MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma

Abstract

Context: MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome.

Objective: To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome.

Design, setting, and patients: MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort.

Main outcome measures: MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point. RESULTS Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome.

Conclusions: Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

Figure 1. High miR-21 Expression in Tumors and Poor Survival in Patients With Typical Adenocarcinoma Histology

This analysis excludes patients with either mucinous adenocarcinoma or adenosquamous carcinoma histology. A, MicroRNA microarrays were used to measure microRNA expression levels of tumors and nontumorous tissues. Tissues with undetectable expression of miR-21 based on microarray data were excluded. High miR-21 expression was classified according to the highest tertile (2.6-fold to 7.9-fold higher than nontumor). B, The association of high miR-21 expression in tumors with poor prognosis is validated in an independent cohort. Expression levels of miR-21 were measured by quantitative reverse transcription polymerase chain reaction. High expression is based on the highest tertile (3.3-fold to 8.7-fold higher than nontumor). Log-rank P values are from Kaplan-Meier analysis.

Figure 2. miR-21 Expressed at Higher Levels in Colon Adenocarcinomas With Increasing Expression in More Advanced Tumors

A, MicroRNA microarrays were used to measure miR-21 expression levels in the Maryland test cohort. Dot plots represent miR-21 log₂ (tumor:nontumor ratios) for paired tissues as calculated from microRNA microarrays from the original cohort. Values greater than 0 indicate tumors with expression values higher than nontumorous tissue. Tissue types have been ordered from TNM stage I to stage IV tumors. Bars indicate median value. The Cuzick nonparametric test for trend was used to evaluate trends. B, miR-21 is expressed at higher levels in more advanced tumors. Dot plots represent miR-21 relative threshold cycle values from quantitative reverse transcription polymerase chain reaction for adenoma and tumor expression levels, each has been normalized to paired nonadenoma or nontumorous tissue, respectively. Relative threshold cycle values greater than 0 indicate expression at levels higher than nontumorous (or nonadenoma) tissue. Tissue types have been ordered from adenoma to stage I through IV tumors. Horizontal bars indicate median expression value. The Cuzick nonparametric test for trend was used to evaluate trends.

Figure 3. In Situ Hybridization of miR-21 in Colon Tumors

In situ hybridization for miR-21 was optimized to distinguish high (brown) and low expression of miR-21. The 3' DIG-labeled probe was hybridized and detected with a polyclonal anti-DIG antibody (DakoCytomation) using amplification with the GenPoint Tyramide Signal Amplification System (DakoCytomation) using Vector NovaRed (Vector Laboratories) as the substrate. The slide was counterstained with Mayer's hematoxylin. A, Colonic epithelial cells in human tumor express higher levels of miR-21 compared with adjacent nontumorous tissue. C, Colonic epithelial cells in tumor tissue express significant amounts of miR-21, at high magnification. E, Nontumor tissue shows no significant expression of miR-21 at the same magnification. B, D, F, The scramble control probe shows no significant staining at low or high magnification in serial sections of tumor and nontumor tissue, as expected.

Figure 4. 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

This analysis includes all patients with TNM stage II or III cancer except those with mucinous adenocarcinoma or adenosquamous carcinoma histologies. A, For the 119 patients with stage II or III cancer, high miR-21 expression is associated with poor survival for those who received chemotherapy (P=.003). B, Among the 52 patients with stage II cancer, associations between high miR-21 expression and prognosis were not statistically significant in 14 (26.9%) of the individuals who received chemotherapy (P=.11). C, For all 67 patients with TNM stage III cancer, high miR-21 expression was significantly associated with poor survival among those who received chemotherapy (P=.007).