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Analytical Validation of Multiplex Biomarker Assay to Stratify Colorectal Cancer Samples into Molecular Subtypes

By Chanthirika Ragulan, Katherine Eason, Elisa Fontana, Gift Nyamundanda, Yatish Patil, Pawan Poudel, Rita T. Lawlor, Maguy Del Rio, Koo Si-Lin, Tan Wah Siew, Francesco Sclafani, Ruwaida Begum, Larissa S. Teixeira Mendes, Pierre Martineau, Aldo Scarpa, Iain Beehuat Tan, David Cunningham, Anguraj Sadanandam

Posted 16 Aug 2017
bioRxiv DOI: 10.1101/174847 (published DOI: 10.1038/s41598-019-43492-0)

Previously, we classified colorectal cancers (CRCs) into five CRCA subtypes with different prognoses and potential treatment responses, using a 786-gene signature. We merged our subtypes and those described by five other groups into four consensus molecular subtypes (CMS) that are similar to CRCA subtypes. Here we demonstrate the analytical development and application of a custom NanoString platform-based biomarker assay to stratify CRC into subtypes. To reduce costs, we switched from the standard protocol to a custom modified protocol (NanoCRCA) with a high Pearson correlation coefficient (>0.88) between protocols. Technical replicates were highly correlated (>0.96). The assay included a reduced robust 38-gene panel from the 786-gene signature that was selected using an in-laboratory developed computational pipeline of class prediction methods. We applied our NanoCRCA assay to untreated CRCs including fresh-frozen and formalin-fixed paraffin-embedded (FFPE) samples (n=81) with matched microarray or RNA-Seq profiles. We further compared the assay results with CMS classification, different platforms (microarrays/RNA-Seq) and gene-set classifiers (38 and 786 genes). NanoCRCA classified fresh-frozen samples (n=39; not including those showing a mixture of subtypes) into all five CRCA subtypes with overall high concordance across platforms (89.7%) and with CMS subtypes (84.6%), independent of tumour cellularity. This analytical validation of the assay shows the association of subtypes with their known molecular, mutational and clinical characteristics. Overall, our modified NanoCRCA assay with further clinical assessment may facilitate prospective validation of CRC subtypes in clinical trials and beyond.

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