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

Previously, we classified colorectal cancers (CRCs) into five CRCAssigner (CRCA) subtypes with different prognoses and potential treatment responses, later consolidated into four consensus molecular subtypes (CMS). Here we demonstrate the analytical development and validation of a custom NanoString nCounter platform-based biomarker assay (NanoCRCA) to stratify CRCs into subtypes. To reduce costs, we switched from the standard nCounter protocol to a custom modified protocol. The assay included a reduced 38-gene panel that was selected using an in-house machine-learning pipeline. We applied NanoCRCA to 413 samples from 355 CRC patients. From the fresh frozen samples (n = 237), a subset had matched microarray/RNAseq profiles (n = 47) or formalin-fixed paraffin-embedded (FFPE) samples (n = 58). We also analyzed a further 118 FFPE samples. We compared the assay results with the CMS classifier, different platforms (microarrays/RNAseq) and gene-set classifiers (38 and the original 786 genes). The standard and modified protocols showed high correlation (> 0.88) for gene expression. Technical replicates were highly correlated (> 0.96). NanoCRCA classified fresh frozen and FFPE samples into all five CRCA subtypes with consistent classification of selected matched fresh frozen/FFPE samples. We demonstrate high and significant subtype concordance across protocols (100%), gene sets (95%), platforms (87%) and with CMS subtypes (75%) when evaluated across multiple datasets. Overall, our NanoCRCA assay with further validation may facilitate prospective validation of CRC subtypes in clinical trials and beyond.

Molecular profiling of colorectal pulmonary metastases and primary tumours: implications for targeted treatment.

This study aimed to molecularly characterise colorectal pulmonary metastases (PM) and investigate whether their molecular profiles were concordant with those of the primary tumour. Clinical data and archival formalin fixed paraffin embedded tissue samples were retrospectively collected from patients who underwent ≥ 1 pulmonary metastasectomies for colorectal cancer between 1997-2012. Primary tumour and metastatic samples were analysed using a targeted capture sequencing panel of 46 cancer-associated genes. The 5-year progression-free and overall survival rates for the 81 patients in this study were 32% (95% CI 22-42%) and 77% (95% CI 66-85%) respectively. Fifty-four patients had samples available from ≥ 1 PM, and sequencing data were successfully obtained from 33 PM from 24 patients. The most frequently mutated genes were APC (71%), KRAS (58%) and TP53 (46%). Seventy-three percent of the 15 patients with matched primary and PM samples and 6 of the 7 patients (86%) with data from ≥ 2 PM had concordant molecular profiles. The concordance for KRAS and NRAS was 100%. At our institutions, patients with resectable colorectal PM had a favourable prognosis. RAS mutations were commonly detected in PM and the molecular profiles of colorectal PM were highly concordant with the primary tumour.