Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies

Abstract

Colorectal cancer (CRC) biopsies underpin accurate diagnosis, but are also relevant for patient stratification in molecularly-guided clinical trials. The consensus molecular subtypes (CMSs) and colorectal cancer intrinsic subtypes (CRISs) transcriptional signatures have potential clinical utility for improving prognostic/predictive patient assignment. However, their ability to provide robust classification, particularly in pretreatment biopsies from multiple regions or at different time points, remains untested. In this study, we undertook a comprehensive assessment of the robustness of CRC transcriptional signatures, including CRIS and CMS, using a range of tumour sampling methodologies currently employed in clinical and translational research. These include analyses using (i) laser-capture microdissected CRC tissue, (ii) eight publically available rectal cancer biopsy data sets (n = 543), (iii) serial biopsies (from AXEBeam trial, NCT00828672; n = 10), (iv) multi-regional biopsies from colon tumours (n = 29 biopsies, n = 7 tumours), and (v) pretreatment biopsies from the phase II rectal cancer trial COPERNCIUS (NCT01263171; n = 44). Compared to previous results obtained using CRC resection material, we demonstrate that CMS classification in biopsy tissue is significantly less capable of reliably classifying patient subtype (43% unknown in biopsy versus 13% unknown in resections, p = 0.0001). In contrast, there was no significant difference in classification rate between biopsies and resections when using the CRIS classifier. Additionally, we demonstrated that CRIS provides significantly better spatially- and temporally- robust classification of molecular subtypes in CRC primary tumour tissue compared to CMS (p = 0.003 and p = 0.02, respectively). These findings have potential to inform ongoing biopsy-based patient stratification in CRC, enabling robust and stable assignment of patients into clinically-informative arms of prospective multi-arm, multi-stage clinical trials. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: biopsy; colorectal cancer; consensus molecular subtypes; gene expression profiling; intrinsic subtypes; molecular stratification; transcriptional signatures.

Figure 1

Patient stratification using CRC cell intrinsic signatures. (A) Left: using the CMS classifier, each sample will be assigned an individual score for CMS1, CMS2, CMS3, and CMS4. Box plots showing the relative CMS ratio for CMS1–4 in patient‐matched central tumour (CT) and invasive front (IF) samples (n = 20). Right: dot plot comparing normalised random forest posterior probability scores for IF front region of stromal and epithelial CMS subtypes (p = 0.0001, Student's t‐test). (B) Dot plot of normalised Pearson similarity scores for each gene signature. (C) Table showing clustering concordance by gene signature. (D) Caleydo (Stratomex) integrative visualisation of CRIS and CMS concordance between matched CT and IF regions.

Figure 2

Molecular subtyping of rectal cancer biopsies. (A) Bar charts showing the proportions, average, and total numbers of each CMS and CRIS group across the eight rectal cancer biopsy datasets. (B) Caleydo (Stratomex) integrative visualisation of CMS and CRIS across the eight rectal cancer biopsy datasets.

Figure 3

Temporal stability of molecular subtypes in serial biopsies. Caleydo (Stratomex) integrative visualisation of CRIS and CMS concordance in serial rectal cancer biopsies from the AXEBeam trial (n = 10).

Figure 4

Spatial stability of molecular subtypes in multi‐regional biopsies. Pie charts showing the concordant classification of multi‐regional biopsies from seven surgical specimens in the BOSS study into CRIS (left) and CMS (right) subtypes.

Figure 5

Molecular subtyping and tumour content in biopsy material from the phase II COPERNICUS clinical trial. (A) Bar charts showing the percentage of patients from each subtype, CMS (left) and CRIS (right), in the COPERNICUS cohort. (B) Dot plots comparing the tumour percentage between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) (Student's t‐test, p = 0.003). (C) Representative H&E images of CMS1 (left), CMS2/3 (middle), and CMS4 (right) biopsies (×10 original magnification).

Figure 6

Proposed model of stromal heterogeneity confounding CMS subtyping in colorectal cancer biopsies depicting the molecular classification for Patient 6 from the BOSS analysis in Figure 4.