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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

Abstract

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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References

  1. 1.

    , & Intra-tumour heterogeneity: a looking glass for cancer? Nat. Rev. Cancer 12, 323–334 (2012).

  2. 2.

    , , , & Intratumor heterogeneity: seeing the wood for the trees. Sci. Transl. Med. 4, 127ps10 (2012).

  3. 3.

    et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011).

  4. 4.

    Cancer genomics: one cell at a time. Genome Biol. 15, 452–464 (2014).

  5. 5.

    Protocols for the in situ PCR-amplification and detection of mRNA and DNA sequences. Nat. Protoc. 2, 2782–2795 (2007).

  6. 6.

    , , , & Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol. Int. 57, 594–599 (2007).

  7. 7.

    , , & In situ detection of unexpected patterns of mutant p53 gene expression in non–small cell lung cancers. Oncogene 20, 2579–2586 (2001).

  8. 8.

    et al. Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity. Cell Rep. 6, 514–527 (2014).

  9. 9.

    et al. Genetic and phenotypic diversity in breast tumor metastases. Cancer Res. 74, 1338–1348 (2014).

  10. 10.

    , , , & Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. J. Clin. Invest. 120, 636–644 (2010).

  11. 11.

    et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol. Ther. 3, 772–775 (2004).

  12. 12.

    Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).

  13. 13.

    et al. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12, 395–402 (2007).

  14. 14.

    et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6, 117–127 (2004).

  15. 15.

    , , , & The predictive role of phosphatase and tensin homolog (PTEN) loss, phosphoinositol-3 (PI3) kinase (PIK3CA) mutation, and PI3K pathway activation in sensitivity to trastuzumab in HER2-positive breast cancer: a meta-analysis. Curr. Med. Res. Opin. 29, 633–642 (2013).

  16. 16.

    , , & Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells. Breast Cancer Res. 16, R9 (2014).

  17. 17.

    & Intrinsic and acquired resistance to HER2-targeted therapies in HER2 gene-amplified breast cancer: mechanisms and clinical implications. Crit. Rev. Oncog. 17, 1–16 (2012).

  18. 18.

    et al. PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups. Breast Cancer Res. 14, R28 (2012).

  19. 19.

    et al. Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab. Br. J. Cancer 108, 1807–1809 (2013).

  20. 20.

    et al. Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow. BMC Cancer 14, 456–467 (2014).

  21. 21.

    et al. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast cancer. Clin. Cancer Res. 17, 667–677 (2011).

  22. 22.

    , , & Automated genotyping using the DNA MassArray technology. Methods Mol. Biol. 170, 103–116 (2001).

  23. 23.

    et al. PI3K pathway activation in high-grade ductal carcinoma in situ—implications for progression to invasive breast carcinoma. Clin. Cancer Res. 20, 2326–2337 (2014).

  24. 24.

    et al. Frequent mutational activation of the PI3K-AKT pathway in trastuzumab-resistant breast cancer. Clin. Cancer Res. 18, 6784–6791 (2012).

  25. 25.

    et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J. Clin. Oncol. 31, 3997–4013 (2013).

  26. 26.

    & Digital PCR. Proc. Natl. Acad. Sci. USA 96, 9236–9241 (1999).

  27. 27.

    et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011).

  28. 28.

    et al. Genotypic intratumoral heterogeneity in breast carcinoma with HER2/neu amplification: evaluation according to ASCO/CAP criteria. Am. J. Clin. Pathol. 131, 678–682 (2009).

  29. 29.

    , , , & Marked intratumoral heterogeneity of c-myc and cyclinD1 but not of c-erbB2 amplification in breast cancer. Lab. Invest. 82, 1419–1426 (2002).

  30. 30.

    , & Intratumoral heterogeneity of HER2/neu in breast cancer—a rare event. Breast J. 13, 122–129 (2007).

  31. 31.

    et al. Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines. Arch. Pathol. Lab. Med. 133, 611–612 (2009).

  32. 32.

    et al. Intratumoral heterogeneity of HER2/neu expression and its consequences for the management of advanced breast cancer. Ann. Oncol. 19, 595–597 (2008).

  33. 33.

    et al. Analysis of intratumoral heterogeneity and amplification status in breast carcinomas with equivocal (2+) HER-2 immunostaining. Am. J. Clin. Pathol. 124, 273–281 (2005).

  34. 34.

    , , , & The equivocally amplified HER2 FISH result on breast core biopsy: indications for further sampling do affect patient management. Am. J. Clin. Pathol. 129, 383–390 (2008).

  35. 35.

    , , & Intratumoral heterogeneity of HER-2/neu in invasive mammary carcinomas using fluorescence in-situ hybridization and tissue microarray. Int. J. Surg. Pathol. 14, 279–284 (2006).

  36. 36.

    et al. Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance. Mod. Pathol. 25, 938–948 (2012).

  37. 37.

    et al. Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition. Am. J. Clin. Pathol. 136, 266–274 (2011).

  38. 38.

    , , & HER-2/neu (c-erbB-2) evaluation in primary breast carcinoma by fluorescent in situ hybridization and immunohistochemistry with special focus on intratumor heterogeneity and comparison of invasive and in situ components. Appl. Immunohistochem. Mol. Morphol. 12, 14–20 (2004).

  39. 39.

    et al. Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification. Genome Biol. 16, 107–127 (2015).

  40. 40.

    Measuring Biological Diversity (Blackwell, 2004).

  41. 41.

    et al. Evolutionary pathways in BRCA1-associated breast tumors. Cancer Discov. 2, 503–511 (2012).

  42. 42.

    et al. Mutant PIK3CA accelerates HER2-driven transgenic mammary tumors and induces resistance to combinations of anti-HER2 therapies. Proc. Natl. Acad. Sci. USA 110, 14372–14377 (2013).

  43. 43.

    & A K-means clustering algorithm. Appl. Stat. 28, 100–108 (1979).

  44. 44.

    et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7, 561–573 (2005).

  45. 45.

    et al. High-throughput oncogene mutation profiling in human cancer. Nat. Genet. 39, 347–351 (2007).

  46. 46.

    et al. In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics. Oncotarget 4, 2407–2418 (2013).

  47. 47.

    , & The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51–74 (2007).

  48. 48.

    et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).

  49. 49.

    et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014).

  50. 50.

    et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch. Pathol. Lab. Med. 134, 907–922 (2010).

  51. 51.

    et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch. Pathol. Lab. Med. 138, 241–256 (2014).

  52. 52.

    et al. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature 514, 54–58 (2014).

  53. 53.

    , , & PCR-amplification of GC-rich regions: 'slowdown PCR'. Nat. Protoc. 3, 1312–1317 (2008).

  54. 54.

    et al. Immunophenotypic and genomic characterization of papillary carcinomas of the breast. J. Pathol. 226, 427–441 (2012).

  55. 55.

    & An Introduction to the Bootstrap (Chapman & Hall, 1993).

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Acknowledgements

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

Affiliations

  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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Contributions

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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DOI

https://doi.org/10.1038/ng.3391

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