Nobuyoshi Yamazaki

Fecal miR-106a Is a Useful Marker for Colorectal Cancer Patients with False-Negative Results in Immunochemical Fecal Occult Blood Test

Background: Immunochemical fecal occult blood test (iFOBT) is widely used for colorectal cancer screening; however, its sensitivity is insufficient. We recently reported a fecal microRNA (miRNA) test (FmiRT) to detect colorectal cancer. In this study, we investigated a new colorectal cancer screening method combining iFOBT and FmiRT to improve the sensitivity compared with iFOBT alone. Methods: In total, 117 colorectal cancer patients and 107 healthy volunteers were enrolled. Ten-milligram fecal samples were collected and iFOBT was conducted. Fecal RNA was extracted from residuum of iFOBT and then the expression of 14 kinds of miRNA was analyzed for the FmiRT using real-time reverse transcription PCR. Results: Levels of fecal miR-106a expression in iFOBT+ patients and iFOBT− patients were significantly higher than in healthy volunteers (P = 0.001). The sensitivity and specificity of FmiRT using miR-106a were 34.2% and 97.2%, and those of iFOBT were 60.7% and 98.1%, respectively. The overall sensitivity and specificity of the new screening method combining iFOBT and FmiRT were 70.9% and 96.3%, respectively. One quarter of colorectal cancer patients with false-negative iFOBT seemed to be true positive upon adding FmiRT using fecal miR-106a. Conclusions: Fecal miR-106a is a good molecular marker to identify colorectal cancer patients from among those with negative iFOBT results. FmiRT combined with iFOBT may improve the sensitivity to detect colorectal cancer. Impact: We have shown the usefulness of fecal miR-106a to detect the colorectal cancer patients among those with negative iFOBT results. Cancer Epidemiol Biomarkers Prev; 22(10); 1844–52. ©2013 AACR.

Application of the Fecal MicroRNA Test to the Residuum from the Fecal Occult Blood Test

OBJECTIVE Though the fecal occult blood test is used for colorectal cancer screening worldwide, it does not have a particularly high sensitivity for detecting colorectal cancer. Here we investigated the applicability of the fecal microRNA test to fecal samples that had been used for a previous fecal occult blood test and stored under various conditions. METHODS Five colorectal cancer patients and five healthy volunteers were enrolled. Fecal samples were stored for 0-5 days at 4°C, room temperature or 37°C. Total RNA was extracted from the fecal occult blood test residuum and microRNA expression was analyzed by real-time reverse transcription polymerase chain reaction. RESULTS There were no remarkable differences either in colorectal cancer patients or in controls with regard to the concentration of RNA extracted from the fecal occult blood test residuum in any of the storage groups compared with the samples prepared on day 0 (Group 0). Ribosomal RNA stored at room temperature or 37°C degraded rapidly. In contrast, the ribosomal RNA stored at 4°C remained intact for at least 5 days. The microRNAs in samples stored at 4°C and room temperature were conserved; however, the microRNAs stored at 37°C were significantly degraded compared with Group 0 (P < 0.05). In the residuum stored at 4°C up to 5 days, the relative quantification of miR-106a normalized with miR-24 in colorectal cancer patients was significantly higher than those in healthy volunteers (P < 0.05). In contrast, the quantification of normalized miR-106a was remarkably low in samples stored at room temperature and 37°C. CONCLUSIONS Fecal microRNA of sufficient quality for reverse transcription polymerase chain reaction analysis was extracted from the fecal occult blood test residuum stored at 4°C for up to 5 days.

High expression of miR-181c as a predictive marker of recurrence in stage II colorectal cancer

INTRODUCTION A standard treatment for stage II colorectal cancer (CRC) is surgical resection without adjuvant chemotherapy. However, the recurrence rate of these patients is approximately 20%. To date, there are no robust biomarkers suitable for predicting recurrence in stage II CRC patients. In this study, microRNAs (miRNAs) extracted from CRC tissues were examined for a possible biomarker to predict recurrence in stage II CRC patients. RESULTS From the comprehensive analysis, 15 miRNAs were selected as candidates for further study. Regarding let-7a, -7d, -7e, miR-23c, -26b, -128a, -151-5p, and -181c, recurrence rates in training cohort patients with higher expression of these miRNAs isolated from their frozen tissues samples were significantly higher than those with lower expression (P < 0.05). According to multivariate analysis, the higher expression of miR-181c was detected as an independent predictive factor of recurrence (P = 0.001, OR: 9.43, 95% CI: 2.57–34.48). Results were similar in miR-181c extracted from FFPE tissues obtained from the training cohort (P = 0.003, OR: 7.46, 95% CI: 1.97–28.57). In the validation cohort using FFPE tissues, the recurrence rate in patients with higher miR-181c expression was significantly higher than those with lower miR-181c expression (P < 0.001). MATERIAL AND METHODS Comprehensive analysis using a highly sensitive miRNA chip was initially performed to select candidate miRNAs associated with recurrence. Candidate miRNAs were analyzed by real-time RT-PCR using RNA from frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. CONCLUSIONS Higher expression of miR-181c may be a useful recurrence predictor of stage II CRC patients.

New molecular diagnosis and screening methods for colorectal cancer using fecal protein, DNA and RNA

Several screening methods for reducing the mortality rate of colorectal cancer (CRC) have been reported in recent decades. Fecal occult blood tests (FOBTs) are widely used for CRC screening and immunochemical FOBTs perform better than guaiac FOBTs; however, the sensitivity and specificity of immunochemical FOBTs remain unsatisfactory. To resolve this problem, novel fecal molecular methods based on fecal protein, DNA and RNA analyses have been developed. Regarding fecal proteins, several marker proteins indicating intestinal bleeding and cancer cell-specific proteins have been investigated. Regarding fecal DNA, numerous gene mutation and gene methylation analyses have been reported. Consequently, fecal DNA analysis was recommended as a CRC screening method in 2008. In addition, gene expression analyses of CRC-specific genes and miRNAs in fecal RNA have been investigated over the last decade. This review article summarizes molecular methods using fecal samples for CRC screening, focusing on reports within the last 5 years.

MicroRNA analysis of colorectal cancer using fecal and tissue samples

Recently, several microRNAs (miRNAs) have been reported as promising biomarkers for cancer detection and tumor recurrence risk. Due to its stability, miRNA can be accessed from samples stored in severe conditions, such as feces or formalin-fixed paraffin-embedded (FFPE) tissue. Fecal miRNA extracted from the residuum of fecal occult blood tests (FOBTs) was assessed to determine whether a combination of this fecal miRNA test (FmiRT) with FOBT could improve the false-negative rate of colorectal cancer (CRC) screening compared with FOBT alone. Expression of miR-106a in patients with both positive and negative FOBTs was significantly higher than in healthy volunteers. To identify a high-risk group for recurrence, miRNAs were extracted from FFPE samples of patients with stage II CRC. Tumor recurrence occurred at a significantly higher rate in patients with increased miR-181c expression than in those with lower expression. The recurrence rate in patients with stage II CRC with higher expression of miR-181c was similar to that of patients with stage III CRC who had been treated by surgical resection alone. As miRNAs are stable in several severe storage conditions, such as in fecal and FFPE samples, they could be valuable, accessible biomarkers for CRC, for use both in cancer screening and as predictors of recurrence.

Abstract 1491: Tissue miRNA as a predictive marker for recurrence of Dukes B colorectal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background:Colorectal cancer (CRC) diagnosed at an early stage is curable, thus, a standard treatment for Dukes B CRC which invades deeply through the muscularis propria without lymph node metastasis and distant metastasis is surgically resectable without any additional chemotherapies. However, the recurrence rate of Dukes B after curative resection is approximately 20%. Although clinical and pathological factors have been investigated in order to characterize the high risk of the recurrence in Dukes B CRC, there were no robust biomarkers suitable for detecting the high risk group. Several microRNAs (miRNAs) have appeared to be associated with CRC and some are under evaluation for a biomarker to detect CRC in serum or fecal samples. In this study, miRNAs isolated from CRC tissue were investigated for a proper biomarker for extracting the high risk group in Dukes B CRC. Methods: Patients with histologically confirmed Dukes B CRC were enrolled in this study. Tissue miRNAs extracted from the cancer tissue and normal adjacent tissue of 5 patients with recurrence were compared with those from 5 patients without recurrence, using a high-sensitivity miRNA chip, in order to select the candidate miRNAs. The candidate miRNAs associated with recurrence were then analyzed by real-time RT PCR using tissue miRNA of 14 patients with recurrence and 66 patients without recurrence. The corresponding relapse-free survival (RFS) was analyzed using the Kaplan-Meier method. Results: From the comprehensive analysis, using a high-sensitivity miRNA chip, the following 14 miRNAs were selected as candidates for further study; let-7a, -7d, -7e, miR-18b, -23c, -128a, -146b-5p, -148b, -151-5p, -181c, -221, -222, -361-5p, and -500a*. The expressions of let-7d, miR-18b, -23c, -148b, -151-5p, -181c, and -361-5p in the recurrent group were significantly higher than those in the recurrence-free group, using univariate analysis (P < 0.05). These seven miRNAs and two histopathological factors, namely pathological type and tdepth of umor invasion, were studied using multivariate analysis. Three miRNAs, miR-23c (P = 0.04, odds ratio [OR]: 21.0, 95% confidence interval [CI]: 2.65-167.4), miR-151-5p (P = 0.01, OR: 7.8, 95% CI: 1.57-38.4), and miR-181c (P = 0.006, OR: 10.0, 95% CI: 1.94-51.9), were detected as independent predictive factors of recurrence. All patients with low expression of these 3 miRNAs (N = 25) survived over 60 months without recurrence. Meanwhile, recurrent tumor occurred within 36 months in all patients with high expression of these 3 miRNAs (N = 3). Conclusion: The miRNAs selected in the present study may be a operationally useful predictors of CRC recurrence. Furthermore, there is a possibility to improve prediction performance by combining several miRNA levels. Citation Format: Nobuyoshi Yamazaki, Yoshikatsu Koga, Norio Saito, Yasuhiro Matsumura. Tissue miRNA as a predictive marker for recurrence of Dukes B colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1491. doi:10.1158/1538-7445.AM2014-1491