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

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.

Multi-cancer detection and tissue of origin determination based on 5-hydroxymethylcytosine biomarkers in circulating cell-free DNA.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3123-3123
Author(s):  
Zhou Zhang ◽  
Wenju Chang ◽  
Jiabin Cai ◽  
Yijiu Ren ◽  
Qiancheng You ◽  
...  
Keyword(s):  
Cancer Detection ◽  
Classification Model ◽  
Protein Markers ◽  
Training Set ◽  
Cell Free Dna ◽  
Free Dna ◽  
Cancer Types ◽  
Tissue Of Origin ◽  
Testing Set ◽  
Circulating Cell Free Dna

3123 Background: Early detection may reduce cancer mortality. Systematic screening programs are available only for a limited number of cancers (e.g., colorectal cancer). The majority of common cancers are detected after the onset of signs and symptoms, making treatment difficult or less effective. We describe here a multi-cancer epigenetic approach for simultaneous cancer detection of common cancers (̃70% of adult cancers) and determination of tissue of origin (TOO) using circulating cell-free DNA (cfDNA) from plasma. Methods: A total of 2241 cancer cases, including patients with newly diagnosed primary colorectal, gastric, esophageal, liver, lung, and breast cancer (stages I-III or equivalent) and 2289 non-cancer controls were recruited from participating hospitals in China. Study participants were randomly assigned into a training set (70%) and a testing set (30%), and patients were matched for cancer types and stages. Plasma samples were collected before radical treatment or surgery. The 5hmC-Seal, a highly sensitive chemical labeling technique, was used to profile genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA from ̃5mL of plasma per person, followed by the next-generation sequencing, data summarization at gene-level, and normalization. We applied the elastic net regularization to establish a predictive rule based on the multivariable logistic regression model for cancer detection in the training set as well as a multiclass classification model for determining TOO. The final solution for simultaneous cancer detection and TOO determination was established by integrating the 5hmC-based models and protein markers (e.g., AFP). Overall sensitivity and specificity were computed and reported in the testing set of 670 cancer cases and 686 non-cancer controls. Results: For the primary scenario (i.e., stages I-III or equivalent), at specificity of 95%, the overall sensitivity achieved 79.3% for detecting a cancer patient in all six cancer types in the testing set, except stage I lung cancer, for which the multi-cancer detection solution showed a sensitivity of 51%. Notably, for individuals with a negative result from conventional protein markers (e.g., AFP, CEA), the 5hmC-only models showed 67.6% sensitivity at 98.2% specificity in the testing set, representing significant improvement. In the testing set, among the 500 cancer patients who were detected from the multi-cancer detection solution, 435 patients were assigned a TOO; of those, 362 (83.2%) TOO were correctly determined. Conclusions: The 5hmC-Seal in cfDNA shows the potential as a non-invasive tool that could be integrated into a screening program for simultaneous detection of common cancers and TOO localization. This approach can be expanded to additional cancer types and is currently undergoing validation in prospectively recruited cohorts.

scholarly journals Alterations of 5-hydroxymethylation in circulating cell-free DNA reflect molecular distinctions of subtypes of non-Hodgkin lymphoma

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Brian C.-H. Chiu ◽  
Chang Chen ◽  
Qiancheng You ◽  
Rudyard Chiu ◽  
Girish Venkataraman ◽  
...  
Keyword(s):  
B Cell Lymphoma ◽  
Cell Free Dna ◽  
Invasive Approach ◽  
Non Invasive ◽  
Signature Genes ◽  
Non Hodgkin Lymphoma ◽  
Free Dna ◽  
Genome Wide ◽  
Circulating Cell Free Dna

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.

scholarly journals 5-Hydroxymethylcytosines of Circulating Cell-Free DNA and Prognosis in Diffuse Large B-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2985-2985
Author(s):  
Brian C.-H. Chiu ◽  
Zhou Zhang ◽  
Qiancheng You ◽  
Elizabeth Stepniak ◽  
Paige Bracci ◽  
...  
Keyword(s):  
Overall Survival ◽  
B Cell ◽  
Cell Lymphoma ◽  
Prognostic Significance ◽  
B Cell Lymphoma ◽  
Cell Free Dna ◽  
Free Dna ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Circulating Cell Free Dna

Abstract Background: Elevated levels of circulating cell-free DNA (cfDNA) have been associated with poor prognosis and relapse in patients with diffuse large B-cell lymphoma (DLBCL). However, the tumor-specific molecular targets in cfDNA that have prognostic significance remain unclear. We investigated the association between 5-hydroxymethylcytosine (5hmC), a mark of active demethylation and gene activation, in cfDNA from plasma and prognosis in DLBCL. Methods: We used the 5hmC-Seal, a highly sensitive chemical labeling technique integrated with next-generation sequencing (NGS), to profile 5hmC in plasma cfDNA samples from Caucasian patients at the University of Chicago who were newly diagnosed with DLBCL (n=43) or follicular lymphoma (FL, n=28), the two most common histological subtypes of non-Hodgkin lymphoma (NHL), in 2011-13. Baseline clinical, laboratory, and vital status data were abstracted from medical records. Patients were followed through December 31, 2017 and those who relapsed after completion of treatment, lost to follow-up, or died were censored. We profiled 5hmC with 1-2 ng of cfDNA extracted from ~2 ml of plasma for library construction and the NGS. We obtained ~25 million reads per sample, providing a depth of coverage ~600X in terms of gene bodies. We normalized read counts and identified differential 5hmC markers using DESeq2. Cox proportional hazards model were used to estimate the association between 5hmC markers and overall survival. Results: We found that in cfDNA from DLBCL patients, 5hmC markers were enriched within gene bodies and depleted in CpG islands. The cfDNA-based 5hmC profiles at diagnosis differed between DLBCL and FL, and in DLBCL, the 5hmC profiles differed between Ann Arbor stage (stage 1/2 vs stage 3/4), lactate dehydrogenase (LDH) level (normal vs elevated), and cell-of-origin (germinal center B-like and activated B-cell-like DLBCL). In addition, genome-wide 5hmC distribution patterns in cfDNA samples are highly correlated with those found in cfDNA-paired tumor tissues, supporting the tumor relevance of cfDNA in a patient. Next, we evaluated the prognostic significance of cfDNA-based 5hmC in DLBCL using a two-step approach. In the discovery phase (7 DLBCL patients with relapse and 12 age- and sex-matched patients without relapse within two years following treatment), a substantial number of 5hmC markers were associated with relapse (449 gene bodies at 5% false discovery rate [FDR]). These relapse-associated 5hmC signatures showed high sensitivity, specificity, and overall accuracy (area under curve [AUC]=0.91) in predicting relapse in the independent validation set (relapse=5, no relapse=13). Finally, we identified a panel of 128 5hmC markers (fold change >20% and p-value <0.05) that were associated with 4-year overall survival. Conclusion: These findings suggest that 5hmC signatures in cfDNA at the time of DLBCL diagnosis correlate with standard clinical prognostic indices and hold promise as non-invasive markers for prognosis and survival. Disclosures Smith: BMS: Consultancy; Portola: Honoraria.

scholarly journals Identification of Somatic Mutations in Papanicolaou Smear DNA and Plasma Circulating Cell-Free DNA for Detection of Endometrial and Epithelial Ovarian Cancers: A Pilot Study

2020 ◽  
Vol 10 ◽  
Author(s):  
Xuan Jiang ◽  
Weihua Li ◽  
Jiaxin Yang ◽  
Shuzhen Wang ◽  
Dongyan Cao ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Somatic Mutations ◽  
Pap Smear ◽  
Hot Spot ◽  
Gene Mutations ◽  
Gene Panel ◽  
Pap Smears ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

ObjectivesThe aim of this study was to identify tumor-derived DNA from Papanicolaou (Pap) smear and plasma specimens collected from patients with endometrial cancer or atypical hyperplasia (EC/AH) or epithelial ovarian cancer (OC).MethodsTumor tissues, peripheral blood, and Pap smear samples were collected from patients with EC/AH and patients with epithelial OC. Somatic mutations of tumor specimens in EC/AH and OC were examined by whole-exome sequencing using a 127-driver gene panel from The Cancer Genome Atlas (TCGA). A nine-gene EC/AH panel and an eight-gene OC panel were established based on the identified significantly mutated genes in the EC/AH and OC tumor specimens. Circulating single-molecule amplification and resequencing technology (cSMART) was applied to evaluate somatic mutations in Pap smear DNA and plasma circulating cell-free DNA (ccfDNA) using the EC/AH and OC gene panels.ResultsIn EC/AH group, there existed 22 tumors and 14 of the 22 tumors contributed hot spot mutations for the EC/AH nine-gene panel. In the Pap smear subgroup, all 21 Pap smears tested positive. Nine out of 11 (81.8%) identified the same gene mutations with their matched tumors and the remaining 10 Pap smears all tested positive. In the plasma subgroup, 10 out of 26 (38.5%) plasmas tested positive. One out of 13 (7.7%) identified the same gene mutation with its matched tumor and 5 out of the remaining 13 plasmas (38.5%) tested positive. In OC group, there existed 17 tumors and 16 of the 17 tumors contributed hot spot mutations for the OC eight-gene panel. In the Pap smear subgroup, all 11 Pap smears tested positive. Five out of 10 (50.0%) identified the same gene mutations with their matched tumors and the remaining one Pap smear also tested positive. In the plasma subgroup, all 22 plasmas tested positive. Ten out of 14 (71.4%) identified the same gene mutation with their matched tumors and the remaining 4 plasmas all tested positive.ConclusionsTumor-derived DNA can be detected in Pap smears and plasmas from patients with EC/AH or epithelial OC. Using a small gene-panel, early detection of EC/AH and OC might be promising. However, the value of plasma ccfDNA for EC/AH requires further investigation.

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

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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