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

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.

[1]  D. Cane,et al.  The nonsense-mediated decay RNA surveillance pathway. , 2007, Annual review of biochemistry.

[2]  Carlos L Arteaga,et al.  Intrinsic and acquired resistance to HER2-targeted therapies in HER2 gene-amplified breast cancer: mechanisms and clinical implications. , 2012, Critical reviews in oncogenesis.

[3]  J. Buhmann,et al.  Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry , 2014, Nature Methods.

[4]  M. Gönen,et al.  Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. , 2010, The Journal of clinical investigation.

[5]  Raymond R Tubbs,et al.  Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines. , 2009, Archives of pathology & laboratory medicine.

[6]  D. Knowles,et al.  Intratumoral Heterogeneity of HER-2/neu in Invasive Mammary Carcinomas Using Fluorescence In-Situ Hybridization and Tissue Microarray , 2006, International journal of surgical pathology.

[7]  K. Polyak,et al.  Intra-tumour heterogeneity: a looking glass for cancer? , 2012, Nature Reviews Cancer.

[8]  Eunyoung Kang,et al.  Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance , 2012, Modern Pathology.

[9]  I. Bièche,et al.  Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab , 2013, British Journal of Cancer.

[10]  C. Perou,et al.  Mutant PIK3CA accelerates HER2-driven transgenic mammary tumors and induces resistance to combinations of anti-HER2 therapies , 2013, Proceedings of the National Academy of Sciences.

[11]  Hiroyuki Konishi,et al.  The PIK3CA gene is mutated with high frequency in human breast cancers , 2004, Cancer biology & therapy.

[12]  U. Frey,et al.  PCR-amplification of GC-rich regions: 'slowdown PCR' , 2008, Nature Protocols.

[13]  Aleix Prat Aparicio.  Comprehensive molecular portraits of human breast tumours , 2012 .

[14]  John M S Bartlett,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. , 2014, Archives of pathology & laboratory medicine.

[15]  D. Visscher,et al.  Analysis of intratumoral heterogeneity and amplification status in breast carcinomas with equivocal (2+) HER-2 immunostaining. , 2005, American journal of clinical pathology.

[16]  C. Cantor,et al.  Automated genotyping using the DNA MassArray technology. , 2001, Methods in molecular biology.

[17]  Steven J. M. Jones,et al.  Comprehensive molecular portraits of human breast tumours , 2013 .

[18]  Rosette Lidereau,et al.  PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups , 2012, Breast Cancer Research.

[19]  Anne Vincent-Salomon,et al.  Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification , 2015, Genome Biology.

[20]  U. Surti,et al.  The equivocally amplified HER2 FISH result on breast core biopsy: indications for further sampling do affect patient management. , 2008, American journal of clinical pathology.

[21]  Alastair I. Bartlett,et al.  Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition. , 2011, American journal of clinical pathology.

[22]  Satoshi Ikeda,et al.  Detection of gene point mutation in paraffin sections using in situ loop‐mediated isothermal amplification , 2007, Pathology international.

[23]  A. Solow,et al.  Measuring biological diversity , 2006, Environmental and Ecological Statistics.

[24]  S. Nofech-Mozes,et al.  Intratumoral Heterogeneity of HER2/neu in Breast Cancer—A Rare Event , 2007, The breast journal.

[25]  S. Jeffrey,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 , 2014, BMC Cancer.

[26]  Carlo Rago,et al.  Mutant PIK3CA promotes cell growth and invasion of human cancer cells. , 2005, Cancer cell.

[27]  A. Vincent-Salomon,et al.  Intratumoral heterogeneity of HER2/neu expression and its consequences for the management of advanced breast cancer. , 2007, Annals of oncology : official journal of the European Society for Medical Oncology.

[28]  J. Troge,et al.  Tumour evolution inferred by single-cell sequencing , 2011, Nature.

[29]  K. Kinzler,et al.  Digital PCR. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Barrett,et al.  PIK3CA Mutations May Be Discordant between Primary and Corresponding Metastatic Disease in Breast Cancer , 2010, Clinical Cancer Research.

[31]  J. A. Hartigan,et al.  A k-means clustering algorithm , 1979 .

[32]  Robin L. Jones,et al.  Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity. , 2014, Cell reports.

[33]  Charles Swanton,et al.  Intratumor Heterogeneity: Seeing the Wood for the Trees , 2012, Science Translational Medicine.

[34]  S. Chandarlapaty,et al.  PI3K Pathway Activation in High-Grade Ductal Carcinoma In Situ—Implications for Progression to Invasive Breast Carcinoma , 2014, Clinical Cancer Research.

[35]  N. Navin.  Cancer genomics: one cell at a time , 2014, Genome Biology.

[36]  O. Bagasra.  Protocols for the in situ PCR-amplification and detection of mRNA and DNA sequences , 2007, Nature Protocols.

[37]  Anthony Rhodes,et al.  American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. , 2010, Archives of pathology & laboratory medicine.

[38]  Yaohui Wang,et al.  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 , 2013, Current medical research and opinion.

[39]  Ming Tan,et al.  PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. , 2004, Cancer cell.

[40]  U. Lehmann,et al.  Marked Intratumoral Heterogeneity of c-myc and CyclinD1 But Not of c-erbB2 Amplification in Breast Cancer , 2002, Laboratory Investigation.

[41]  S. Chandarlapaty,et al.  Frequent Mutational Activation of the PI3K-AKT Pathway in Trastuzumab-Resistant Breast Cancer , 2012, Clinical Cancer Research.

[42]  M. Kenward,et al.  An Introduction to the Bootstrap , 2007 .

[43]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[44]  M. Gönen,et al.  Evolutionary pathways in BRCA1-associated breast tumors. , 2012, Cancer discovery.

[45]  Alexander van Oudenaarden,et al.  Genetic and phenotypic diversity in breast tumor metastases. , 2014, Cancer research.

[46]  Mats Nilsson,et al.  In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics , 2013, Oncotarget.

[47]  J. Reis-Filho,et al.  Immunophenotypic and genomic characterization of papillary carcinomas of the breast , 2012, The Journal of pathology.

[48]  M. Birrer,et al.  In situ detection of unexpected patterns of mutant p53 gene expression in non-small cell lung cancers , 2001, Oncogene.

[49]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[50]  C. Arteaga,et al.  Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells , 2014, Breast Cancer Research.

[51]  J. Bergh,et al.  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 , 2004, Applied immunohistochemistry & molecular morphology : AIMM.

[52]  S. Gabriel,et al.  High-throughput oncogene mutation profiling in human cancer , 2007, Nature Genetics.

[53]  S. Bersani,et al.  Genotypic intratumoral heterogeneity in breast carcinoma with HER2/neu amplification: evaluation according to ASCO/CAP criteria. , 2009, American journal of clinical pathology.

[54]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[55]  Steven J. M. Jones,et al.  Comprehensive molecular portraits of human breast tumors , 2012, Nature.