Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection.

BACKGROUND: Detecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge. METHODS: To address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq. MESA enables simultaneous inference of four epigenetic modalities: cfDNA methylation, nucleosome occupancy, nucleosome fuzziness, and windowed protection score for regions surrounding gene promoters and polyadenylation sites. RESULTS: When applied to 690 cfDNA samples from 3 colorectal cancer clinical cohorts, MESA's novel modalities, which include nucleosome fuzziness, and genomic features, including polyadenylation sites, improve cancer detection beyond the traditional epigenetic markers of promoter DNA methylation. CONCLUSIONS: Together, MESA stands as a major advancement in the field by utilizing comprehensive and complementary epigenetic profiles of cfDNA for effective non-invasive cancer detection.

A multi-analyte cell-free DNA-based blood test for early detection of hepatocellular carcinoma.

The limited performance of guideline-recommended abdominal ultrasound and serum alpha-fetoprotein (AFP) highlights the urgent, unmet need for new biomarkers for more accurate detection of early hepatocellular carcinoma (HCC). To this end, we have conducted a prospective clinical validation study to evaluate the performance of the HelioLiver Test, a multi-analyte blood test combining cell-free DNA methylation patterns, clinical variables, and protein tumor markers. A blinded, multicenter validation study was performed with 247 subjects, including 122 subjects with HCC and 125 control subjects with chronic liver disease. The performance of the HelioLiver Test was compared with AFP and the GALAD score as established HCC surveillance blood tests. The performance of the HelioLiver Test (area under the receiver operating characteristic curve [AUROC] = 0.944) was superior to both AFP (AUROC = 0.851; p < 0.0001) and GALAD (AUROC = 0.899; p < 0.0001). Using a prespecified diagnostic algorithm, the HelioLiver Test showed sensitivities of 85% (95% confidence interval [CI], 78%-90%) for HCC of any stage and 76% (95% CI, 60%-87%) for early stage (American Joint Committee on Cancer [AJCC] I and II) HCC. In contrast, AFP (≥20 ng/mL) alone and the GALAD score (≥-0.63) showed lower sensitivities of 62% (95% CI, 54%-70%) and 75% (95% CI, 67%-82%) for HCC overall, and 57% (95% CI, 40%-71%) and 65% (95% CI, 49%-79%) for early stage (AJCC I and II) HCC, respectively. The specificities of the HelioLiver Test (91%; 95% CI, 85%-95%), AFP (97%; 95% CI, 92%-99%), and the GALAD score (94%; 95% CI, 88%-97%) were similar for control subjects. The HelioLiver Test showed superior performance for HCC detection compared to with both AFP and the GALAD score and warrants further evaluation in HCC surveillance settings.

Design, synthesis, and evaluation of liver-specific gemcitabine prodrugs for potential treatment of hepatitis C virus infection and hepatocellular carcinoma.

Many successful anti-viral and anti-cancer drugs are nucleoside analogs, which disrupt RNA and/or DNA synthesis. Here, we present liver-specific prodrugs of the chemotherapy drug gemcitabine (2',2'-difluorodeoxycytidine) for the treatment of hepatitis C virus (HCV) infection and hepatocellular carcinoma. The prodrugs were synthesized by introducing aromatic functional moieties to the cytosine 4-NH2 group of gemcitabine via amide bonds. The chemical modification was designed to i) enable passive diffusion across cellular membrane, ii) protect the prodrugs from inactivating deamination by cellular enzymes, and iii) allow release of active gemcitabine after amide hydrolysis by high levels of carboxylesterases in the liver. We found that many of our prodrugs exhibited similar toxicity as gemcitabine toward liver- and kidney-derived cancer cell lines but were 24- to 620-fold less cytotoxic than gemcitabine in breast- and pancreas-derived cancer cells, respectively. The prodrugs also inhibited an HCV replicon with IC50 values ranging from 10 nM-1.7 µM. Moreover, many of the prodrugs had therapeutic index values of >10,000 and have synergetic effects when combined with other Food and Drug Administration-approved anti-HCV small molecule drugs. These characteristics support the development of gemcitabine prodrugs as liver-specific therapeutics.

Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

Erratum to: ENOX2-based early detection (ONCOblot) of asbestos-induced malignant mesothelioma 4-10 years in advance of clinical symptoms.

[This corrects the article DOI: 10.1186/s12014-016-9103-3.].

ENOX2-based early detection (ONCOblot) of asbestos-induced malignant mesothelioma 4-10 years in advance of clinical symptoms.

BACKGROUND: Malignant mesothelioma is an aggressive, almost uniformly fatal tumor, caused primarily by exposure to asbestos. In this study, serum presence of mesothelioma-specific protein transcript variants of ecto-nicotinamide adenine dinucleotide oxidase disulfide-thiol exchanger 2 (ENOX2), a recently identified marker of malignancy, were investigated using the ONCOblot tissue of origin cancer detection test. METHODS: Sequential serum samples collected from asbestos-exposed individuals prior to the development of frank mesothelioma were assayed for ENOX2 presence by 2-D gel immunoblot analysis to determine how long in advance of clinical symptoms mesothelioma-specific ENOX2 transcript variants could be detected. RESULTS: Two mesothelioma-specific ENOX2 protein transcript variants were detected in the serum of asbestos-exposed individuals 4-10 years prior to clinical diagnosis of malignant mesothelioma (average 6.2 years). Either one or both ENOX2 protein transcript variants indicative of malignant mesothelioma were absent in 14 of 15 subjects diagnosed with benign pleural plaques either with or without accompanying asbestosis. CONCLUSIONS: In a population of asbestos-exposed subjects who eventually developed malignant mesothelioma, ENOX2 protein transcript variants characteristic of malignant mesothelioma were present in serum 4-10 years in advance of clinical symptoms. As with all biomarker studies, these observations require validation in a larger, independent cohort of patients and should include prospective as well as retrospective sampling.

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

The X-family DNA polymerases λ (Polλ) and ß (Polß) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polß, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polß preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.