MicroRNA Expression Patterns in Colon Tumors
The characteristics of the patients with incident colon adenocarcinoma in the test cohort (from Baltimore) and the validation cohort (from Hong Kong) are shown in . The median follow-up time was 68.0 months for the Baltimore cohort and 84.6 months for the Hong Kong cohort. The 2 cohorts were similar in TNM staging, tumor histology, and cancer-specific mortality rates. The 5-year survival rate was 57.5% for the US cohort and 49.5% for the Hong Kong cohort and were not significantly different from one another (P=.49, Kaplan-Meier test). In addition to the racial, geographic, and cultural differences between these 2 cohorts, the Baltimore cohort was considerably older (average 64.6 years vs 55.8 years) had a higher percentage of men (79% vs 50%).
Characteristics of Study Population and Tumorsa
We compared microRNA profiles of 84 pairs of colon tumor and adjacent nontumorous tissues in the Baltimore cohort using microRNA microarrays.31
Tumor microRNA profiles were distinctly different from nontumor profiles. Using class comparison analysis in BRB array tools, 37 independent microRNAs were found to be differentially expressed in tumors (P
<.001 with a false-discovery rate<0.5%; ). Twenty-six microRNAs were expressed at higher levels in tumors with miR-21
enriched the most at 1.8-fold. Global microRNA profiles distinguish between tumor and paired nontumorous tissue with 89% accuracy using either the 3 nearest neighbors or nearest centroid class prediction algorithms within BRB array tools (10-fold cross validation repeated 100 times), suggesting a systematic change in microRNA expression patterns during tumor formation.
MicroRNAs That Are Differentially Expressed in Tumors Compared With Nontumorous Tissue
We next performed a preliminary analysis to identify whether any of the 37 differentially expressed (P<.001) microRNAs were associated with cancer survival. We analyzed individual microRNA T:N expression ratios for associations with poor prognosis in the Maryland test cohort. Tumor:nontumor microRNA expression ratios were classified as high based on highest tertile. We searched for any microRNA for which high T:N ratios were associated with cancer survival (P<.05) using Cox regression. Five microRNAs satisfied these criteria. High expression of miR-20a (hazard ratio [HR], 2.2;95% confidence interval [CI], 1.1-4.6; P=.03), miR-21 (HR, 2.5; 95% CI, 1.2-5.0; P=.01), miR-106a (HR, 2.3; 95% CI, 1.1-4.5; P=.02), miR-181b (HR, 2.0; 95% CI, 1.0-3.9; P=.04), and miR-203 (HR, 3.1; 95% CI, 1.5-6.4; P=.003) were each associated with poor survival and were selected for further analysis.
To validate overall differences in microRNA expression between tumor and nontumorous tissue, we measured the expression levels of these 5 microRNAs with quantitative RT-PCR in tumor and paired nontumorous tissue in the independent validation cohort of 113 patients with incident colon cancer recruited from Hong Kong, China (). MiR-20a (2.3-fold), miR-21 (2.8-fold), miR106a (2.4-fold), miR-181b (1.4-fold), and miR-203 (1.8-fold) were all expressed at higher levels in tumors (P<.001, Wilcoxon matched-pairs test; ). Most tumors (89% for miR-20a, 87% for miR-21, 90% for miR-106a, 71% for miR-181b, and 74% for miR-203) had higher expression of these microRNAs than paired nontumorous tissue. Expression patterns for these 5 microRNAs distinguish tumor vs paired nontumor status with 96% or 98% accuracy based on 3 nearest neighbors or nearest centroid algorithms, respectively (10-fold cross validation, repeated 100 times).
Expression of MicroRNAs in Colon Adenocarcinoma Tumors and Colon Adenomas
High miR-21 Expression and Prognosis
Colon adenocarcinomas from 89% to 93% of the patients in this study were of a typical histology. A minority of tumors were of mucinous adenocarcinoma (8 of 84 patients [10%] in the US cohort; 7 of 113 patients [6%] in the Hong Kong cohort), adenosquamous carcinoma (1 of 84 patients [1%] in the US cohort), or signet ring cell carcinoma (1 of 113 patients [1%] in the Hong Kong cohort) histologies (). Different subtypes of adenocarcinomas can be associated with different clinical outcomes, including survival prognosis.33
To remove potential confounding associated with histology, we excluded all patients (9 in the US cohort; 8 in the Hong Kong cohort) with mucinous adenocarcinomas, adenosquamous carcinomas, and signet ring cell carcinomas from the initial analysis.
We found high T:N expression ratios for miR-20a, miR-21, miR-106a, miR-181b, and miR-203 to be associated with poor survival in the Maryland test cohort. These associations could be due to microRNA expression levels in the tumor tissue, the surrounding nontumorous tissue, or a combination of both. To distinguish these possibilities, we analyzed the association of microRNA expression in tumors and paired nontumorous tissues separately. High expression levels in tumors (based on highest tertile) for miR-20a (HR, 2.7; 95% CI, 1.3-5.8; P=.01), miR-21 (HR, 2.5; 95% CI, 1.2-5.2; P=.01), miR-106a (HR, 2.4; 95% CI, 1.2-5.1; P=.02), miR-181b (HR, 3.2; 95% CI, 1.6-6.7; P=.002), and miR-203 (HR, 3.3; 95% CI, 1.5-7.1; P=.001) were each associated with a poor survival in the Maryland test cohort. No significant association with microRNA expression in nontumorous tissue was observed for any of the 5 microRNAs.
The associations with microRNA expression and survival in the test cohort were made in the context of a microarray experiment in which we were evaluating the dichotomized expression of 37 microRNAs. To validate these findings, we used quantitative RT-PCR to measure tumor- and nontumor-expression levels for these 5 microRNAs in the Hong Kong validation cohort and analyzed associations with prognosis. We dichotomized high and low expression for each microRNA based on highest tertiles, consistent with our methods in the test cohort. High miR-21 tumor expression was associated with poor prognosis in the Hong Kong validation cohort (P=.001, Kaplan-Meier log-rank test) while expression in nontumorous tissue was not (), consistent with associations in the Maryland test cohort. We did not find statistically significant associations with prognosis and expression of miR-20a, miR-106a, miR-181b, or miR-203 in this cohort.
High miR-21 Expression in Tumors and Poor Survival in Patients With Typical Adenocarcinoma Histology
Multivariate Cox proportional hazards analysis was used to further evaluate the association of miR-21 expression in tumors with prognosis in both the Maryland test cohort and the Hong Kong validation cohort () to evaluate the potential formiR-21 expression as a prognostic biomarker. The dichotomized miR-21 expression values were not associated with age, sex, race, or tumor location (Fisher exact test). In univariate analysis for the Maryland test cohort, high expression of miR-21 in tumors (HR, 2.5; 95% CI, 1.2-5.2; P=.01) and TNM staging (HR, 3.5; 95% CI, 1.6-7.9; P=.002) was associated with prognosis while age, sex, race, and tumor location were not. In the final multivariate model, which included miR-21 expression and TNM staging, high miR-21 expression in tumors was associated with a poor survival prognosis independent of tumor staging (HR, 2.7; 95% CI, 1.3-5.5; P=.008; ).
Univariate and Multivariate Cox Regression Analysis of miR-21 Expression Levels and Overall Cancer Survival in Subjects With Colon Adenocarcinomaa
In the Hong Kong validation cohort, the dichotomized values for miR-21 expression in tumors were not significantly associated with age, sex, tumor histology, or tumor location (Fisher exact test). High miR-21 expression in tumors (HR, 2.4; 95% CI, 1.4-3.9; P=.002) and TNM staging (HR, 4.7; 95% CI, 2.4-9.5; P<.001) were significantly associated with survival in univariate models while age, sex, and tumor location were not (). In the final multivariate Cox regression model, including miR-21 expression and TNM staging, high miR-21 expression in tumors was associated with poor survival prognosis (HR, 2.4; 95% CI, 1.4-4.1; P=.002) independent of other clinical covariates, consistent with findings in the Maryland test cohort.
miR-21 Expression in Colon Adenomas
Adenomas represent a precursor stage for colon adenocarcinomas.34
We tested miR-20a, miR-21, miR-106a, miR-181b
, and miR-203
expression levels by quantitative RT-PCR in 18 pairs of adenoma and adjacent nonadenoma tissue that were acquired from the Cooperative Human Tissue Network. Using only 18 pairs of tissues reduced the power to detect differences in these analyses and may result in false-negatives. However, miR-21
was significantly enriched at 1.6-fold higher (P
= .006, Wilcoxon matched-pairs test; ). Adenoma tissue expressed higher levels of in 15 of 18 matched pairs.
Tumor Stages and miR-21 Expression
If miR-21 expression is causal to the progression of colon cancer, expression of miR-21 may be associated with more advanced stages of the disease. Patients were stratified based on the diagnosis of adenoma and TNM staging, in which adenoma was considered the least advanced and TNM stage IV was most advanced. Adenomas expressed lower levels of miR-21 than tumors from the validation cohort (P<.001, Mann-Whitney test). More advanced tumors expressed higher levels of miR-21 using either microarray data from the Maryland test cohort (P=.04, test for trend) or the quantitative RT-PCR data from the Hong Kong validation cohort (test for trend, P<.001; ).
miR-21 Expressed at Higher Levels in Colon Adenocarcinomas With Increasing Expression in More Advanced Tumors
miR-21 Expression in Colonic Epithelial Cells
Although we found that high expression of miR-21 in tumors was associated with a worse survival outcome, these experiments did not identify the cells within a tumor that expressed miR-21. To identify these cells, we used in situ hybridization to visualize miR-21 expression in tumor and adjacent nontumor tissue (). The miR-21 is expressed at higher levels in colonic epithelial cells in human tumor tissue, consistent for a role for miR-21 overexpression within tumor cells during colon carcinogenesis.
In Situ Hybridization of miR-21 in Colon Tumors
miR-21 Expression Levels and Therapeutic Outcome
We analyzed associations with miR-21 expression and therapeutic outcomes in stage II and III cancer patients treated with adjuvant chemotherapy. Information on the administration of adjuvant chemotherapy was available for 47 of 65 stage II or III patients in the Maryland test cohort and for all patients in the Hong Kong validation cohort. In both cohorts chemotherapy regimens were primarily fluorouracil based (in forms of either intravenous 5-fluorouracil or oral drugs including tegafur with uracil [UFT]) with or without generics. Only patients with typical adenocarcinoma histology were used for this analysis, leaving 20 of 42 stage II or III patients who had received chemotherapy in the Maryland cohort. For those who received chemotherapy, high miR-21 expression in tumors predicted worse overall survival (HR, 4.3; 95% CI, 1.1-16.4; P= .03) giving preliminary support that high miR-21 is associated with poor therapeutic outcome.
For the Hong Kong validation cohort, 77 individuals with stage II or III cancer with typical adenocarcinoma histology were used for this analysis. Among the 36 patients who received adjuvant chemotherapy, high miR-21 expression in tumors was associated with a poor response to therapy (HR, 3.5; 95% CI, 1.1-11.6; P= .04), consistent with observations in the Maryland cohort. Additionally, among the 25 patients with stage III cancer who received adjuvant chemotherapy, high miR-21 expression was associated with poor survival (HR, 3.9; 95% CI, 1.2-12.9; P=.03). Analyses using cancer relapse as an end point resulted in similar associations with high miR-21 expression in tumors predicting a more rapid disease recurrence in patients with TNM stage III cancer who received adjuvant chemotherapy (HR, 3.5; 95% CI, 1.0-11.5; P=.04).
Both cohorts showed similar associations of high miR-21 expression in tumors with therapeutic outcomes, although neither cohort was large enough to perform a fully stratified analysis. Although these are 2 different populations, the similarity of the associations with miR-21 expression, prognosis, and therapeutic outcome allowed for pooled analysis using both cohorts. Kaplan-Meier analysis of the pooled cohorts demonstrated that high miR-21 expression was associated with a poor prognosis in either stage II (P=.02) or stage III (P=.004) patients () further indicating its potential as a prognostic biomarker. Receipt of adjuvant chemotherapy was beneficial for patients with either stage II or stage III cancer, although this was only significant for stage III patients. For individuals who received adjuvant therapy, high miR-21 expression was associated with a poor therapeutic outcome in patients with stage II or III cancer (P=.003, Kaplan-Meier log rank) or in patients with stage III cancer alone (P=.007, Kaplan-Meier log rank; ). Multivariate Cox regression demonstrated that high miR-21 expression predicted poor prognosis (HR, 3.0; 95% CI, 1.7-5.4; P<.001) and treatment with adjuvant chemotherapy was associated with better survival (HR, 0.4; 95% CI, 0.2-0.8; P=.004) independent of other clinical covariates ().
Combined Analysis of Maryland Test Cohort and Hong Kong Validation Cohort Examining Associations Between miR-21 Expression in Tumors and Receipt of Adjuvant Chemotherapy With Prognosis
Univariate and Multivariate Cox Regression Analysis of miR-21 Expression, Receipt of Adjuvant Chemotherapy, and Cancer Survival in Patients With Stage II or III With Adenocarcinoma in both Maryland and Hong Kong Cohortsa