Genome-wide 5hmC profiles to enable cancer detection and tissue of origin classification in breast, colorectal, lung, ovarian, and pancreatic cancers.
Authors: Haan David , Bergamaschi Anna , Ning Yuhong , Gibb William , Kesling Michael , Pitea Adriana , Nabiyouni Maryam , Ellison Chris , Malta Roger , Nguyen Albert , Guler Gulfem , McCarthy Erin , Phillips Tierney , Scott Aaron , Hazen Kyle , Sheard Judy , Peters Melissa , Bethel Kelly , Volkmuth Wayne , Levy Samuel 
Published: June 2021

3044 Background: Epigenomics assays have recently become popular tools for identification of molecular biomarkers, both in tissue and in plasma. In particular 5-hydroxymethyl-cytosine (5hmC) method, has been shown to enable the epigenomic regulation of gene expression and subsequent gene activity, with different patterns, across several tumor and normal tissues types. In this study we show that 5hmC profiles enable discrete classification of tumor and normal tissue for breast, colorectal, lung ovary and pancreas. Such classification was also recapitulated in cfDNA from patient with breast, colorectal, lung, ovarian and pancreatic cancers. Methods: DNA was isolated from 176 fresh frozen tissues from breast, colorectal, lung, ovary and pancreas (44 per tumor per tissue type and up to 11 tumor tissues for each stage (I-IV)) and up to 10 normal tissues per tissue type. cfDNA was isolated from plasma from 783 non-cancer individuals and 569 cancer patients. Plasma-isolated cfDNA and tumor genomic DNA, were enriched for the 5hmC fraction using chemical labelling, sequenced, and aligned to a reference genome to construct features sets of 5hmC patterns. Results: 5hmC multinomial logistic regression analysis was employed across tumor and normal tissues and identified a set of specific and discrete tumor and normal tissue gene-based features. This indicates that we can classify samples regardless of source, with a high degree of accuracy, based on tissue of origin and also distinguish between normal and tumor status.Next, we employed a stacked ensemble machine learning algorithm combining multiple logistic regression models across diverse feature sets to the cfDNA dataset composed of 783 non cancers and 569 cancers comprising 67 breast, 118 colorectal, 210 Lung, 71 ovarian and 100 pancreatic cancers. We identified a genomic signature that enable the classification of non-cancer versus cancers with an outer fold cross validation sensitivity of 49% (CI 45%-53%) at 99% specificity. Further, individual cancer outer fold cross validation sensitivity at 99% specificity, was measured as follows: breast 30% (CI 119% -42%); colorectal 41% (CI 32%-50%); lung 49% (CI 42%-56%); ovarian 72% (CI 60-82%); pancreatic 56% (CI 46%-66%). Conclusions: This study demonstrates that 5hmC profiles can distinguish cancer and normal tissues based on their origin. Further, 5hmC changes in cfDNA enables detection of the several cancer types: breast, colorectal, lung, ovarian and pancreatic cancers. Our technology provides a non-invasive tool for cancer detection with low risk sample collection enabling improved compliance than current screening methods. Among other utilities, we believe our technology could be applied to asymptomatic high-risk individuals thus enabling enrichment for those subjects that most need a diagnostic imaging follow up.