Introduction: Tumor heterogeneity is a major underlying cause of therapy resistance and disease recurrence. Recent evidence shows that resistant clones are nearly always pre-existing in a cancer, but are present at low frequencies. Moreover, genetic analysis of clonal composition has been largely at the level of ‘bulk’ sequencing, which does not inform of the arrangement of tumor subclones in space. This spatial information contains crucial clues as to the mechanisms of subclone emergence. To address this gap in our knowledge, we have used Basescope™, a novel RNA in situ hybridization (ISH) technology to detect somatic single nucleotide variants (SNVs) within key treatment-resistance genes in sections of archival formalin fixed paraffin embedded (FFPE) colorectal cancer (CRC) and adenoma (CRA) samples.
Experimental procedures: Basescope™ probes were designed against common CRC mutations in KRAS, BRAF and PIK3CA, genes that are all implicated in modulating targeted therapy response, and also for the corresponding wildtype genes. Publically available cell lines containing the mutations of interest were used to confirm the sensitivity and specificity of the Basescope™ probes. We then performed RNA-ISH on a series of FFPE colorectal tumors (n=24) using novel Basescope™ technology. The presence of detected mutations was confirmed by DNA sequencing. In additional analyses we multiplexed Basescope™ technology with conventional immunohistochemistry and DNA ISH to study concurrent DNA- and protein-level alterations. Stained sections containing subclonal mutations were digitized and used to create 2D maps depicting the precise location of mutant and wild-type subclones. These were then used to test and refine mathematical models of subclone expansion.
Summary of novel results: Our data reports the application of Basescope™ technology for the in situ detection of SNVs in human CRC. We have used custom designed Basescope™ probes to visualize common mutations, and mapped the spatial arrangement of these mutant subclones. We found mutant subclones in 10 samples analyzed, and noted that these subclones appeared generally contiguous in space. Mathematical modeling of these data reveals the patterns of clonal selection and expansion within CRCs.
Conclusions: This study is the first to use RNA-ISH sensitive to specific somatic SNVs to visualize the subclonal architecture of CRC, specifically of clones involved in treatment failure. Our results are suggestive of a complex clonal architecture within this common cancer type. Furthermore, we have confidently detected mutant subclones comprising less than 5% of the total lesion - these would not be reliably detected by standard methodology, and would likely be responsible for the failure of targeted therapy. More generally, we envisage that Basescope™ technology will be a useful tool in basic research, and furthermore is readily transferrable to clinical applications.
Citation Format: Ann-Marie Baker, Mindy Wang, Xiao-Jun Ma, Courtney Anderson, Weini Huang, Enric Domingo, Annabelle Lewis, John Bridgewater, Marnix Jansen, Nicholas A. Wright, Manuel Rodriguez-Justo, Emily Park, Ian Tomlinson, Trevor A. Graham. Visualization of treatment resistant subclones in colorectal cancer by mutation specific RNA in situ hybridization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2017-3953