The implementation of CRC subtypes along with other molecular features in clinic is challenging due to lack of easy-to-use and low cost assays suitable for FFPE tissues. Based on our CRCAssigner and Consensus Molecular Subtype (CMS), we validated a small gene panel for nCounter assay (Nanostring Technologies) to classify CRC fresh-frozen samples (ESMO 2017 Abstr 4633). Here, we tested nCounter and MiSeq platforms (Illumina; targeted mutational panel) in archival FFPE samples.
Tissues from 36 chemorefractory patients treated at the Royal Marsden were collected. Tumour-enriched areas were macrodissected, RNA/DNA was extracted, and nCounter assay was performed. RNA technical (same extraction, n = 6) and biological (same block, different extractions, n = 3) replicates were generated. Hotspot BRAF, KRAS, NRAS, PIK3CA, TP53 mutations (MT) were sequenced.
Out of 26 untreated primaries, 8 were enterocyte/transit-amplifying (TA) (CMS2, 30%), 7 goblet-like (CMS3, 27%), 8 stem-like (CMS4, 30%), 0 inflammatory (CMS1), and 3 mixed subtypes. BRAF/RAS MT were mutually exclusive and detected in all goblet-like (CMS3) samples. Interestingly, PIK3CA mutation was exclusively present in differentiated (goblet-like and enterocyte) subtypes, whereas TP53 mutation was detected in all the subtypes. Among 10 pre-treated samples, 3 were inflammatory (CMS1, 30%), 3 TA (CMS2, 30%), 3 stem-like (CMS4, 30%), and 1 mixed subtypes. Pearson correlation coefficients were 0.96 and 0.88 (high reproducibility) for technical and biological replicates, respectively.
With the caveat of small numbers, the subtype distribution in chemorefractory patients is different compared to early stage patients assessed within the CMS consortium. The enrichment for less differentiated subtypes in pre-treated samples suggests potential treatment-induced changes in tumours. Overall, nCounter assay along with MiSeq platform was able to classify standard archival diagnostic CRC FFPE samples into subtypes, which warrants further improvement and validation for clinical practice.
Clinical trial identification
Legal entity responsible for the study
The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research
NIHR Biomedical Research Centre at The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, London; Cancer Research UK; Rosetrees Trust UK
D. Cunningham: Research Funding to institution: Amgen, AstraZeneca, Bayer, Celgene, MedImmune, Merck Serono, Merrimack, Sanofi. I. Chong: Research funding: Janssen. C. Peckitt: Advisory roles with Sanofi. D. Watkins: Funding for meeting attendance from Amgen. I. Chau: Honoraria: Amgen, Bayer, Pfizer, Taiho Pharmaceutical. Consulting or advisory role: Bristol-Myers Squibb, Gilead Sciences, Lilly, Merck Serono, MSD, Sanofi. Research funding: Janssen-Cilag, Merck Serono, Novartis, Roche, Sanofi. N. Starling: Research funding to institution: AstraZeneca, Verastem. A. Sadanandam: Entitled to a share of royalties received by the licensor for a patent patent number PCT/IB2013/060416. Research funding from Bristol-Myers Squibb for pancreatic cancer. All other authors have declared no conflicts of interest.