Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection

Abstract

Cell-free DNA (cfDNA)–based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions

“…PanIN regions with sufficient DNA in the eight slabs used for combined 3D anatomic and genomic analysis were also analyzed by whole exome sequencing in addition to the targeted sequencing described above. Mutation Capsule (MC) technology was applied to profile a FFPE DNA sample with both hybridization-based exome sequencing and amplification-based deep sequencing, as recently described 25 . Approximately 200ng of the previously fragmented DNA was subjected to end-repair, dA-tailing, and ligation to a customized adapter with random DNA barcodes as a unique identifier (UID) tag 26 .…”

“…To further examine the clonality of single PanIN lesions and the genetic heterogeneity among spatially distinct PanINs in the same slab, we complemented our multi-region targeted sequencing data with whole exome sequencing (WES) and ultra-deep sequencing of KRAS hotspots using the Mutation Capsule approach when sufficient DNA remained following targeted sequencing 25,26 . In total, 57 samples from 24 lesions (20 exclusively LG, 2 with LG and HG dysplasia, 2 PDAC) within the reconstructed tissue blocks were analyzed by WES and deep sequencing of KRAS utilizing Mutation Capsule technology (Supplementary Tables 4 and 5).…”

Comparing a PD-L1 inhibitor plus chemotherapy to chemotherapy alone in neoadjuvant therapy for locally advanced ESCC: a randomized Phase II clinical trial

“…The ctDNA fraction was calculated based on the number and fraction of the mutations detected in plasma samples to determine the fraction of ctDNA among cell-free DNA (cfDNA) [21]. The detailed methods of ctDNA detection were shown in Additional File 1 [20][21][22]. We focused on ctDNA clearance before the operation, which means that the ctDNA fraction was zero at preO.…”

Disease Diagnosis Based on Nucleic Acid Modifications

“…Also through cfMeDIP-seq, Nassiri et al showed that the profile of DNA methylation from plasma reveals very specific features for diagnosing and precisely distinguishing common primary intracranial neoplasms that possess cell-of-origin lineages as well as gliomas originating from extracranial cancer types that may metastasize to the brain with healthy controls, which can be arduous to differentiate using standard-of-care imaging approaches . Very recently, cfDNA methylation has even been combined with its mutations to realize the detection of hepatocellular carcinoma (HCC) with both excellent sensitivity and specificity …”

“…57 Very recently, cfDNA methylation has even been combined with its mutations to realize the detection of hepatocellular carcinoma (HCC) with both excellent sensitivity and specificity. 58 In addition, profiling DNA methylation for cancer diagnosis and classification in solid tumors has been confirmed as a potentially practical tool. Hou et al performed a genetic and morphological investigation on a range of brain cancers that had been histologically verified by institutions as papillary glioneuronal tumors.…”