The association of miR-126-3p,miR-126-5p and miR-664-3p expression profiles with outcomes of patients with metastatic colorectal cancer treated with bevacizumab

Abstract

MicroRNAs regulate the expression of genes involved in several important cancer-related processes including cell adhesion, proliferation, and tumour angiogenesis. Bevacizumab is routinely used in the treatment of patients with metastatic colorectal cancer, but, so far, no reliable biomarker predicting response to bevacizumab has been established. The aim of our retrospective study was to evaluate the association of miR-126-3p, miR-126-5p and miR-664-3p tumour expression levels with outcomes of patients with metastatic colorectal cancer treated with bevacizumab. The study included 63 patients. For the assessment of microRNA expression, gene-specific TaqMan assays were used. The median progression-free survival and overall survival for patients with low tumour expression of miR-126-3p were 8.8 and 20.6 months versus 13.5 months and median overall survival was not reached for patients with high expression (p = 0.0064 and p = 0.0027), respectively. The median progression-free survival and overall survival for patients with low tumour expression of miR-126-5p were 9.0 and 22.2 months versus 12.0 and 23.4 months for patients with high expression (p = 0.2113 and 0.6858), respectively. The median progression-free survival and overall survival for patients with low tumour expression of miR-664-3p were 9.1 and 22.5 months versus 8.8 and 23.4 months for patients with high expression (p = 0.2542 and p = 0.1922), respectively. The multivariable Cox proportional hazards model revealed that miR-126-3p expression was significantly associated with progression-free survival (hazard ratio = 0.28, p = 0.0053) and also with overall survival (hazard ratio = 0.18, p = 0.0046). In conclusion, the results of this study suggest that the expression of miR-126-3p in the tumour tissue was associated with outcome of metastatic colorectal cancer patients treated with bevacizumab.

Keywords

Colorectal cancer bevacizumab chemotherapy microRNA miR-126-3p miR-126-5p miR-664-3p

Introduction

Colorectal cancer (CRC) is one of the most common cancer-related causes of morbidity and mortality in developed countries.^1,2 The introduction of targeted agents based on monoclonal antibodies against vascular endothelial growth factor (VEGF) or epidermal growth factor receptor (EGFR) significantly improved survival of patients with metastatic CRC (mCRC). The mechanism of action of bevacizumab is based on blockade of angiogenesis by inhibition of the most important angiogenic growth factor, VEGF. The efficacy and safety of bevacizumab in the treatment of patients with mCRC have been proved in randomized phase III clinical trials as well as observational studies previously,^3–7 but, so far, no reliable biomarker predicting response to bevacizumab has been established. Several candidate predictive biomarkers, including angiopoietin-2, soluble VEGFR-1 and intramural expression of VEGFR-2, circulating levels of short VEGF-A isoforms, specific KRAS mutation types or serum carcinoembryonic antigen (CEA), have been studied but not sufficiently validated for routine clinical use.^8–12

MicroRNAs (miRs) are small noncoding RNAs (containing approximately 22 nucleotides) emerging as new players in epigenetic regulation of gene expression at post-transcriptional level. MiRs regulate the expression of genes involved in several important cancer-related processes including cell adhesion, proliferation, and tumour angiogenesis.¹³ During recent years, many efforts were made to explore the association between various miRs and cancer diagnostics, prognosis, and treatment response in order to find potential biomarkers to be used in clinical practice.^14–16

The aim of our retrospective study was to evaluate the association of miR-126-3p, miR-126-5p and miR-664-3p expression levels with outcomes of patients with mCRC treated with bevacizumab.

MiR-126-3p has multiple functions with important roles in angiogenesis, tumour growth and invasion and vascular inflammation. It is encoded by intron 7 of the epidermal growth factor (EGF)-like domain 7 (Egfl7) gene, and its expression appears to be highly enriched in endothelial cells.^17–19 Because of this endothelial-specific expression, miR-126-3p is one of the commonly studied miRs in angiogenesis, and it is considered essential to the regulation of blood vessel integrity.^18,19 The VEGF-A messenger RNA (mRNA) is a miR-126-3p target and previous studies have suggested its role in regulation of VEGF-A-mediated signal transduction.^18–21 MiR-126 is downregulated in several malignant tumours and is often referred to as a tumour suppressor.^22–25 MiR-126-5p plays roles that are relevant to cancer progression like promotion of endothelial proliferation or regulation of leukocyte adhesion and transmigration.^26,27 MiR-664-3p has been shown to be involved in several aspects of carcinogenesis including angiogenesis. Among its most important targets is neuroligin 1, which plays an important role in regulation of angiogenesis. Recently, neuroligin and its binding partner neurexin have been shown to be widely expressed in the vascular system and involved in angiogenesis.^28,29

Patients and methods

Patients and treatment

We retrospectively analysed clinical data of 63 adult patients with histologically confirmed mCRC treated with bevacizumab-based therapy between years 2009 and 2015 at Department of Oncology and Radiotherapeutics, Faculty of Medicine and University Hospital in Pilsen, Czech Republic. Bevacizumab (Avastin; F. Hoffmann-La Roche Ltd., Basel, Switzerland) was administered in combination with chemotherapy or as a single agent in a standard approved doses (5.0 mg/kg every 14 days or 7.5 mg every 21 days). The chemotherapy consisted of following schedules: fluorouracil and leucovorin in combination with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) or alone (FUFA), capecitabine alone, oxaliplatin alone and irinotecan alone. None of the patients had previously received antiangiogenic therapies. The protocol of our study was approved by the independent ethics committee of the Faculty of Medicine and University Hospital in Pilsen and complied with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice Guidelines, the Declaration of Helsinki and local laws. The patients signed informed consent with the inclusion and subsequent analysis of their data.

Clinical monitoring

Clinical data were obtained retrospectively from the hospital information system. Physical examination and routine laboratory tests were performed every 2 weeks; computed tomography (CT) or positron emission tomography (PET; PET/CT) was performed every 3 months of the treatment. The objective tumour response was assessed by the attending physician using Response Evaluation Criteria in Solid Tumors (RECIST).³⁰

Assessment of miR-126-3p, miR-126-5p and miR-664-3p expression

Matched samples of tumour and macroscopically healthy mucosa from the same resected part of colon (sampled as far from the tumour site as possible) underwent total RNA isolation by TRI Reagent^® RT (MRC, Cincinnati, OH, USA) using manufacturer’s protocol. Total RNA concentration was measured by Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and the samples were diluted to 2 ng/µL for reverse transcription.

For the assessment of miR expression, gene-specific TaqMan assays (Thermo Fisher Scientific) were used. Targets for the analysis were hsa-miR-126-3p (assay ID 002228), hsa-miR-126-5p (000451), hsa-miR-664-3p (002897), hsa-miR-16 (000391) and hsa-miR-345 (002186), with the last two miRs used as reference genes based on the previously published data³¹ and analysis of expression stability using our data and combination of four algorithms (BestKeeper, NormFinder, Genorm and Comparative Delta Ct method).^32–35 Reverse transcription was done separately for each miR using the target-specific primers from individual TaqMan Assays in combination with TaqMan^® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). Relative expression of miRs was measured in triplicates in 10 µL reactions on the CFX96 real-time polymerase chain reaction (PCR) system (Bio-Rad, Hercules, CA, USA) with TaqMan^® Universal Master Mix II, with UNG (Thermo Fisher Scientific). All reactions were tested for nonspecific signals.

Statistical analysis

Standard frequency tables and descriptive statistics were used to characterize sample data set. Changes of miR-126-3p, miR-126-5p and miR-664-3p expression in tumour tissue (relative to healthy tissue) were tested for statistical significance using Wilcoxon signed-rank test. Correlations between relative miR expressions were assessed by means of Spearman’s correlation coefficient. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method, and all point estimates were accompanied by 95% confidence intervals. PFS was determined from the date of bevacizumab initiation until the date of first documented progression or death. OS was determined from the date of bevacizumab initiation until the date of death, regardless of its cause. Patients who had not progressed or had not died were censored at the date of last follow-up with respect to their PFS or OS, respectively. Point estimates of observed survival times (or survival quantiles) were calculated from the Kaplan-Meier estimates of the survival functions using linear interpolation between the complete observations to the required time (or proportion surviving). Estimates of median follow-up were calculated using the inverse Kaplan–Meier method.³⁶ Statistical significance of the differences in Kaplan–Meier estimates according to miR-126-3p, miR-126-5p and miR-664-3p expression was assessed using the log-rank test. Multivariable Cox proportional hazards model was then used to evaluate the effect of miR-126-3p, miR-126-5p and miR-664-3p expression in the context of other potential prognostic factors of PFS and OS. All reported p values are two-tailed and the level of statistical significance was set at α = 0.05. Statistical processing and testing were performed using STATISTICA data analysis software system (Version 12; StatSoft, Inc, 2013; www.statsoft.com).

Results

Patient characteristics

The study included 63 patients. The median age was 62.5 (range = 39.1–78.7) years. Of them, 43 (68.3%) patients were male, 36 (57.1%) had a primary tumour localized in the colon, 41 (65.1%) had metastatic disease at diagnosis, and 48 (76.2%) received the bevacizumab-containing regimen in the first line. The baseline patient characteristics are summarized in Table 1. The median PFS of the whole cohort was 9.1 months (95% CI = 7.9–10.8), and the median OS was 23.4 months (95% CI = 18.6–31.0). In the whole sample, 55 complete and 8 censored observations were present with respect to PFS, and 47 terminated and 17 censored observations concerning OS. Median follow-up with respect to OS was 47.6 months.

Table 1.

Baseline characteristics of patients.

Characteristic	Category	n (%)
Gender	Female	20 (31.7)
	Male	43 (68.3)
Age	<70 years	48 (76.2)
	≥70 years	15 (23.8)
Localization of primary tumour	Rectum	26 (41.3)
Localization of primary tumour	Colon	36 (57.1)
	Sigma	1 (1.6)
Line of therapy	1	48 (76.2)
	2	9 (14.3)
	3	1 (1.6)
	4	2 (3.2)
	Missing data	3 (4.8)
Resectability	Non-resectable	32 (50.8)
	Potentially resectable	11 (17.5)
	Missing data	20 (31.7)
Objective response rate	PR	23 (36.5)
Objective response rate	CR	7 (11.1)
	SD (at least 6 weeks)	14 (22.2)
	PD	11 (17.5)
	Not assessed or missing data	8 (12.7)
KRAS mutation status	Non-mutated (WT-KRAS)	17 (27.0)
	Mutated (MT-KRAS)	21 (33.3)
	Not assessed or missing data	25 (39.7)

PR: partial response; CR: complete response; SD: stable disease; PD: progressive disease.

Expression of miR-126-3p, miR-126-5p and miR-664-3p in the tumour tissue

Low tumour expression of miR-126-3p and miR-126-5p in comparison with healthy tissue was assessed in 51 (80.9%) and 53 (84.1%) patients, respectively (p < 0.0001 and p < 0.0001). Low tumour expression of miR-664-3p in comparison with healthy tissue was assessed in 23 (36.5%) patients (p = 0.0132). The expression of assessed miRs is shown in Figure 1. Relative tumour expressions of miR-126-3p and miR-126-5p were significantly and strongly correlated (Spearman’s r_s = 0.711, p < 0.0001), while only weak correlation was observed between miR-664-3p and miR-126-3p (r_s = 0.274, p = 0.0297), and no significant correlation between miR-664-3p and miR-126-5p (r_s = 0.107, p = 0.4058). The results of correlation analysis are shown in Figure 2.

Figure 1.

Expression of miR-126-3p, miR-126-5p and miR-664-3p in the tumour tissue.

Figure 2.

Correlation analysis of the expression of miR-126-3p and miR-126-5p (a), miR-126-3p and miR-664-3p (b), and miR-664-3p and miR-126-5p (c). Grey lines correspond to an expression ratio of 1, that is, no expression change between the tumour tissue and healthy tissue. Dashed line represents identity of expression ratios on both axes (y = x).

Association of miR-126-3p, miR-126-5p and miR-664-3p expression with survival

The median PFS for patients with low tumour expression of miR-126-3p was 8.8 months compared to 13.5 months for patients with high expression (p = 0.0064; Figure 3(a)). The median OS for patients with low tumour expression of miR-126 was 20.6 months, while the median OS was not reached for patients with high expression (p = 0.0027; Figure 3(d)). The median PFS and OS for patients with low tumour expression of miR-126-5p were 9.0 and 22.2 months compared to 12.0 and 23.4 months for patients with high expression (p = 0.2113 and p = 0.6858, respectively; Figure 3(b) and (e), respectively). The median PFS and OS for patients with low tumour expression of miR-664-3p were 9.1 and 22.5 months compared to 8.8 and 23.4 months for patients with high expression (p = 0.2542 and p = 0.1922, respectively; Figure 3(c) and (f), respectively). The PFS and OS data are summarized in Tables 2 –4.

Figure 3.

Progression-free survival (PFS) and overall survival (OS) according to (a and d) miR-126-3p, (b and e) miR-126-5p and (c and f) miR-664-3p expression.

Table 2.

Progression-free survival (PFS) and overall survival (OS) according to miR-126-3p expression.

	Expression of miR-126-3p in tumour		p value (log-rank test)
	< Healthy tissue (n = 51)	≥ Healthy tissue (n = 12)	p value (log-rank test)
Median PFS (95% CI)	8.8 months (7.1–10.0)	13.5 months (3.4–38.2)	0.0064
3-month PFS (95% CI)	79.8% (68.8–90.8)	78.7% (44.7–96.1)
6-month PFS (95% CI)	65.5% (52.4–78.6)	65.8% (39.0–92.6)
12-month PFS (95% CI)	24.0% (12.0–36.1)	58.1% (30.2–86.0)
Median OS (95% CI)	20.9 months (17.8–27.2)	Not reached	0.0027
12-month OS (95% CI)	81.0% (70.2–91.8)	75.5% (51.2–99.8)
24-month OS (95% CI)	40.1% (25.8–54.5)	66.1% (39.3–92.9)
36-month OS (95% CI)	24.9% (11.8–38.0)	57.6% (29.1–86.2)

CI: confidence interval.

Table 3.

Progression-free survival (PFS) and overall survival (OS) according to miR-126-5p expression.

	Expression of miR-126-5p in tumour		p value (log-rank test)
	< Healthy tissue (n = 53)	≥ Healthy tissue (n = 10)	p value (log-rank test)
Median PFS (95% CI)	9.0 months (7.9–10.4)	12.0 months (2.9–15.4)	0.2113
3-month PFS (95% CI)	80.5% (69.9–91.2)	74.5% (47.7–100)
6-month PFS (95% CI)	66.8% (54.1–79.5)	58.9% (28.5–89.4)
12-month PFS (95% CI)	27.1% (14.8–39.3)	49.7% (18.8–80.7)
Median OS (95% CI)	22.2 months (18.1–29.7)	23.4 months (6.9–36.8)	0.6858
12-month OS (95% CI)	81.7% (71.3–92.2)	70.6% (42.5–98.8)
24-month OS (95% CI)	44.9% (30.8–59.1)	49.4% (18.4–80.5)
36-month OS (95% CI)	31.5% (17.9–41.1)	37.7% (6.3–69.2)

CI: confidence interval.

Table 4.

Progression-free survival (PFS) and overall survival (OS) according to miR-664-3p expression.

	Expression of miR-664-3p in tumour		p value (log-rank test)
	< Healthy tissue (n = 23)	≥ Healthy tissue (n = 40)	p value (log-rank test)
Median PFS (95% CI)	9.1 months (5.7–11.6)	8.8 months (5.4–11.0)	0.2542
3-month PFS (95% CI)	81.8% (66.1–97.6)	79.2% (66.7–91.8)
6-month PFS (95% CI)	68.4% (49.2–87.6)	63.4% (48.5–78.3)
12-month PFS (95% CI)	24.4% (5.8–43.0)	33.9% (19.2–48.6)
Median OS (95% CI)	22.5 months (17.8–31.6)	23.4 months (17.2–38.1)	0.1922
12-month OS (95% CI)	82.9% (67.3–98.5)	78.1% (65.3–90.9)
24-month OS (95% CI)	43.3% (21.8–64.7)	46.7% (30.5–62.8)
36-month OS (95% CI)	26.0% (6.0–46.0)	35.3% (19.4–51.2)

CI: confidence interval.

Baseline clinical parameters were assessed together with miR-126-3p, miR-126-5p and miR-664-3p expression in multivariable model. The multivariable Cox proportional hazards model revealed that miR-126-3p expression was significantly associated with PFS (hazards ratio (HR) = 0.28, p = 0.0053) and also with OS (HR = 0.18, p = 0.0046; Table 5).

Table 5.

Multivariable Cox proportional hazards model for progression-free and overall survival.

Parameter	Category	Progression-free survival (p = 0.0157)		Overall survival (p = 0.0054)
Parameter	Category	HR (95% CI)	p value	HR (95% CI)	p value
miR-126-3p expression in tumour	< Healthy tissue	1	0.0053	1	0.0046
miR-126-3p expression in tumour	≥ Healthy tissue	0.28 (0.11–0.69)	0.0053	0.18 (0.05–0.59)	0.0046
miR-126-5p expression in tumour	< Healthy tissue	1	0.6329	1	0.1606
miR-126-5p expression in tumour	≥ Healthy tissue	0.81 (0.34–1.94)	0.6329	2.10 (0.75–5.90)	0.1606
miR-664-3p expression in tumour	< Healthy tissue	1	0.8633	1	0.8326
miR-664-3p expression in tumour	≥ Healthy tissue	0.95 (0.51–1.75)	0.8633	1.08 (0.52–2.23)	0.8326
Gender	Female	1	0.0316	1	0.3432
Gender	Male	2.03 (1.06–3.88)	0.0316	1.38 (0.71–2.69)	0.3432
Age	<70 years	1	0.1781	1	0.0991
Age	≥70 years	1.61 (0.80–3.22)	0.1781	1.87 (0.89–3.94)	0.0991
Line of therapy	First line	1	0.2240	1	0.0184
Line of therapy	Second or higher line	1.57 (0.76–3.29)	0.2240	2.76 (1.19–6.41)	0.0184
Localization of primary tumour	Rectum or sigma	1	0.2036	1	0.5562
Localization of primary tumour	Colon	1.46 (0.81–2.62)	0.2036	1.21 (0.64–2.28)	0.5562

HR: hazard ratio; CI: confidence interval.

Discussion

MiRs have emerged as important endogenous regulators of tumourigenesis, disease progression and metastasis in various cancers, including CRC.³⁷ They function as inhibitors of target genes activity by binding to their mRNAs and hindering their translation.³⁸ Each miR achieves functional specificity by targeting a core network of genes and has potential effects on multiple signalling pathways.³⁹ Increasing evidence suggests that aberrant miR expression is clearly associated with the initiation and progression of certain cancers, and miRs may act as oncogenes and/or tumour suppressor genes via regulation of expression of genes involved in crucial cancer-related processes including cell adhesion, proliferation, and also tumour angiogenesis.¹³ In the present retrospective study, we focused on the association of miR-126-3p, miR-126-5p and miR-664-3p expression levels with outcomes of patients with mCRC treated with bevacizumab.

Even though, the prognostic role of miR-126-3p expression in patients with CRC has been recently suggested, little is known about its possible predictive role in patients with mCRC treated with antiangiogenic therapies, including bevacizumab.^40,41 In our study, we observed a significantly shorter PFS and also OS for patients with low expression of miR-126-3p in the tumour tissue compared to those with high expression (p = 0.0064 and p = 0.0027, respectively). The multivariable Cox proportional hazards model revealed that miR-126-3p expression was significantly associated with PFS (HR = 0.28, p = 0.0053) and also with OS (HR = 0.18, p = 0.0046), being evaluated as the most significant prognostic factor with respect to both survival indicators. Similar results have been recently published by Hansen et al.,⁴² who reported significantly shorter PFS for patients with low tumour expression of miR-126-3p in a translational analysis performed on previously untreated mCRC patients enrolled in a phase III Nordic ACT trial. Based on our results, together with those reported by Hansen et al., we suggest that the aberrant activation of angiogenesis mainly mediated by VEGF in tumours with high miR-126-3p expression could promote high sensitivity to VEGF-targeted agents represented by bevacizumab.

Compared to the miR-126-3p, very little is known about the miR-126-5p, which is processed from the same precursor. To our knowledge, there is no relevant study of miR-126-5p effect on the CRC progression, and in our set of patients, we did not observe any differences among patients with opposite change of miR-126-5p relative expression. High correlation of miR-126-3p and miR-126-5p expression but different relation to clinical parameters may show the specific effects of miR-126-3p on OS and PFS, although this implication is somewhat limited by the size of our cohort.

Very little data have been published also about miR-664-3p in the field of CRC. In our study, we did not observe any difference in PFS nor OS in association with miR-664-3p expression. The results of our study did not confirm the data published by Boisen et al.,⁴³ who reported improved outcome of CAPEOX plus bevacizumab in patients with high tumour expression of miR-664 in a retrospective study.

The principal limitations of this study are the retrospective nature and limited number of patients with resulting heterogeneity, especially regarding chemotherapy backbone regimens. This study also did not include a control group not treated with bevacizumab, and therefore, it cannot be concluded with certainty whether the expression change of miR-126-3p in tumour tissue is a predictive factor exclusively for bevacizumab-based therapy or a prognostic factor associated with mCRC in general. This question should be answered in prospective randomized trials in the future. Nevertheless, it is the first study focusing on the role of miR-126-3p, miR-126-5p and miR-664-3p, all related to regulation of angiogenesis, in patients with mCRC treated with bevacizumab. In conclusion, the results of the present retrospective study suggest that the expression of miR-126-3p in the tumour tissue was significantly associated with survival of mCRC patients treated with bevacizumab-based therapy. We have not demonstrated association of expression of miR-126-5p or miR-664-3p in the tumour tissue with patients’ outcome. Prospective studies on the predictive role of miR expression profile should be performed to confirm these results.

Footnotes

Acknowledgements

The authors would like to thank all patients voluntarily taking part in the study.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.F. has received honoraria from Astra Zeneca, Roche and Novartis for consultations and lectures unrelated to this project. T.B. has received honoraria from Roche for consultations and lectures unrelated to this project. A.P. has received honoraria from GSK, Roche and Bayer for consultations and lectures unrelated to this project. O.F., P.P., P.H., V.L., J.B., O.V., O.T. and O.S. declare that they have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence this work.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Sustainability Program I (NPU I; No. LO1503) provided by the Ministry of Education, Youth and Sports of the Czech Republic and by the grant of Ministry of Health of the Czech Republic – Conceptual Development of Research Organization (Faculty Hospital in Pilsen – FNPl, 00669806).

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