Abstract PR05: The effect of chemotherapy on HER2+ breast cancer heterogeneity measured by STAR-FISH: Detection of PIK3CA mutation and HER2 amplification at single-cell level in situ
Authors: Janiszewska Michalina , Liu Lin , Almendro Vanessa , Kuang Yanan , Paweletz Cloud , Weigelt Britta , Sakr Rita A. , King Tari A. , Chandarlapaty Sarat , Reis-Filho Jorge S. , Hanker Ariella B. , Arteaga Carlos L. , Yeon Park So , Michor Franziska , Polyak Kornelia 
Published: May 2016

Abstract
Current therapies in HER2-positive breast cancer are effective in only a subset of cases and part of the resistance is attributed to single nucleotide mutation H1047R in PIK3CA. Conventional PIK3CA mutation detection methods require isolation of DNA from the tumor bulk, which requires relatively large amount of tissue and may not detect mutations in rare cancer cells.
We developed a novel method, Specific-To-Allele PCR-FISH (STAR-FISH), which allows for in situ detection of point mutation and gene amplification at single cell level. The assay consists of in situ PCR steps with mutation specific primers, followed by hybridization of a fluorescently labeled DNA probe homologous to PCR primer overhangs and probes for genomic regions of interest. The STAR-FISH signals present in intact formalin-fixed paraffin embedded (FFPE) samples are imaged and quantified in each individual nucleus within a tissue, with false discovery rate at 0.001, which facilitates identification of sub-populations of cells with different genetic makeup. The method was validated against FACS, immunofluorescence, droplet digital PCR, and MassArray; high correlation of the results was observed (R2=0.901 -0.9037, p&lt;0.001).
We applied STAR-FISH for PIK3CA hot-spot mutation and HER2 amplification to FFPE samples of HER2 positive breast tumors from 22 patients. For each case a chemotherapy naïve core needle biopsy and a post-neoadjuvant chemotherapy sample upon tumor resection were collected. STAR-FISH analysis was performed on 3-5 regions of each sample, to account for intratumor heterogeneity. Long-term patient survival data after adjuvant treatment, mostly with trastuzumab, were available for all the patients.
High-sensitivity of STAR-FISH allowed us to detect rare single cells carrying PIK3CA mutation in most of the pre-treatment samples. After adjuvant chemotherapy the frequency of these cells was significantly increased. Since the STAR-FISH signals are quantified in each individual nucleus, subpopulations of cells with PIK3CA mutation or HER2 amplification or both features can be distinguished. Based on frequencies of cells within each of these subpopulations we calculated Shannon diversity index for each pre- and post-chemotherapy sample. The index was significantly increased after treatment. However, only topologic and not overall changes in diversity predicted poor long-term survival of the patients.
In addition to analyzing the frequency of cells with PIK3CA mutation, HER2 amplification or both changes, STAR-FISH also assesses the spatial distribution of genetically distinct subtypes. We have found that cells with PIK3CA mutation, irrespective of their HER2 status, are much more dispersed within tumors after neaodjuvant chemotherapy, whereas cells with HER2 amplification and wild-type PIK3CA cluster together. These results suggest that PIK3CA mutant cells are more migratory and invasive, in agreement with prior studies of cell lines and animal models.
STAR-FISH provides a unique view into genetic intratumor heterogeneity since thousands of cells within different regions of a single tumor biopsy can be analyzed within their tissue environment. Application of this novel in situ method allowed us to detect rare cells with PIK3CA mutation, pre-existing in the majority of treatment-naïve tumors and increasing in frequency after neoadjuvant chemotherapy. Moreover, STAR-FISH data revealed the correlation of chemotherapy-induced changes in intratumor heterogeneity with long-term survival of HER2+ breast cancer patients and support the significance of tumor diversity in situ analyses.
Citation Format: Michalina Janiszewska, Lin Liu, Vanessa Almendro, Yanan Kuang, Cloud Paweletz, Britta Weigelt, Rita A. Sakr, Tari A. King, Sarat Chandarlapaty, Jorge S. Reis-Filho, Ariella B. Hanker, Carlos L. Arteaga, Park So Yeon, Franziska Michor, Kornelia Polyak. The effect of chemotherapy on HER2+ breast cancer heterogeneity measured by STAR-FISH: Detection of PIK3CA mutation and HER2 amplification at single-cell level in situ. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR05.