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Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples

Nature Medicine volume 23, pages 376–385 (2017)Cite this article

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Abstract

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

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Figure 1: Schematic representation of the formalin-fixed paraffin-embedded (FFPE) single-cell sequencing platform.
Figure 2: Establishment of a whole-genome copy-number profiling method for single nuclei derived from FFPE samples.
Figure 3: Validation of the whole-genome CN method for FFPE-derived single nuclei using samples from case 2, a synchronous DCIS and invasive breast cancer.
Figure 4: Sequencing of FFPE single nuclei from samples of case 3 and phylogeny reconstruction of progression from DCIS to invasive breast cancer.
Figure 5: Whole-genome single-nucleus copy-number information for case 4, a suboptimal, overly damaged FFPE sample.

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Acknowledgements

We thank the M. Wigler lab (CSHL) for kindly providing access to necessary equipment, and M. Schatz, T. Garvin and R. Aboukhalil (CSHL) for their assistance with Ginkgo, an interactive, online platform for the analysis of CNAs from single cells. We thank the CSHL Flow Cytometry Shared Resources, which is supported in part by the National Cancer Institute Cancer Center Shared Grant award number CA045508. We thank S. Turcan and J. Taranda for critically reviewing the manuscript. This work was funded in part by a Susan G. Komen Investigator-Initiated Research Grant (IIR13265578; J.B.H., J.S.R.-F., T.A.K. and B.W.), and by the MSKCC Single-Cell Sequencing Initiative (T.B.) and the William and Joyce O'Neil Research Fund (T.B.). S.P. was funded in part by a Susan G. Komen Postdoctoral Fellowship grant (PDF14298348). J.S.R.-F. is funded in part by the Breast Cancer Research Foundation. Research reported in this publication was supported in part by the Cancer Center Support Grant of the US National Institutes of Health–National Cancer Institute (P30CA008748; MSKCC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

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Author notes

  1. Timour Baslan, Tari A King & James B Hicks

    Present address: Present addresses: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA (T.B.), Dana-Farber Cancer Institute and Brigham and Women's Hospital, Boston, Massachusetts, USA (T.A.K.) and USC Dana and David Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, California, USA (J.B.H.).,

  2. Luciano G Martelotto and Timour Baslan: These authors contributed equally to this work.

  3. Britta Weigelt, James B Hicks and Jorge S Reis-Filho: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Pathology, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York, USA

    Luciano G Martelotto, Felipe C Geyer, Kathleen A Burke, Lee Spraggon, Salvatore Piscuoglio, Charlotte K Y Ng, Arnaud da Cruz Paula, Hannah Y Wen, Britta Weigelt & Jorge S Reis-Filho

  2. Cold Spring Harbor Laboratory (CSHL), Cold Spring Harbor, New York, USA

    Timour Baslan, Jude Kendall, Pamela Moody, Sean D'Italia, Linda Rodgers, Hilary Cox, Asya Stepansky & James B Hicks

  3. Department of Molecular and Cellular Biology, Stony Brook University, New York, New York, USA

    Timour Baslan

  4. Molecular Cytogenetics, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Kalyani Chadalavada & Gouri Nanjangud

  5. Instituto Português de Oncologia, Porto, Portugal

    Arnaud da Cruz Paula

  6. Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Michail Schizas & Tari A King

  7. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Larry Norton

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Contributions

J.S.R.-F., B.W. and J.B.H. conceived and supervised the study; L.G.M. developed the single-cell FFPE methodology, and designed and conducted the experiments; T.B. and J.K. conducted the bioinformatics and statistical analyses of the single-cell data; K.A.B. and C.K.Y.N. performed the bioinformatics analysis of WES data; M.S. performed single-cell data preprocessing for use in the Ginkgo platform; S.P. prepared the FFPE and frozen blocks for cell lines; L.S. prepared the sequencing libraries for the FFPE and frozen cell lines and performed confocal microscopy of sorted nuclei; K.C. and G.N. conducted the FISH experiments, which were analyzed by K.C., G.N. and F.C.G.; T.A.K. and H.Y.W. provided tumor samples; J.S.R.-F. and F.C.G. reviewed and microdissected the histological samples; P.M., L.R. and S.D'I. performed flow cytometric analysis and sorting; T.B. prepared the LP–WGS libraries; H.C., A.S. and A.d.C.P. prepared the WES libraries; L.G.M., T.B., J.K., L.N., B.W., J.B.H. and J.S.R.-F. analyzed, discussed and interpreted the data; L.G.M., T.B., J.K., B.W., J.B.H. and J.S.R.-F. wrote the manuscript. All authors reviewed and approved the manuscript for submission.

Corresponding authors

Correspondence to Britta Weigelt, James B Hicks or Jorge S Reis-Filho.

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Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–10 and Supplementary Tables 1–3 (PDF 8826 kb)

Supplementary Table 4

Whole-Exome Sequencing Statistics And Somatic Mutations Identified In The Dcis And Idc Of Case 3 By Whole-Exome Sequencing (XLSX 42 kb)

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Martelotto, L., Baslan, T., Kendall, J. et al. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples. Nat Med 23, 376–385 (2017). https://doi.org/10.1038/nm.4279

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