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In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer

Nature Genetics volume 47, pages 12121219 (2015) | Download Citation


Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR–FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell–based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.

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We thank E. Winer, I. Krop, B. Vogelstein and members of the Polyak and Michor laboratories for their critical reading of the manuscript and useful discussions. We thank A. Marusyk and D. Tabassum for their help with the xenograft assays, R. Witwicki for help with data processing, L. Cameron in the Dana-Farber Cancer Institute Confocal Microscopy center for her technical support, A. Richardson (Dana-Farber Cancer Institute) for providing slides from a human breast tumor with known status for the PIK3CA mutation encoding p.His1047Arg, and H. Russness and I. Rye (Oslo University Hospital) for providing the BAC probe for HER2. This work was supported by the Dana-Farber Cancer Institute Physical Sciences–Oncology Center (U54CA143798 to F.M.), the European Molecular Biology Organization (EMBO; M.J.), the Swiss National Science Foundation (M.J.), the American Cancer Society (CRP-07-234-06-COUN to C.L.A.) and the Breast Cancer Research Foundation (K.P.).

Author information


  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Michalina Janiszewska
    • , Vanessa Almendro
    • , Yanan Kuang
    • , Cloud Paweletz
    •  & Kornelia Polyak
  2. Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Michalina Janiszewska
    • , Vanessa Almendro
    •  & Kornelia Polyak
  3. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Michalina Janiszewska
    • , Vanessa Almendro
    •  & Kornelia Polyak
  4. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Lin Liu
    •  & Franziska Michor
  5. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Lin Liu
    •  & Franziska Michor
  6. Belfer Institute of Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Yanan Kuang
    •  & Cloud Paweletz
  7. Breast Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Rita A Sakr
    •  & Tari A King
  8. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Britta Weigelt
    •  & Jorge S Reis-Filho
  9. Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee, USA.

    • Ariella B Hanker
    •  & Carlos L Arteaga
  10. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Sarat Chandarlapaty
    •  & Jorge S Reis-Filho
  11. Department of Medicine, Breast Cancer Research Program, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee, USA.

    • Carlos L Arteaga
  12. Department of Pathology, Seoul National University College of Medicine, Seoul, South Korea.

    • So Yeon Park
  13. Broad Institute, Cambridge, Massachusetts, USA.

    • Kornelia Polyak
  14. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Kornelia Polyak


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M.J. developed the STAR-FISH method and performed the experiments and data analyses. V.A. assisted with image acquisition and analyses. L.L. performed mathematical modeling and data analysis. S.Y.P. provided tumor samples. Y.K. and C.P. performed the digital PCR experiment and data analysis. R.A.S., B.W., T.A.K., S.C. and J.S.R.-F. provided patient samples and performed the Sequenom MassARRAY experiment. A.B.H. and C.L.A. provided data and tissues from transgenic models of HER2-positive breast cancer. K.P. and F.M. supervised the study. All authors helped to design the study and write the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Franziska Michor or Kornelia Polyak.

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