Microsatellite status and associated characteristics
Microsatellite analysis of the tumour samples from 120 Bethesda-positive patients examined in this study revealed an MSI-H phenotype in 47 (39.2%) cases, 73 (60.8%) were non-MSI-H (64 MSS, nine MSI-L). Patients in the MSI-H group (median 39 years) were ‘younger' than patients in the MSS collective (median 43 years, exact Wilcoxon's rank sum test: P=0.02); the median age at time of first diagnosis was 42 years ().
Characteristics of the 120 included colorectal cancer patients
Clinical criteria were distributed unequally among MSI-H and MSS group (Fisher's exact test: P=0.01). The most obvious discrepancy was observed for Amsterdam-positive cases, which were much more frequent in the MSI-H (14/47, 30.1%) than in the MSS group (7/73, 9.6%, Fisher's exact test: P=0.006, ).
The trend towards a more advanced tumour stage observed in the MSS CRC group (Fisher's exact test: P<0.001, ) is reflected by the higher frequency of patients treated with 5-FU chemotherapy (Fisher's exact test: P=0.04, ).
For comparison of tumour localisation with microsatellite status, only patients with single carcinomas (n=100) were evaluated. As expected, right-sided location correlated closely with the MSI-H phenotype. Among 30 carcinomas in the proximal colon, 20 were MSI-H (66.7%), in the group of tumors with distal location, only 18 of 70 tumours (25.7%) displayed the MSI-H phenotype (Fisher's exact test: P<0.001, ).
In all, 27 of 47 MSI-H carcinomas had a CLR (57.4%), whereas CLRs were found in only 14 from 71 MSS tumours (19.7%). Thus, CLR frequency was significantly elevated in MSI-H colorectal cancers (Fisher's exact test: P<0.001, ).
Patients' overall survival
The median follow-up was 33 months from the date of first diagnosis, 29 patients died within the follow-up period. The survival distribution of MSI-H patients differed significantly from that of MSS patients (log-rank test: P<0.001, ). Kaplan–Meier estimates of the survival time distributions are presented in . The estimated 5-year survival rate of MSI-H patients was 88% (CI95: 77–100%) compared to 56% (CI95: 42–75%) in the MSS group (; log-rank test: P<0.001). An improved overall survival was also observed in patients with CLR+CRC (, P=0.04). No significant differences were observed in groups with or without 5-FU chemotherapy (not shown). Additionally, we tested the prognostic value of microsatellite status together with UICC stage, presence of CLR, and age at diagnosis and application of 5-FU chemotherapy by using a proportional hazards regression model. Here, UICC stage and age at diagnosis were the only statistically significant prognostic factors for overall survival ().
Figure 2 Kaplan–Meier estimates of overall survival. (A and B) Patients of all tumour stages stratified by microsatellite status (A) and presence of Crohn's like reaction (CLR, B). (C and D) Overall survival of patients with T3 stage colon cancer depending (more ...)
Cox's proportional hazards regression for survival
The MSI-H phenotype was rarely detected in stage UICC IV: only six out of 43 (13.9%) tumours with distant metastases (UICC IV) were MSI-H, compared to 41 out of 77 (53.2%) in the collective with UICC I-III (Fisher's exact test: P<0.001, ). For presence of CLR, the observed tendency was similar. From 42 UICC IV cases that could be scored for lymphocyte infiltration at primary tumour site, only seven (16.7%) primary tumours had a marked peritumoural lymphocyte infiltration with CLR, whereas in the collective with UICC grade I–III the relation of CLR-positive tumours was 35 out of 76 (46.1%, Fisher's exact test: P=0.001).
Comparison of overall survival after stratification by microsatellite status and presence of CLR is shown in for tumours infiltrating the lamina muscularis propria (all other T stages did not reveal significant results). A lower risk was observed for patients with MSI-H T3 carcinomas (log-rank test: P=0.003, ) but this result could not be confirmed by multiple proportional hazards regression analysis ().
Metastasis frequency in correlation to microsatellite status and CLR
These results raised the question whether the longer overall survival of patients with MSI-H tumours was linked to the occurrence of organ metastases. To address this question in more detail, we stratified the tumours by depth of infiltration and compared the occurrence of metastases in the MSI-H vs the MSS group. From 76 T3 carcinomas, 35 (46.1%, CI95: 0.35–0.57) were MSI-H, hence the MSI-H frequency was comparable to the MSI-H frequency observed overall in all included patients (39.2%, CI95: 0.31–0.48).
T3 cancers with MSI-H had organ metastases defining UICC IV stage in 4/35 cases (11.4%), while those with MSS phenotype in almost half of the cases (20 out of 41, 48.8%, ). The increased frequency of metastases in MSS T3 cases was highly significant (Fisher's exact test: P<0.001, ).
Frequency of metastases in patients with T3 colorectal cancers stratified by microsatellite status and presence of Crohn's like reaction
To clarify whether this observation was related to morphological features of an antitumoural immune response, T3 tumours were grouped according to the presence of a CLR. The comparison of M stage in T3 carcinomas with or without CLR revealed a trend towards a lower frequency of metastases in cases with CLR (six out of 29 M1 in CLR+ T3 tumours vs 17 out of 45 M1 in CLR−, P=0.13, ).
In summary, we observed a markedly reduced frequency of organ metastases in T3 colorectal cancers that exhibited either the MSI-H phenotype, a CLR or both.
In regard to lymph node metastases, differences were neither significant after stratification for microsatellite status nor for CLR presence (P=0.28 and 0.51, respectively, ).
To test microsatellite status and CLR as predictors of UICC tumour stage, a logistic regression model was applied (). For CLR, the statistically significant correlation was limited to the regression analysis that included tumours of all T stages (OR 0.44, P=0.04). In contrast, MSI-H was significantly associated with a lower UICC stage when looking at all tumours (OR 0.33, P=0.006) and also in the T3 subgroup (OR 0.24, P=0.006). These data show that the improved overall survival of patients with MSI-H CRC included in this study is closely linked to lower rates of distant metastases, even after stratification for local depth of invasion of the primary tumour.
Ordinal logistic regression for UICC