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Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics

Abstract

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

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Fig. 1: EPINUC decodes the combinatorial epigenetic states of plasma cfNucleosomes.
Fig. 2: Multiplexed single-molecule detection of cancer-associated protein biomarkers, mutant p53 and DNA methylation.
Fig. 3: EPINUC reveals significant epigenetic and biomarker alterations in the plasma of individuals with CRC.
Fig. 4: EPINUC differentiates healthy individuals from individuals with CRC and identifies the tumor tissue of origin.

Data availability

The datasets generated and analyzed during this study are summarized in Supplementary Tables 1, 3 and 4. BED files of plasma-sequenced reads are available at the Zenodo repository at https://doi.org/10.5281/zenodo.6627498. Image analysis pipelines are available at the Zenodo repository at https://doi.org/10.5281/zenodo.6627723. Data from public repositories used in the study (cBioportal database for CRC primary tumor RNA expression) can be found at https://www.cbioportal.org/study/summary?id=coadread_tcga. Source data are provided with this paper.

Code availability

Code for performing overlap analysis is available at https://github.com/Vadim-Fed/EPINUC-overlap. Code for performing PPS analysis is available at https://github.com/8080labs/ppscore.

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Acknowledgements

We thank R. Rozenzweing, O. Fasust, M. Maurer and R. Irwin for their contributions in establishing a protocol for MBD2 labeling. We thank L. Segev for help with writing and integrating the μManager scripts for performing EPINUC–seq. We are grateful for the important comments made by I. Ulitsky while reading the manuscript. E.S. is an incumbent of the Lisa and Jeffrey Aronin Family Career Development chair. This research was supported by grants from the European Research Council (ERC801655 and ERC_PoC_963863), Emerson Collective, The Israeli Science Foundation (1881/19), The Israel Cancer Research Fund: Research Career Development Award, The German-Israeli Foundation for Scientific Research and Development and Minerva. We also obtained generous support from the Swiss Society Institute for Cancer Prevention Research and the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics.

Author information

Authors and Affiliations

Authors

Contributions

V.F., N.E. and E.S. designed the study and wrote the manuscript. V.F. and N.E. performed the experiments and analyzed the data. N.F. and O.B. assisted in the experiments, G.R. assisted with data analysis. B.B.Z., Y.M., T.P., A.H., J.E.C., A.S., M.T., A.G., M.M. and A.Z. collected the plasma samples of individuals with early- and late-stage CRC. D.J., A.S. and E.A. contributed to the development of single-nucleosome imaging technology and sequencing experiments described in this study.

Corresponding author

Correspondence to Efrat Shema.

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Competing interests

Yeda Research and Development Co., Ltd., and SeqLL, Inc., have filed a provisional patent application related to aspects of this publication, and E.S., N.E., V.F., A.S., K.A. and D.J. are named inventors. SeqLL, Inc., has a patent application related to this work (US2016/047747), on which E.S. and D.J. are inventors. E.A., A.S. and D.J. own equity in SeqLL, Inc., where D.J. is an officer and director.

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Nature Biotechnology thanks Shixin Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Table 1

Clinical data.

Supplementary Table 2

Antibody inventory.

Supplementary Table 3

EPINUC results.

Supplementary Table 4

ENCODE tissue accession codes used for EPINUC overlap.

Source data

Source Data Fig. 1

Unprocessed gels.

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Fedyuk, V., Erez, N., Furth, N. et al. Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01447-3

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