Serum miR-21 as a Diagnostic and Prognostic Biomarker in CRC
Serum miR-21 as a Diagnostic and Prognostic Biomarker in CRC
Background The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC).
Methods To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan–Meier analyses and Cox regression models. All statistical tests were two-sided.
Results Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03).
Conclusions Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.
Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. In the United States, CRC is the third most common cancer, with more than 143000 new cases and more than 52000 deaths each year (1). Several CRC screening tests, including fecal occult-blood testing and colonoscopy, have been available for years and have aided in reducing the mortality associated with this disease. However, compliance with these screening tests has been far from adequate. Patients with metastatic disease frequently receive expensive cytotoxic chemotherapeutic regimens coupled with targeted monoclonal antibodies but with relatively modest benefits. Without a priori knowledge of which patients will experience tumor recurrence, there is inevitable overtreatment with agents associated with toxic side effects. These limitations underscore the need for novel biomarkers, particularly noninvasive biomarkers in serum or plasma, for diagnosis, prognosis, and prediction of response to chemotherapy.
MicroRNAs (miRNAs) are a class of small noncoding RNAs that play a central role in the regulation of mRNA expression. The discovery that miRNA expression is frequently dysregulated in a cancer-specific manner provides an opportunity to develop these RNAs as biomarkers for cancer detection. Although most previous studies on miRNA expression have been performed on tissue specimens, some studies have shown diagnostic and prognostic potential for circulating miRNAs because tumor-derived miRNAs can be present in blood and appear to be stably protected from endogenous ribonuclease activity in the circulation. Nonetheless, it is unclear whether expression profiles of circulating miRNAs reflect miRNA profiles of tumor tissues and to the best of our knowledge, no systematic investigation of the relationship between miRNA profiles in body fluids vs matched primary CRCs has thus far been undertaken. This is critical because increased expression of circulating miRNAs could be indicative of miRNAs secreted from a tumor, raising the overall diagnostic specificity of the biomarker.
MiR-21 is an oncogenic miRNA that modulates the expression of multiple cancer-related target genes such as PTEN, TPM1, and PDCD and has been shown to be overexpressed in various human tumors. In addition, miR-21 expression is upregulated in CRC tissues, is elevated during tumor progression, and is also associated with poor survival and response to chemotherapy. The clinical significance of circulating miR-21 levels in CRC remains unclear at this time. Although an earlier study was unable to use plasma miR-21 as a biomarker because of low levels of detection using a direct amplification method, a more recent study demonstrated statistically significantly elevated plasma miR-21 expression in CRC patients using TaqMan-based approaches. On the other hand, miR-31 is another miRNA frequently overexpressed in CRC tissues and has been shown to be associated with tumor prognosis. Additionally, both miR-21 and miR-31 are frequently upregulated, even in premalignant lesions such as colonic adenomas, which are the target lesions of CRC screening. In light of these observations, we hypothesized that these two miRNAs might be good candidates for exploration as circulating biomarkers for the early detection and prognosis of CRC, assuming that the expression pattern for these miRNAs in serum mirrors that in the neoplastic tissues.
We have systematically investigated the expression of miR-21 and miR-31 in a two-phase study. In the first phase, we determined whether cultured CRC cells secrete these miRNAs into the culture medium, establishing their secretory potential. We then performed quantitative analyses of these miRNAs in a subset of serum samples from CRC patients and healthy control subjects to determine the feasibility of their detection in the circulation. In the second phase, using a large validation cohort comprised of matched serum and tissue samples from patients with colorectal neoplasia and serum from healthy control subjects, we evaluated the clinical significance of these miRNAs as potential biomarkers for diagnosis and prognosis of CRC patients.
Abstract and Introduction
Abstract
Background The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC).
Methods To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan–Meier analyses and Cox regression models. All statistical tests were two-sided.
Results Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03).
Conclusions Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.
Introduction
Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. In the United States, CRC is the third most common cancer, with more than 143000 new cases and more than 52000 deaths each year (1). Several CRC screening tests, including fecal occult-blood testing and colonoscopy, have been available for years and have aided in reducing the mortality associated with this disease. However, compliance with these screening tests has been far from adequate. Patients with metastatic disease frequently receive expensive cytotoxic chemotherapeutic regimens coupled with targeted monoclonal antibodies but with relatively modest benefits. Without a priori knowledge of which patients will experience tumor recurrence, there is inevitable overtreatment with agents associated with toxic side effects. These limitations underscore the need for novel biomarkers, particularly noninvasive biomarkers in serum or plasma, for diagnosis, prognosis, and prediction of response to chemotherapy.
MicroRNAs (miRNAs) are a class of small noncoding RNAs that play a central role in the regulation of mRNA expression. The discovery that miRNA expression is frequently dysregulated in a cancer-specific manner provides an opportunity to develop these RNAs as biomarkers for cancer detection. Although most previous studies on miRNA expression have been performed on tissue specimens, some studies have shown diagnostic and prognostic potential for circulating miRNAs because tumor-derived miRNAs can be present in blood and appear to be stably protected from endogenous ribonuclease activity in the circulation. Nonetheless, it is unclear whether expression profiles of circulating miRNAs reflect miRNA profiles of tumor tissues and to the best of our knowledge, no systematic investigation of the relationship between miRNA profiles in body fluids vs matched primary CRCs has thus far been undertaken. This is critical because increased expression of circulating miRNAs could be indicative of miRNAs secreted from a tumor, raising the overall diagnostic specificity of the biomarker.
MiR-21 is an oncogenic miRNA that modulates the expression of multiple cancer-related target genes such as PTEN, TPM1, and PDCD and has been shown to be overexpressed in various human tumors. In addition, miR-21 expression is upregulated in CRC tissues, is elevated during tumor progression, and is also associated with poor survival and response to chemotherapy. The clinical significance of circulating miR-21 levels in CRC remains unclear at this time. Although an earlier study was unable to use plasma miR-21 as a biomarker because of low levels of detection using a direct amplification method, a more recent study demonstrated statistically significantly elevated plasma miR-21 expression in CRC patients using TaqMan-based approaches. On the other hand, miR-31 is another miRNA frequently overexpressed in CRC tissues and has been shown to be associated with tumor prognosis. Additionally, both miR-21 and miR-31 are frequently upregulated, even in premalignant lesions such as colonic adenomas, which are the target lesions of CRC screening. In light of these observations, we hypothesized that these two miRNAs might be good candidates for exploration as circulating biomarkers for the early detection and prognosis of CRC, assuming that the expression pattern for these miRNAs in serum mirrors that in the neoplastic tissues.
We have systematically investigated the expression of miR-21 and miR-31 in a two-phase study. In the first phase, we determined whether cultured CRC cells secrete these miRNAs into the culture medium, establishing their secretory potential. We then performed quantitative analyses of these miRNAs in a subset of serum samples from CRC patients and healthy control subjects to determine the feasibility of their detection in the circulation. In the second phase, using a large validation cohort comprised of matched serum and tissue samples from patients with colorectal neoplasia and serum from healthy control subjects, we evaluated the clinical significance of these miRNAs as potential biomarkers for diagnosis and prognosis of CRC patients.
