 levels and radiological responses to predict responses to systemic therapy. Methods: In a laboratory blinded, prospective multicenter pilot study, 40 non-resectable PDAC patients, treated with (m)FOLFIRINOX, CAPIRI, or gemcitabine +/- nab-paclitaxel) are currently enrolled. CA19-9 was determined in the local center’s laboratory. Tumor cfDNA was measured with a copy-number instability (CNI) scoring assay, determined by next generation sequencing in a centralized laboratory. The CNI score assesses the amount of cfDNA with somatic macro-alterations originating from malignant neoplasms. The difference of the values before commencing therapy (baseline) and prior to cycle 2 (either rising or falling) was calculated as a predictor of standardized radiological evaluation of chemotherapeutic efficacy. Results: 37 patients (3 drop-outs) had data for baseline and cycle 2, of which CA19-9 was elevated and evaluable in 29 patients. The direction from baseline to cycle 2 of CA19-9 and CNI scores were in agreement in 18/29 patients. 9 of 11 cases with discordant CNI score and CA19-9 had treatment response data, and CNI correlated with 7/9 (78%); in contrast 7/9 had rising CA19-9, when response was stable disease or better (22% concordance). In the 27 patients with available imaging, CNI predicted better (n = 18) than CA19-9 (n = 10) (p = 0.03 Fisher’s exact). Conclusions: This comparative study on cfDNA versus CA19-9 suggest that cfDNA CNI quantitation is a potentially more reliable blood based marker for early real-time assessment of efficacy in systemic PDAC therapy than CA19-9, compared to standard of care imaging. The better prediction after the first cycle might be due to the very short in vivo half-life of cfDNA ( < 1hr) compared to about one week for CA19-9. These results justify a larger prospective validation trial.

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Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA

10.1101/422675 ◽

2018 ◽

Cited By ~ 1

Author(s):

Francois Collin ◽

Yuhong Ning ◽

Tierney Phillips ◽

Erin McCarthy ◽

Aaron Scott ◽

...

Keyword(s):

Pancreatic Cancer ◽

Early Stage ◽

Training Data ◽

Ductal Adenocarcinoma ◽

Data Sets ◽

Cell Free Dna ◽

Non Invasive ◽

Pancreatic Cancers ◽

Free Dna ◽

Circulating Cell Free Dna

AbstractPancreatic cancers are typically diagnosed at late stage where disease prognosis is poor as exemplified by a 5-year survival rate of 8.2%. Earlier diagnosis would be beneficial by enabling surgical resection or earlier application of therapeutic regimens. We investigated the detection of pancreatic ductal adenocarcinoma (PDAC) in a non-invasive manner by interrogating changes in 5-hydroxymethylation cytosine status (5hmC) of circulating cell free DNA in the plasma of a PDAC cohort (n=51) in comparison with a non-cancer cohort (n=41). We found that 5hmC sites are enriched in a disease and stage specific manner in exons, 3’UTRs and transcription termination sites. Our data show that 5hmC density is reduced in promoters and histone H3K4me3-associated sites with progressive disease suggesting increased transcriptional activity. 5hmC density is differentially represented in thousands of genes, and a stringently filtered set of the most significant genes points to biology related to pancreas (GATA4, GATA6, PROX1, ONECUT1) and/or cancer development (YAP1, TEAD1, PROX1, ONECUT1, ONECUT2, IGF1 and IGF2). Regularized regression models were built using 5hmC densities in statistically filtered genes or a comprehensive set of highly variable 5hmC counts in genes and performed with an AUC = 0.94-0.96 on training data. We were able to test the ability to classify PDAC and non-cancer samples with the Elastic net and Lasso models on two external pancreatic cancer 5hmC data sets and found validation performance to be AUC = 0.74-0.97. The findings suggest that 5hmC changes enable classification of PDAC patients with high fidelity and are worthy of further investigation on larger cohorts of patient samples.

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An integrative analysis of genome-wide 5-hydroxymethylcytosines in circulating cell-free DNA detects non-invasive diagnostic markers for gliomas

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab049 ◽

2021 ◽

Author(s):

Jiajun Cai ◽

Chang Zeng ◽

Wei Hua ◽

Zengxin Qi ◽

Yanqun Song ◽

...

Keyword(s):

Clinical Outcomes ◽

Idh1 Mutation ◽

Healthy Individuals ◽

Cell Free Dna ◽

Mutation Status ◽

Non Invasive ◽

Free Dna ◽

Genome Wide ◽

Validation Set ◽

Circulating Cell Free Dna

Abstract Background Gliomas, especially the high-grade glioblastomas (GBM), are highly aggressive tumors in the central nervous system (CNS) with dismal clinical outcomes. Effective biomarkers, which are not currently available, may improve clinical outcomes through early detection. We sought to develop a non-invasive diagnostic approach for gliomas based on 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA). Methods We obtained genome-wide 5hmC profiles using the 5hmC-Seal technique in cfDNA samples from 111 prospectively enrolled patients with gliomas and 111 age-, gender-matched healthy individuals, which were split into a training set and a validation set. Integrated models comprised of 5hmC levels summarized for gene bodies, long non-coding RNAs (lncRNAs), cis-regulatory elements, and repetitive elements were developed using the elastic net regularization under a case-control design. Results The integrated 5hmC-based models differentiated healthy individuals from gliomas (AUC [area under the curve] = 84%; 95% confidence interval [CI], 74-93%), GBM patients (AUC = 84%; 95% CI, 74-94%), WHO II-III glioma patients (AUC = 86%; 95% CI, 76-96%), regardless of IDH1 (encoding isocitrate dehydrogenase) mutation status or other glioma-related pathological features such as TERT, TP53 in the validation set. Furthermore, the 5hmC biomarkers in cfDNA showed the potential as an independent indicator from IDH1 mutation status and worked in synergy with IDH1 mutation to distinguish GBM from WHO II-III gliomas. Exploration of the 5hmC biomarkers for gliomas revealed relevance to glioma biology. Conclusions The 5hmC-Seal in cfDNA offers the promise as a non-invasive approach for effective detection of gliomas in a screening program.

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5-Hydroxymethylcytosines in circulating cell-free DNA and overall survival in patients with multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.8032 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 8032-8032

Author(s):

Brian C Chiu ◽

Zhou Zhang ◽

Benjamin Avi Derman ◽

Jason Karpus ◽

Spencer Langerman ◽

...

Keyword(s):

Multiple Myeloma ◽

Overall Survival ◽

Gene Panel ◽

Marker Genes ◽

Training Set ◽

Cell Free Dna ◽

Free Dna ◽

Genome Wide ◽

Testing Set ◽

Circulating Cell Free Dna

8032 Background: The epigenetic mark 5-methylcytosines (5mC) have been associated with poor prognosis and survival in multiple myeloma (MM), but the prognostic role of 5-hydroxymethylcytosines (5hmC) as marks of tissue-specific enhancers generated from 5mC through active demethylation is unknown. We showed that 5hmC can be profiled in circulating cell-free DNA (cfDNA) and is associated with relapse/death in another lymphoproliferative disorder diffuse large B-cell lymphoma. To date, no study has investigated genome-wide 5hmC profiles in cfDNA for its prognostic significance in MM. Methods: A total of 354 newly diagnosed MM patients at the University of Chicago Medical Center were prospectively enrolled between 2010-2017. Blood samples were collected at the time of diagnosis. Patients were followed through 31 December 2020 (avg. follow-up = 77.8 mths). We collected baseline clinical, laboratory, and cytogenetic data from electronic medical records. Vital status was ascertained in 351 of the 354 patients (deaths = 73) using the National Death Index. We profiled genome-wide 5hmC in cfDNA using the 5hmC-Seal and next-generation sequencing. The 5hmC-Seal data were mapped to the human genome reference (hg19) and annotated to gene bodies. Overall survival (OS) was defined as time from diagnosis until death from any cause. We used Cox proportional hazards model and the elastic net regularization to identify genes with modified 5hmC levels that are associated with OS. Patients were randomly divided into a training set (n = 264) and testing set (n = 87). Results: The median age at diagnosis was 61.8 years and 47% (n = 165) were males. We used the differential 5hmC enrichment levels of a preliminary four-gene marker panel (i.e., YPEL1, VIPR2, PLAC8L1, and CYP2D6) to compute a weighted prognostic score (wp-score). In the training set (deaths = 55), MM patients with high wp-score had worse OS (Hazard Ratio [HR] = 2.2, 95% Confidence Interval [CI]: 1.3-3.9; p = 0.004). The same trend was observed in the testing set (deaths = 18) (HR = 3.5, 95% CI: 1.1-10.6; p = 0.02). The 5hmC-based wp-score remained significantly associated with OS after controlling for standard prognostic factors, suggesting that 5hmC-based wp-score for this 4-gene panel is an independent prognostic factor for MM. We also explored population-specific 5hmC and wp-score. We found that 5hmC profiles in cfDNA differ between Blacks (n = 117) and Whites (n = 234). In addition, 5hmC marker genes associated with OS differ between Blacks (13 genes) and Whites (20 genes). Conclusions: Our results suggest that 5hmC in cfDNA at the time of diagnosis correlate with OS in MM and the 5hmC marker genes associated with OS differ between Blacks and Whites. These findings suggest that a plasma-derived cfDNA 5hmC-modified gene panel holds promise as a noninvasive approach for predicting prognosis in MM and may be integrated in clinical practice to improve precision care.

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