Prognostic value of BRAF/MIR-17 signature and B-Raf protein expression in patients with colorectal cancer: A pilot study

Abstract

Background: Despite the recent improvement in colorectal cancer (CRC) treatment, it still has a poor prognosis with a low survival rate. Genetic and epigenetic mechanisms have proved to play a substantial role in CRC tumorigenesis and progression. According to Gene Ontology and TargetScan analyses, the B-Raf proto-oncogene (BRAF) gene is one of the microRNA-17 (miR-17) targets. We aimed to explore the prognostic value of B-Raf protein and BRAF/microRNA-17 (MIR-17) gene expression signature in CRC archived samples.

Methods: B-Raf protein expression was identified by immunohistochemistry, while gene expression studies were quantified by real-time qPCR in 53 paired archived CRC specimens.

Results: The BRAF showed higher expressions in CRC specimens relative to non-cancer tissues (p = 0.006). MIR17 expression was inversely and significantly correlated with both B-Raf protein (r = -0.79, p < 0.001) and gene expression (r = -0.35, p = 0.010) and showed a significant direct correlation with a high rate of relapse (p = 0.020). BRAF/miR-17 expression in CRC was associated inversely with tumor size, high grade of colonic carcinoma, lymph node metastasis, and carcinoma subtype. Spearman correlation and Kaplan-Meier survival curve analyses revealed that disease-free survival and overall survival were inversely and significantly correlated with positive B-Raf protein expression (r = -0.31 and -0.35, p = 0.023 and 0.011, respectively) and directly correlated with log BRAF/MIR17 ratio (r = 0.50 and 0.41, p < 0.001 and = 0.003, respectively). Cox hazard regression analysis revealed the BRAF/MIR17 ratio could predict both types of patients' survival, among other variables.

Conclusion: BRAF/MIR17 ratio could have prognostic utility in patients with CRC. Further larger-scale studies are warranted to confirm this utility.

Keywords: BRAF; IHC; colorectal cancer; gene expression; miR-17; prognosis; qPCR.

Figure 1

Role of BRAF and MIR‐17 genes in cancer hallmarks. Hallmark annotations were derived from The Cancer Hallmarks Analytics Tool (CHAT) web browser, which analyzes the strength of association between genes of interest and the hallmarks of cancer in the literature based on a text‐mining analysis of 26 million PubMed abstracts. npmi: normalized pointwise mutual information of h and q: npmi (h,q) = pmi (h,q)/‐log2 p(h,q). The "npmi" metric normalizes "pmi" by dividing by the negative log probability of co‐occurrence ‐log2 p(h,q), which constrains its values to the range [−1, 1]. Zero is a frequency of co‐occurrence matching random (no association, as for "pmi") and 1 for complete co‐occurrence (perfect association). [Data source: http://chat.lionproject.net]

Figure 2

Immunohistochemistry of BRAF protein in colorectal cancer specimens. Immunohistochemical staining for B‐Raf protein (x400) showed (A) negative staining in colonic adenoma, (B) another colonic adenoma showed weak cytoplasmic staining with non‐specific nuclear staining, (C) grade 2 colonic adenocarcinoma showed moderate cytoplasmic staining, (D) grade 3 colonic adenocarcinoma showed strong cytoplasmic staining, (E) mucinous adenocarcinoma showed negative staining, (F) moderately differentiated adenocarcinoma with lymphovascular emboli showed strong cytoplasmic staining, and (G) protein intensity in the overall analysis of CRC samples

Figure 3

The relative expression level of BRAF and MIR17 genes in colorectal cancer specimens compared to control tissues. Values are presented as median (Q1‐Q3). The Mann‐Whitney U test was applied for p‐value calculation

Figure 4

Correlation analysis for gene and protein expression in colorectal cancer. Spearman's correlation coefficient is shown

Figure 5

Association between BRAF/miR‐17 ratio and clinicopathological features. Kruskal‐Wallis and Mann‐Whitney U tests were applied. The significance level was set at p < 0.05

Figure 6

Prognostic value of the BRAF/miR‐17 ratio for detecting survival. (A‐B) Association of BRAF/MIR17 expression ratio with survival. (C‐D) Spearman correlation analysis between gene expression and survival. (E‐F) Receiver characteristic operator curve to discriminate between prolonged and short survivors. (G‐H) Kaplan‐Meier survival curve for overall and disease‐free survival. LogRank (Mantel‐Cox) test was used