MicroRNA-126 and epidermal growth factor-like domain 7-an angiogenic couple of importance in metastatic colorectal cancer. Results from the Nordic ACT trial

Abstract

Background: This study investigated the clinical importance of linked angiogenetic biomarkers to chemotherapy, combined with the anti-vascular endothelial growth factor A (anti-VEGF-A), as a first-line treatment in patients with metastatic colorectal cancer (mCRC).

Methods: A total of 230 patients from a randomised phase III study were included. The primary microRNA-126 (pri-miRNA-126) A24G single-nucleotide polymorphism and the mature miRNA-126 were analysed by PCR using genomic DNA (full blood) and formalin-fixed paraffin-embedded tissue sections, respectively. The epidermal growth factor-like domain 7 (EGFL7) protein was visualised and quantified using immunohistochemistry.

Results: High tumour expression of miRNA-126 was significantly related to a longer progression-free survival. The independent prognostic value of miRNA-126 was confirmed using a Cox regression analysis (hazard ratio=0.49, 95% confidence interval=0.29-0.84, P=0.009). Although not significant, a relationship between EGFL7 expression and response rates is suggested, with EGFL7 expression at the invasive front being lower in responding patients than in the non-responders (P=0.063).

Conclusion: The results validate the previous findings on the prognostic value of miRNA-126 in mCRC and may suggest a relationship between treatment efficacy and EGFL7 expression. As miRNA-126 may target VEGF-A as well as EGFL7, the results may provide predictive information in relation to next-generation anti-angiogenetics.

Trial registration: ClinicalTrials.gov NCT00598156.

Figures

Figure 1

(A) Illustration of an endothelial cell, demonstrating the interaction of the investigated parameters. (B) Diagram illustrating the investigated parameters: pri-miRNA-126 SNP; mature miRNA-126; EGFL7 protein, and sample availability. EGFL7 analyses of samples (tumour resections) from four patients were not possible as all the available tissue was used in the initial qPCR profiling of miRNA-126. Thus, immunohistochemical staining was performed on 122 resections and 47 biopsies, respectively. EGFL7 tumour was consequently scored in 169 samples, whereas EGFL7 invasive front was scored in 122 samples. Abbreviations: Pri-miRNA-126, primary microRNA-126; miRNA-126, microRNA-126; EGFL7, epidermal growth factor-like domain 7; SNP, single nucleotide polymorphism; pre-miRNA-126, precursor microRNA-126; Dicer, The RNAse III enzyme responsible for excising the miRNA duplex from the pre-miRNA hairpin.

Figure 2

(A) Dark brown staining of endothelial-derived structures along with a larger caldesmon-positive (red staining) blood vessel. (B, C) Two examples of clusters of tumour cells with signs of cytoplasmic EGFL7 expression and unspecific background staining of the non-endothelial stromal compartment.

Figure 3

Box plot illustrating the distribution of EGFL7 expression at the invasive front according to response rates (n=122, P=0.06). Boxes indicate the range from lower to upper quartile values, with the line inside the box representing the median. The vertical lines mark the highest and lowest value observed within a distance of 1.5 times the inter-quartile range from the bottom and the top of the boxes, respectively. Dots are outliers.

Figure 4

Progression-free survival curves according to (A) pri-miRNA-126 SNP genotypes (green=GG, red=AG, blue=AA); (B) miRNA-126 expression levels (red=above median, blue=below median); (C) EGFL7 expression in the tumour (red=above median, blue=below median); (D) EGFL7 expression at the invasive tumour front (red=above median, blue=below median).N reflects the number of patients randomised to maintenance treatment (patients with progressive disease were excluded per protocol) and thus differs from the base-line numbers presented in Table 1.