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Purpose: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. We profiled 5-hmC with Nano-hmC-Seal technology using 10 ng of plasma-derived cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma to determine its utility as a biomarker. Experimental Design: For the Discovery cohort, 100 5-hmC profiles were generated from 34 well children and 32 patients (27 high-risk, 2 intermediate-risk, and 3 low-risk) at various time points during the course of their disease. An independent Validation cohort encompassed 5-hmC cfDNA profiles (n = 29) generated from 21 patients (20 high-risk and 1 intermediate-risk). Metastatic burden was classified as high, moderate, low, or none per Curie metaiodobenzylguanidine scores and percentage of tumor cells in bone marrow. Genes with differential 5-hmC levels between samples according to metastatic burden were identified using DESeq2. Results: Hierarchical clustering using 5-hmC levels of 347 genes identified from the Discovery cohort defined four clusters of samples that were confirmed in the Validation cohort and corresponded to high, high-moderate, moderate, and low/no metastatic burden. Samples from patients with increased metastatic burden had increased 5-hmC deposition on genes in neuronal stem cell maintenance and epigenetic regulatory pathways. Further, 5-hmC cfDNA profiles generated with 1,242 neuronal pathway genes were associated with subsequent relapse in the cluster of patients with predominantly low or no metastatic burden (sensitivity 65%, specificity 75.6%). Conclusions: cfDNA 5-hmC profiles in children with neuroblastoma correlate with metastatic burden and warrants development as a biomarker of treatment response and outcome.

Background: Biomarkers currently used for prognostic stratification or predicting survival in patients with newly diagnosed multiple myeloma (MM) either lack good sensitivity and specificity or require invasive bone marrow biopsies. Aberrant DNA methylation has been associated with poor prognosis and survival in MM. We recently identified a gene panel modified with 5-hydroxymethylcotosine (5hmC), a mark of gene activation, in circulating cell-free DNA (cfDNA) that is associated with relapse or death in patients with diffuse large B-cell lymphoma, another lymphoproliferative disorder. To date, no study has investigated 5hmC in cfDNA for its prognostic significance in MM. Methods: Starting in 2010, we prospectively enrolled patients who were newly diagnosed with MM at The University of Chicago Medical Center. Blood samples were collected at the time of diagnosis. The current report includes data from 184 MM patients. Mortality was ascertained using medical records and the National Death Index. Follow-up was through December 2017, with an average follow-up length of 41.64 months (deceased, n=31; alive, n=153). We profiled genome-wide 5hmC in cfDNA by first using the nano-hmC-Seal technique to construct DNA libraries, which were subjected to next-generation sequencing, mapped to the human genome, and annotated to ~22,000 gene bodies. Cox proportional hazards model and the elastic net regularization, controlling for age, gender, and population, were used to detect 5hmC marker genes associated with overall survival (OS) after diagnosis. Results: For the 184 patients, median age at diagnosis was 61.7 years and 48% (n=88) were males. The differential 5hmC enrichment levels of a preliminary eight-gene marker panel were used to compute a weighted prognostic score (wp-score). In multivariate Cox models, patients with high wp-score had worse OS (Hazard Ratio [HR] = 8.2; 95% Confidence Interval [CI], 2.9-23.4, p < 0.0001), compared with those in the low risk group. Some of the eight 5hmC-midified marker genes have been previously implicated in MM survival. For example, GTF2I (encoding general transcription factor IIi), a known prosurvival gene in MM, showed a single-gene HR of 0.3 (95% CI, 0.1-0.7, p=0.002). The 5hmC-based wp-score outperformed (sensitivity=0.74, specificity=0.84) standard prognostic indices (e.g., albumin, beta-2 microglobulin, and risk classification by cytogenetics) in predicting patients at risk for inferior survival. We are validating the gene panel in an independent cohort of ~150 MM patients. The results will be presented. Conclusions: These findings suggest that 5hmC in cfDNA at the time of diagnosis correlate with OS and outperform standard clinical prognostic indices. Our novel findings, if confirmed, 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 of MM. This abstract is also being presented as Poster A40. Citation Format: Brian C.-H. Chiu, Zhou Zhang, Jason Karpus, Benjamin Derman, Chang Zeng, Elizabeth Stepniak, Rudy Chiu, John Spinelli, Andrzej Jakubowiak, Chuan He, Wei Zhang. Genome-wide 5-hydroxymethylcytosine profiles in circulating cell-free DNA and survival in patients with multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR03.

Background: Positron emission tomography (PET) is one of the current standard-of-care imaging techniques for evaluating extramedullary disease in multiple myeloma (MM); hypermetabolic focal lesions at diagnosis are associated with poor prognosis. Molecular analysis of circulating cell-free DNA (cfDNA) has potential to not only be a noninvasive test for measuring extramedullary disease to complement imaging, but also provide pathobiologic insights. We previously discovered that signatures of the epigenetic modification 5-hydroxyethylcytosine (5hmC) in cfDNA differ between MM and its precursor conditions and predict relapse risk for MM patients at the time of diagnosis. We evaluated differentially modified 5hmC genes between PET-negative and PET-positive patients at the time of MM diagnosis and assessed concordance of the cfDNA 5hmC profiles with PET. Methods: We prospectively enrolled patients with newly diagnosed MM at The University of Chicago Medical Center from 2010 to 2017. Patients enrolled in the study who underwent standard-of-care PET imaging within 30 days of a corresponding blood draw were evaluated. Blood samples were collected and processed immediately to separate plasma. We profiled 5hmC with DNA extracted from ~2 mL of plasma to construct the 5hmC-seal libraries using the nano-hmC-Seal technology, which then underwent next-generation sequencing. 5hmC sequencing data were mapped to the human genome and annotated to ~22,000 gene bodies. We compared genome-wide 5hmC loci between PET-positive and PET-negative patients with MM. We developed an eight gene-based weighted prognostic score (wp-score) for predicting overall survival by applying the elastic net regularization on Cox proportional hazards model. Next, we evaluated concordance between PET results and wp-score. Results: A total of 71 MM patients (age, 61.2±10.65 year; males n=42) had at least one PET scan performed and had cfDNA sequencing data available; 29 (36%) patients had an initial PET positive for MM and the remaining 52 had a negative PET. We found 14 differentially modified 5hmC genes between PET-positive and PET-negative patients at baseline (p Conclusions: Differential enrichment of 5hmC-modifications on gene body regions identified from 5hmC profiling of plasma cfDNA at the time of MM diagnosis differentiates patterns of PET imaging and adds complementary prognostic information to PET. These novel findings support the investigation of 5hmC in cfDNA as part of a multimodal approach to enhance prognostication in MM and potentially guide initial therapy. Citation Format: Benjamin Derman, Zhou Zhang, Jason Karpus, Chang Zeng, Elizabeth Stepniak, Diana West-Szymanski, Rudy Chiu, John Spinelli, Chuan He, Jagoda Jasielec, Andrzej Jakubowiak, Wei Zhang, Brian Chiu. Concordance of 5-hydroxymethylcytosine-modified genes from circulating cell-free DNA and positron emission tomography in multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A39.