Detection of minor genetically distinct subpopulations within tumors is a key challenge in cancer genomics. two genetic events are not usually present within the same cell. Chemotherapy selects for mutant cells a minor subpopulation in nearly all treatment-na? ve samples and modulates genetic diversity within tumors. Treatment-associated changes in spatial Fumonisin B1 distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant trastuzumab therapy. Our findings support the use of single-cell based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance. Cancer is usually a genetic disease and thus the identification of somatic genetic alterations within tumors has been the focus of clinical oncology. Cancer genome sequencing studies Fumonisin B1 Fumonisin B1 have traditionally been performed on bulk tumors limiting their ability to detect minor subclones which commonly drive therapy resistance1 2 Sequencing of bulk tumors also cannot accurately predict which mutations are present in the same versus in different cells. Sequencing of single malignancy cells overcomes these limitations3 4 but currently this is still laborious expensive and error-prone due the inefficiencies of whole genome amplification and thus not yet suitable for the analysis of large patient cohorts. We developed a novel methodology termed STAR-FISH based on the combination of PCR5-7 and fluorescence hybridization (FISH)8-10 to enable the simultaneous detection of point mutations and copy number variation at the single cell level in intact formalin-fixed paraffin-embedded (FFPE) tissue samples. We designed STAR-FISH for several commonly mutated genes in breast cancer focusing on clinically relevant mutational hotspots. is one of the most commonly mutated genes in breast malignancy11. Mutations in mutation may be used as a predictor of resistance. However the significant heterogeneity for mutation both within different regions of the same tumor and also between different lesions in the same patient20 21 make its accurate detection challenging. We applied STAR-FISH to assess changes in intratumor cellular heterogeneity for amplification and His1047Arg mutation in a cohort of HER2+ breast cancer patients subjected to neoadjuvant chemotherapy followed by adjuvant trastuzumab and correlated these changes with long-term clinical outcome. RESULTS STAR-FISH development and validation GRK6 The first step of STAR-FISH is an PCR using mismatched primers designed to specifically amplify mutant Fumonisin B1 and wild type alleles (Fig. 1a Supplementary Physique 1a Supplementary Table 1 Supplementary Note). The primers contain a 5’ overhang a unique Fumonisin B1 sequence not found in the human genome which serves as a priming site in the second round of PCR. The use of a few amplification cycles in the first round and 30 cycles in the second round of PCR ensures proper amplification of the product with high specificity. PCR products are visualized by hybridization of fluorescently labeled probes complimentary to the 5’ overhang (Fig. 1a). The specificity of the primers for the His1047Arg mutation was first evaluated by PCR using genomic DNA isolated from human breast malignancy cell lines with known mutation status (Fig. 1b). The sensitivity of the assay was tested by performing PCR on defined mixtures of DNA from MDA-MB-231 (wild type) and SUM-185PE cells (homozygous for His1047Arg mutation; Supplementary Physique 1b). Primers for the second round of PCR were tested in the same manner (data not shown). We also developed PCR assays for two other commonly occurring mutations in breast malignancy E542K and R175H mutations (Supplementary Physique 1c d). Physique 1 Outline of the STAR-FISH method and its validation. Scale bars represent 75 μm. (a) Schematic of the STAR-FISH protocol on a cell with heterozygous mutation. In step 1 1 & 2 PCR with a mixture of wild-type (green) and mutant (red) … Next we performed PCR on FFPE tissue slides of xenografts derived from MDA-MB-231 and T-47D breast malignancy cell lines (Fig. 1c and Supplementary Physique 1e) followed by testing of primary human breast tumors with known His1047Arg mutation status (Fig. 1d). Signal for wild type and mutant.