Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition

Patients

The CRC molecular subtypes were identified on a previously analyzed cohort of 188 CRC patients¹⁶ and were validated in 543 stages II and III CRC patients (Table 1, Supporting Information Table S1). Seventy-three percentage of the stage III patients in the validation cohort, for which treatment and outcome data was available, had been treated with 5FU-based chemotherapy, none with oxaliplatin. All tissue samples were collected from patients with appropriate informed consent. The study was performed in accordance with the ethical standards of the Helsinki Declaration and was approved by the Medical Ethical Board of the participating hospitals.

Molecular subtype classification

Unsupervised clustering of whole genome data revealed three mayor groups with distinct gene expression pattern.¹⁶ In this study, three distinct gene expression signatures representative for the molecular subtypes were used for sample classification (Table 2). Signature development and sample classification is described in the Supporting Information Methods. Subtype-related gene expression measurements were performed on custom made Agilent microarrays.^16,17 Microarray data are available in the Gene Expression Omnibus (GSE42284).

Mutation analysis

Mutations in BRAF(V600E), KRAS codons 12, 13 and 61 and PIK3CA exons 9 and 20 were assessed in cDNA by means of Sanger sequencing (Supporting Information Methods). In addition, 615 genes including all genes encoding the kinases (kinome) were analyzed by deep-sequencing on 73 patients (selected to include all subtypes, Supporting Information Table S1). Sample preparation, sequencing procedure (HiSeq2000, Illumina) and sequence data analysis variant calling (somatic point mutations and small insertions and deletions) are described in Supporting Information Methods.

MSI and dMMR phenotype assessment

Assessment of MSI-status was performed according to the local standard at each participating hospital and is described in Supporting Information Methods. In addition, all samples were analyzed for their MSI/dMMR associated gene expression pattern by a previously reported 64-gene signature that accurately identifies CRC tumors that are MSI and/or show a dMMR phenotype¹⁸ (Supporting Information Methods).

Readout of EMT phenotype

Epithelial and mesenchymal characteristics were analyzed based on relative gene expression levels (median centered per gene) of marker genes known to be upregulated in mesenchymal and upregulated in epithelial cells. For each marker, a Student’s t-test was performed for comparison of A versus B+C, B versus A+C and C versus A+B (development cohort).

In addition, gene expression of the previously reported EMT signature from Loboda et al. was investigated in the development cohort.¹⁴ Ninety-six of their top 100 genes that were previously reported to be strongly associated with the EMT program were analyzed for differential expression across the three molecular subtypes, together with a multigene profile readout in which a higher EMT index represented a more mesenchymal phenotype (Supporting Information Methods).

Statistical and survival analysis

All analyses and statistical tests were performed in R 2.14.1 (www.r-project.org), and were considered significant with a p-value <0.05 (two-sided). Association of the subtypes with clinical and molecular makers was analyzed using a Pearson’s Chi-squared test. Survival analysis was performed on the validation cohort using Cox proportional hazard models with two end-points: 10-year distant metastasis-free survival (DMFS) and 10-year cancer-related OS. Average follow-up time was 70 months (range 3–270 months). Investigation of benefit from 5FU-based adjuvant chemotherapy was performed by comparing OS rates of patients with and without chemotherapy. An interaction analysis was performed to analyze the differences in benefit between the subtypes based on 5-year OS. Most (76%) stage II patients were untreated, therefore this analysis was limited to the stage III patients of the validation cohort.

Three intrinsic molecular subtypes

Unsupervised clustering of whole genome expression data revealed three intrinsic CRC molecular subtypes¹⁶ (Fig. 1a). To further characterize and understand the biological and clinical differences between these intrinsic subtypes, we have developed a diagnostic single sample classifier based on three “core” gene profiles representative for each of the subtypes (A-, B- and C-type) (Fig. 1b, Table 2). On the development cohort, 35% was classified as A-type, 52% as B-type and 13% as C-type CRC (Fig. 1c, Table 1), with a very high concordance of 97% compared to the unsupervised clustering method.¹⁶

Subtype classification and characteristics were confirmed in a validation cohort of 543 stages II and III CRC samples (Table 1, Supporting Information Table S1). Twenty-two percent of patients were classified as A-type, 62% as B-type and 17% as C-type. Subtype classification was not associated with tumor stage (p = 0.21) or patient age (p = 0.23), but was significantly associated with tumor grade (p = 5.23e-8), gender (p = 1.07e-3) and microsatellite status (p < 2.2e-16) (both cohorts combined, Table 1). A significant difference in colon tumor location was observed (left vs. right colon, p = 1.04e-15; colon vs. rectum, p = 0.027).

Association with molecular markers

Molecular characterization of the intrinsic subtype indicated that activating BRAF(V600E) mutations were unequally distributed between the subtypes (p = 1.9e-7) (Table 1). Not only A-type (32%, both cohorts) but also C-type patients (16%) were enriched for BRAF mutations, while B-type patients were almost exclusively BRAF wild-type (98%). Subtype classification was also associated with activating PIK3CA mutations (p = 9.23e-4) but not associated with activating KRAS mutations (p = 0.08) (Table 1). Combining the mutually exclusive KRAS and BRAF mutations, a highly significant association was observed with 68% A-type patients harbored an activating BRAF or KRAS mutation (Supporting Information Table S2).

The A-type group showed a significant higher proportion of MSI patients (49%, p < 2.2e-16, both cohorts) while B-type patients were almost exclusively MSS (99%) (Table 1). The association of A-type with MSI-status is in good agreement with other characteristics that have been observed before to be associated with MSI (BRAF mutations, right-sided tumor location, female gender and poor differentiation).¹⁹ The lack of statistically significant association of A-type with stage might be explained by the absence of stages I and IV cancers in the validation cohort. However, the development cohort indicates that A-type patients might be more frequent in early stage cancer, in good agreement with better prognosis of A-type and MSI-patients.

Readout of the MSI/dMMR signature that is able to identify MSI and MSI-like tumors representative of a dMMR phenotype^18,19 reinforced the strong MSI/dMMR characteristics of A-type patients with 68% showing a MSI/dMMR expression profile and confirmed the exclusive MSS and proficient MMR (pMMR) phenotype (99%) of B-type patients (Fig. 2a). Although not enriched for MSI by the hospital assessment, a significant proportion of C-type patients (36%, p = 5.7e-4) showed a dMMR phenotype based on the MSI/dMMR-signature (Fig. 2a).

To validate the dMMR phenotype, 73 samples were deep-sequenced for their complete kinome. A- and C-type patients showed a significantly higher mutation frequency compared to B-type (p = 5.90e-5). On average, B-type patients showed a low mutation frequency of ten mutated genes of the 615 analyzed (1.6%), while the A-type and C-type groups harbored 38 (6.2%) and 26 (4.2%) mutated genes, respectively (Fig. 2b).

These results are in agreement with a recent publication from the Cancer Genome Atlas Network (TCGA) that shows that some “hypermutated” cancers have high levels of microsatellite instability (MSI) while others are not and are classified as MSS/MSI-L by traditional MSI-methods.¹³ This might explain why the C-type group has a high level of dMMR characteristics and high mutation rate but many patients are not classified as MSI by hospital methods.

Epithelial-to-mesenchymal characteristics

Gene expression levels of known epithelial and mesenchymal markers were assessed in the subtypes (Fig. 3a). In C-type cancers, almost all the mesenchymal markers, except FLT1, were significantly up-regulated, and three epithelial markers (CDH1, EGFR and MET) were down-regulated. In B-type, four of the five epithelial markers were up-regulated and three mesenchymal markers (CDH2, FGFR1 and TGFB1) were down-regulated. A-type patients showed a significant reduced expression of two mesenchymal markers (TWIST1 and AXL) but also a reduction in one of the epithelial markers (CDH1). These results indicated that C-type tumors have a more mesenchymal phenotype while A- and B-type tumors can be considered as epithelial.

The difference in epithelial and mesenchymal characteristics was confirmed by the recently published EMT signature by Loboda et al.¹⁴ C-type patients showed a significantly higher (p < 2.2e-16) EMT index representative of a more mesenchymal phenotype (Fig. 3b). Classification of C-type patients by the Loboda signature showed an AUC of 0.92 (Fig. 3c) with mesenchymal genes significant up-regulated in C-type samples and epithelial genes significant up-regulated in A-type or B-type samples (Fig. 3d).

Prognosis

Survival analysis on the validation cohort (n = 543, stages II and III) showed a significant difference in DMFS between the three subtypes (p = 0.004), with hazard ratios (HR) of 2.19 (p = 0.011) for B-type and 2.90 (p = 0.0024) for C-type patients compared to A-type (Fig. 4a). C-type patients showed a significant reduced OS compared to A and B-types with a HR of 2.3 (p = 0.016) (Fig. 4b).

Although MSI status is generally associated with a good prognosis, we observed significant differences in DMFS between the subtypes (p = 0.034) with A-type-MSI patients having a very good prognosis but with C-type-MSI-patients having a poor prognosis (93 and 50% 10-year DMFS, respectively) (Fig. 4c). A multivariate analysis, including MSI, BRAF, stage, gender and NCCN classification,²⁰ confirmed that the subtype classification has independent prognostic value (Supporting Information Table S3) that is additive to MSI and BRAF assessment (Fig. 4d).

Adjuvant chemotherapy benefit

To analyze chemotherapy benefit, outcome of 222 stage III patients were compared. Of these patients, 161 received adjuvant 5FU chemotherapy and 61 received no adjuvant chemotherapy. In accordance with data from clinical trials,²¹ treatment with 5FU-based chemotherapy was beneficial when compared with no additional treatment (HR 0.55, p = 0.033). The benefit from chemotherapy was different in the three molecular subtypes (Figs. 5a–5d). Stage III patients with A- and B-type tumors showed a benefit in OS with a HRs of 0.39 (p = 0.18) and 0.42 (p = 0.014), respectively; the difference not being statistically significant in A-type due to lower number of patients in this type. In contrast to the epithelial-like A- and B-type patients, the mesenchymal-like C-type patients showed no benefit from chemotherapy treatment (HR 1.4, p = 0.542). An interaction analysis between subtypes and chemotherapy with OS as endpoint confirmed that chemotherapy response was significantly different between the molecular subtypes (p = 0.017, Fig. 5d). Since MSI has been associated with nonresponsiveness to 5FU, the analysis for C-type patients was repeated for MSS patients only. C-type-MSS tumors again showed no benefit from chemotherapy (HR 1.08, p = 0.95) (data not shown).

We hypothesized that the difference in chemotherapy benefit might be related to the proliferative activity of the molecular subtypes. Relative gene expression levels of two proliferative markers, Ki-67 (MKI67) and Aurora Kinase A (AURKA), showed a significantly reduced expression of both markers in C-type compared to A- and B-type tumors (MKI67 p = 6.06e-5, AURKA p = 4.53e-6, Student’s t-test) (Fig. 5e). Interestingly, A-type tumors showed the highest expression of MKI67, while AURKA was the strongest proliferative marker for B-type samples.