Abstract
Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope—a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.
Highlights
Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth
The temporal patterns of clone spread and mixing were qualitatively the same in both formulations of the model (Supplementary Fig. 10). This manuscript describes the first application of the novel 1ZZ BaseScope in situ hybridization technology to measure the spatial distribution of point mutations in human archival Formalin-fixed paraffin embedded (FFPE) samples
Our analysis shows the very high sensitivity and specificity of 9 BaseScope probesets, representing common driver mutations in the genes KRAS (6 probesets), BRAF (1 probeset) and PIK3CA (2 probesets)
Summary
Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. ITH is most frequently measured by ‘bulk’ sequencing of tumor biopsies[3], or by single cell sequencing (a limited number of) individual cells sorted by flow cytometry[4] These approaches may fail to identify rare, potentially clinically relevant subclones if they are present at a frequency below the limit of generation sequencing (NGS) detection or if they are incorrectly classified as artefacts. Genome-wide copy-number analysis on a gland-by-gland level revealed occasional extensive clonal mixing within CRCs9, and a targeted DNA copy number analysis suggested a complex 3D tumor architecture in a single extensively mapped case[14] Because these previous genotyping methods have required laborious tissue microdissection, detailed topographical maps of clone spread throughout CRCs are lacking
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