Michalina Janiszewska

Clinical samples of human cancer are most often preserved in FFPE blocks, which maintain tissue architecture, yet are not suitable for single-cell mutation analysis by standard sequencing methods. Point mutations cause treatment resistance, thus their analysis is of high clinical impact. We developed STAR-FISH – Specific-To Allele PCR-FISH – which allows for in situ visualization of sub-populations of cells with point mutation and gene amplification, at single-cell level. STAR-FISH was applied to FFPE samples of HER2 positive breast tumors, chemotherapy naïve core needle biopsy and matched post-neoadjuvant chemotherapy tumor sample, from 22 patients. High sensitivity of STAR-FISH revealed presence of single PIK3CA mutant cells in most of the therapy naïve samples. Single cell analysis of PIK3CA mutation and HER2 amplification co-occurrence showed that after chemotherapy an increase in PIK3CA mutant HER2-nonamplified cell population is predominant. STAR-FISH based diversity scores, assessed within different regions of the same tumor, linked significantly altered (increased or decreased) intratumor heterogeneity after chemotherapy to poor patient survival. Our study demonstrates that single-cell analysis of point mutations within intact tumor samples can uncover clinically relevant changes in intratumor heterogeneity and spatial organization of the tumor. Thus, it may aid patient management and the design of more efficient therapies.