Patient cohort
Colorectal cancers from 308 patients were included (median age 73·7 years; range, 28–91 years). One hundred and sixty-three patients were female and 145 were male, with 296 of the patients of European decent, three Asian and nine Maori. Right-sided tumours were more common (55%) compared to left-sided colon tumours (27%) and rectal tumours made up 18%. Median follow up was 50 months (range, 0·3–172 months). More detailed patient demographics are shown in Supplementary Table S1.
Association of TNM staging with clinical variables
Post-surgical TNM staging based on pathological examination stratified the cohort as follows: 53 stage 1, 128 stage 2 patients, 105 stage 3 and 22 stage 4 patients. Analysis of associations between post-surgical staging of patients and clinicopathological variables (Tables 1 and 2) showed that increasing TNM stage was significantly associated with lymph-node positivity and subsequent development of metastasis, which can be attributed to liver metastases; there was no association with local recurrence.
Table 1. Tumour recurrence, metastasis and lymph-node invasion by post-operative stage and by Consensus Molecular Subtype
|
LR
|
Mets
|
Mets + LR
|
LM
|
LN
|
|
%
|
%
|
%
|
%
|
%
|
TNM1
|
0
|
11.3
|
11.3
|
9.4
|
0
|
TNM2
|
4.7
|
9.4
|
12.5
|
6.2
|
1.6
|
TNM3
|
6.7
|
31.4
|
32.4
|
20
|
97.1
|
TNM4
|
4.5
|
100
|
100
|
81.8
|
86.4
|
P-value
|
0.2407
|
< 0.0001
|
< 0.0001
|
< 0.0001
|
< 0.0001
|
|
|
|
|
|
|
CMS1
|
3.3
|
11.7
|
13.3
|
3.3
|
25
|
CMS2
|
4.1
|
26.2
|
26.9
|
21.4
|
37.9
|
CMS3
|
5.3
|
13.2
|
18.4
|
7.9
|
44.7
|
CMS4
|
0
|
41.2
|
41.2
|
29.4
|
64.7
|
P-value*
|
0.9061
|
0.0211
|
0.0479
|
0.0009
|
0.0171
|
Tumour recurrence, metastasis and lymph-node invasion by Consensus Molecular Subtype (CMS) and by tumour-node-metastasis (TNM) stage given as percentages. P-value* is derived from analysis of classified tumours only and a P-value of < 0.05 is considered significant; LR, local recurrence; Mets, distant metastasis, diagnosed either at surgery or during the follow-up period; Mets + LR, local recurrence and distant metastasis combined; LM, liver metastasis; LN, lymph-node positive.
Table 2. Histological characteristics of colorectal cancer tumours by post-operative stage and by Consensus Molecular Subtype
|
Poorly diff
|
Mucinous
|
LVI
|
EMVI
|
PNI
|
|
%
|
%
|
%
|
%
|
%
|
TNM1
|
11.4
|
7.5
|
11.3
|
0
|
0
|
TNM2
|
18
|
12.5
|
23.4
|
7
|
9.4
|
TNM3
|
18.1
|
9.5
|
46.7
|
26.7
|
18.1
|
TNM4
|
22.7
|
9.1
|
72.7
|
40.9
|
31.8
|
P-value
|
0.5737
|
0.7864
|
< 0.001
|
< 0.001
|
< 0.001
|
|
|
|
|
|
|
CMS1
|
48.3
|
20
|
31.7
|
13.3
|
8.3
|
CMS2
|
9
|
2.8
|
28.8
|
15.2
|
11.7
|
CMS3
|
10.5
|
18.4
|
31.6
|
7.9
|
7.9
|
CMS4
|
17.6
|
11.8
|
35.3
|
23.5
|
23.5
|
P-value*
|
< 0.001
|
0.0001
|
0.8686
|
0.4497
|
0.3334
|
|
|
|
|
|
|
Histological characteristics of colorectal cancer tumours by tumour-node-metastasis (TNM) stage and by Consensus Molecular Subtype (CMS) given as percentages. P-value* is derived from analysis of classified tumours only and a P-value of < 0.05 is considered significant. Poorly diff, poorly differentiated; LVI, lymphovascular invasion; EMVI, extramural venous invasion; PNI, perineural invasion.
CMS subtypes and clinical variables
Of the 308 patients, 60 were classified as CMS1 (19%), 145 as CMS2 (47%), 38 as CMS3 (12%) and 17 as CMS4 (6%) (Supplementary Table S2). Univariate analysis of patient demographic and clinical variables showed that CMS1 tumours were more likely to be right-sided, found in females, poorly-differentiated, with a high proportion of mucinous histology and less likely to be seen in younger patients. CMS2 tumours made up nearly half of our cohort and were predominantly left-sided tumours found in male patients, and showed a negative association with mucinous type. CMS4 tumours were associated with younger age, and presented at an advanced TNM stage with lymph-node positivity. There was no significant difference in the local recurrence rates between subtypes, but CMS2 and CMS4 were associated with higher rates of distant metastases and this association was attributable to liver metastases. A detailed breakdown of associations with CMS subtypes is given in Tables 1, 2 and 3.
Table 3. Patient and tumour characteristics by Consensus Molecular Subtype
|
|
|
CMS1
(n=60)
|
CMS2
(n=145)
|
CMS3 (n=38)
|
CMS4 (n=17)
|
P-value*
|
|
|
(n)
|
%
|
%
|
%
|
%
|
|
Age
|
< 60y
|
48
|
3.3
|
18.6
|
15.8
|
11.8
|
0.0188
|
|
61-80y
|
170
|
48.3
|
53.8
|
60.5
|
52.9
|
|
|
> 80y
|
90
|
48.3
|
27.6
|
23.7
|
35.3
|
|
Gender
|
F
|
163
|
73.8
|
42.8
|
63.2
|
52.9
|
0.0009
|
|
M
|
145
|
26.2
|
57.2
|
36.8
|
47.1
|
|
Site
|
Colon
|
253
|
98.3
|
75.9
|
84.2
|
64.7
|
< 0.0001
|
|
Rectum
|
55
|
1.7
|
24.1
|
15.8
|
35.3
|
|
Side
|
Left
|
172
|
20
|
71
|
50
|
76.5
|
< 0.0001
|
|
Right
|
136
|
80
|
29
|
50
|
23.5
|
|
Stagea
|
1
|
53
|
16.7
|
17.2
|
28.9
|
11.8
|
0.01449
|
|
2
|
128
|
55
|
44.8
|
26.3
|
23.5
|
|
|
3
|
105
|
26.7
|
29.7
|
42.1
|
47.1
|
|
|
4
|
22
|
1.7
|
8.3
|
2.6
|
17.6
|
|
Patient and tumour characteristics by Consensus Molecular Subtype (CMS) given as percentages. N, total number of patients per group; P-value* is derived from analysis of classified tumours only and a P-value of < 0.05 is considered significant; y, years; F, female; M, male; a Post-operative Tumour-Node-Metastasis staging.
Survival analysis
The median follow-up period was 50 months (0·3–172 months) with a median survival of 82 months (95% CI 71·8 – 110·5). Survival curves and proportions at 5 and 10 years are shown in Figure 1. Both progression-free survival (PFS, P = 0·039) and overall survival (OS, P = 0·036) were associated with CSM subtype in the classified samples. The associations were largely due to the difference between CMS subtype 4 and the other classes; the hazard ratios for CMS4 relative to all other classified samples were 2·28 (95% CI 1·28 – 4·05, P = 0·005) and 2·29 (95% CI 1·26 – 4·18, P = 0·007) for PFS and OS, respectively. However, after adjusting for age and sex, there was no significant association between CMS stage and OS (P = 0·11) or PFS (P = 0·12).
10-year overall survival based on TNM staging showed that, when adjusted for age and gender, Stage 1 and 2 show little difference in survival outcome. However, there is some evidence that Stage 3 is associated with increased mortality, while it is quite clear that Stage 4 is associated with increased mortality (OR = 2·8, 95% CI 1·6 – 5·0, P < 0·0005).
Considering all samples, older and male patients were at greater risk of poorer outcomes from CRC (Table 4). Cancers that were rectal, had lymph-node involvement, local recurrence or post-operative metastases posed significantly greater risk, however side did not significantly affect risk. Adjusting for all other covariates showed that there was independent risk associated with lymph-node involvement, local recurrence and post-operative metastases, but not rectal cancers. Including both TNM stage and CMS in models of survival analysis shows that TNM stage significantly explains mortality independently of age and gender, whereas CMS subtype does not. From this we conclude that stratification using CMS does not perform as well as TNM staging as an independent prognostic indicator in our cohort.
Table 4. Hazard ratios for risk factors associated with mortality in colorectal cancer
|
|
HR (95% CI)
|
P
|
PAge,Sex
|
PAll
|
Age (years)
|
60–80
|
2.3 (1.2,4.2)
|
0.0002
|
|
0.0002
|
|
80+
|
3.4 (1.7,6.5)
|
|
|
|
Male
|
|
1.4 (1.0,1.9)
|
0.0640
|
|
0.0175
|
Rectal
|
|
1.5 (1.0,2.1)
|
0.0511
|
0.1341
|
0.1341
|
Right side
|
|
0.9 (0.7,1.3)
|
0.5930
|
0.3881
|
0.7978
|
Lymph node involvement
|
1.5 (1.1,2.1)
|
0.0139
|
0.0046
|
0.0076
|
Local recurrence
|
|
2.2 (1.2,4.1)
|
0.0187
|
0.0120
|
0.0193
|
Post-operative metastasis
|
3.5 (2.4,5.0)
|
<0.0001
|
<0.0001
|
<0.0001
|
TNM Stage
|
2
|
0.8 (0.5,1.3)
|
0.0004
|
0.0000
|
0.0015
|
|
3
|
1.3 (0.8,2.0)
|
|
|
|
|
4
|
3.0 (1.6,5.7)
|
|
|
|
CMS
|
2
|
1.0 (0.6,1.5)
|
0.0949
|
0.2619
|
0.4237
|
|
3
|
1.4 (0.8,2.6)
|
|
|
|
|
4
|
2.2 (1.1,4.5)
|
|
|
|
|
Unclassified
|
1.2 (0.7,2.1)
|
|
|
|
Hazard ratio (HR) with 95% confidence interval (CI) and P-value from analysis of deviance from univariate models. PAge,Sex, P-value from analysis of deviance for Cox proportional hazards model with covariates for age and gender; PAll, P-value from analysis of deviance on Cox proportional hazards model including all covariates; TNM, tumour-node-metastasis; CMS, consensus molecular subtype.
|
Of the 308 patients, 63 patients had relapse of their disease; either local recurrence or distant metastases or both within the follow-up period. There was no significant difference in the median survival after relapse which was 16·5 months, 12·4 months, 33·9 months and 4·6 months for CMS1, CMS2, CMS3 and CMS4 tumours respectively (P = 0·187).
Prognostic effect of CMS in CRC stratified by TNM Stage
There were 17 participants with stage 4 cancer classified by CMS and coincidentally 17 CMS4 patients. These numbers were insufficient to draw robust conclusions for stage 4 or CMS4 when cross tabulated, and were omitted from the analysis. Differential survival by stage (1 to 3) and CMS (1 to 3) was identified by analysis of deviance on a Cox proportional hazard model with interaction between TNM stage and CMS (P = 0.048). Including covariates for age (dichotomous at 80), sex, tumour site and side increased the significance of this effect (P = 0.022). To assess the magnitude of the differences between CMS subtypes within different stage tumours, survival analysis was performed on the data stratified by TNM stage. Median survival times were calculated and Cox proportional hazard models fitted with and without covariates for age, sex, site and side (Table 5). There was a significant difference in survival predicted by CMS for stage 1 tumours. However, this was explained by covariates. For stage 2 tumours, there was a suggestion that CMS subtype 3 has worse survival than CMS1 and 2, which was statistically significant after adjusting for age, sex, site and side. There was no evidence that outcome differed by CMS subtype for stage 3 or 4.
Table 5. Survival time by CMS stratified by TNM stage.
|
|
CMS1
|
CMS2
|
CMS3
|
P-value
|
Padj
|
Stage 1
|
39.8
|
127.9
|
110.6
|
0.035
|
0.170
|
Stage 2
|
108.5
|
121.8
|
50.53
|
0.051
|
0.006
|
Stage 3
|
90.3
|
72.7
|
64.4
|
0.769
|
0.752
|
Stage, TNM stage; CMS, consensus molecular subtype; Padj, P-value adjusted for age, sex, site and side
Differential gene expression and gene-set enrichment analysis in Stage 2 tumours
Differentially expressed genes between Stage 2 patients who died and those who were alive at the end of the follow-up period, were further analysed to identify genes potentially associated with survival in Stage 2 tumours. Differentially up-regulated genes strongly associated with survival in this patient group includes immune-cell related genes, in particular genes coding for B-cell markers, and several known (LRRC4, PKNOX2, FEZF2) and putative tumour suppressor genes (MTO18B, NCAM1 and SCN4B) (Supplementary Table S3). Genes that were significantly up-regulated in patients with poor survival included pro-inflammatory genes (IL17REL, RETNLB) and genes that have been previously associated with progression and poor outcome in CRC (ERBB2, TBLRXR1, TAPBP, CPS1, AGR2) (Supplementary Table S4).
In order to compare biologic pathways and processes potentially associated with survival in this subgroup of patients, we used DEGs as input into an assortment of gene ontology tools. Differentially upregulated biologic pathways associated with survival were predominantly immune pathways, including B-cell activation, IL-12 and PD-1 signalling and T-cell activation. In addition, glutamatergic signalling was differentially enriched in Stage 2 patients still alive at the end of follow-up (Supplementary Table S5). GSEA showed an enrichment of pathways involved in metabolic regulation in Stage 2 tumours, which reflects the association of CMS3 with poor survival, and also differential up-regulation of processes involved in protein and nucleic acid synthesis (Supplementary Table S6).