Aims Integrin αv subunits are involved in tumour angiogenesis and tumour progression in various types of cancers. Clinical trials evaluating agents targeting integrin αv are ongoing. Integrin αv expression has been reported in several cancers in association with tumour progression or poor survival. However, no study has addressed the prognostic influence of integrin αv expression on survival of patients with colorectal cancer (CRC).
Methods Immunohistochemical staining of integrin αv was performed in 198 CRC samples to evaluate its prognostic significance.
Results High expression of integrin αv was observed in 58.1% (115/189) of colorectal adenocarcinoma samples, while only in 11.5% (3/26) of tubular adenoma samples and in none of normal mucosa or hyperplastic polyp samples. It was more frequently found in female patients and less frequently observed in well differentiated tumours. The proportion of cases with high expression of integrin αv showed an increasing trend with increased T stage (p=0.032), N stage (p=0.006) and TNM stage (p=0.001). Patients displaying exuberant expression of integrin αv showed shorter overall survival (p=0.001) and disease-free survival (p=0.004). Elevated integrin αv expression was an independent prognostic factor for overall survival (HR: 2.04, 95% CI 1.16 to 3.56; p=0.013) and disease-free survival (HR: 2.19, 95% CI 1.16 to 4.13; p=0.015).
Conclusions Overexpression of integrin αv is associated with advanced T and N stage and as an independent prognostic factor in CRC.
- Colorectal Cancer
- cell-adhesion molecules
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Colorectal cancer (CRC) is the second most common cause of worldwide cancer-related death in women and the third-ranked cause in men.1 Although CRC has traditionally been known to be more common in Western populations, the incidence of CRC is rapidly rising in Asian countries, such as Korea, China, Japan and Singapore.2 ,3 Therapeutic use of monoclonal antibodies as bevacizumab, cetuximab and pantimumab have shown confer survival benefits in CRC patients.4 This fact has led to evaluating new molecular targets for CRC.
Integrin encompasses a large family of αβ heterodimeric transmembrane adhesion receptors that mediate cell-to-extracellular matrix and cell-to-cell interactions.4 ,5 Integrin-mediated signals are involved in many aspects of cellular functions, which include cell proliferation, apoptosis, migration, invasion, angiogenesis and tumour progression.5–10 Integrin consists of a larger α subunit and a smaller β subunit. Twenty-four different heterodimers of integrin that involve 18 α- and eight β-subunits have been recognised.11 Among them, the integrin αv subunit binds five different β subunits (β1, β3, β5, β6 and β8) and has been linked with tumour angiogenesis and tumour progression.5 ,6 ,9 ,12 Integrin αv is expressed in a variety of human cancers,13–26 and this expression has been associated with tumour progression and poor survival.13 ,15 ,17–21 23–26 In CRC, integrin αv expression has been associated with advanced stage27 and hepatic metastasis.28 ,29 However, the prognostic influence of integrin αv expression on the survival of CRC patients is unknown.
Target agents against integrin αv have been developed and investigated in a variety of cancers, and phase I or II clinical trials are in progress.9 ,12 A role for the fully human monoclonal antibody CNTO 95 against integrin αv in the treatment of CRC was suggested by the demonstration that CNTO 95 applied in combination with dasatinib inhibits adhesion, migration and integrin signal transduction in CRC cell lines.30 While intriguing, the study was a cell-line-based in vitro study. Clinical evidence for the significance of integrin αv is required before integrin αv-based treatment of CRC can be seriously contemplated.
With this goal in mind, the present study conducted immunohistochemical staining of integrin αv in 198 CRC samples and evaluated its association with clinicopathologic parameters and its prognostic significance.
Materials and methods
Patients and tissue samples
We retrospectively collected 198 formalin-fixed and paraffin-embedded (FFPE) tumour samples from patients who had undergone curative surgical resection for primary CRCs with negative resection margin and no neoadjuvant treatment at the Samsung Medical Center, Seoul, Korea, in 2000. Clinicopathologic characteristics obtained from medical records included gender, tumour size, tumour location, histological type, differentiation grade, invasion depth, lymph node metastasis, distant metastasis, operation record, treatment record, recurrence and vital status at the last follow-up date. The median follow-up period was 111 months (range 1–136 months). The pathological tumour-node-metastasis classification was according the 2010 American Joint Committee on Cancer staging manual guidelines. Additionally, we randomly collected FFPE samples of 19 normal colorectal mucosa, 13 hyperplasic polyps and 26 tubular adenomas with low-grade dysplasia, to compare integrin αv expression patterns in these benign lesions and cancers. Normal colorectal mucosa was obtained from patients who underwent colectomy for CRC, and hyperplastic polyps and tubular adenomas were obtained from patients who had polypectomy with no cancer. This study was approved by the institutional review board of Samsung Medical Center.
Tissue microarray construction
H&E-stained tissues were reviewed to confirm the histological diagnosis and to select representative areas for immunostaining. A cylindrical core (3 mm in diameter) was removed from FFPE tissue blocks corresponding to the H&E slides to construct the tissue microarray (TMA). TMA punch was obtained at the area of mostly invading front of carcinoma. Sectioning of microarray blocks produced 4 mm-thick sections after completion of the tissue array.
Sections on microslides were deparaffinised with xylene, hydrated using a diluted alcohol series, and immersed in 0.3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Sections were treated with TE buffer (10 mM Tris and 1 mM EDTA, pH 9.2) at 98°C for 20 min. To reduce non-specific staining, each section was blocked with 4% bovine serum albumin in phosphate buffered saline containing 0.1% Tween 20 (PBST) for 30 min. The sections were incubated with a 1:50 PBST dilution of mouse monoclonal anti-integrin αv antibody (ab16821; Abcam, Cambridge, UK) containing 3 mg/mL goat globulin (Sigma-Aldrich, St Louis, Missouri, USA) for 60 min at room temperature, followed by three successive washes with buffer. Sections were then incubated with an antimouse/rabbit antibody (Envision plus; Dako, Carpinteria, California, USA) for 30 min at room temperature. The chromogen was 3,3′-diaminobenzidine (Dako). Sections were counterstained with Meyer's haematoxylin. Omitting the primary antibody provided negative controls for immunostaining.
Evaluation of results from immunohistochemical staining
The intensity of immunohistochemical staining of epithelial cells was evaluated using a previously described scoring method31 that considers staining intensity and percentage of positive cells. Staining intensity was classified as 1 (weak), 2 (moderate) and 3 (strong). Positive cells were quantified as a percentage of the total number of epithelial cells and were assigned to one of the following five categories: 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). These two numeric variables were then multiplied to generate an immunohistochemistry (IHC) score for each tumour specimen. An IHC score between 0 and 4, and between 5 and 12 indicates low expression and high expression, respectively. Each lesion was separately examined and scored by two pathologists (SHK and SYH) with no information of clinical characteristics and outcome. The pathologists discussed any discrepancies in scores until a consensus was reached.
Statistical analyses were conducted using Pearson's χ2 tests, Fisher's exact tests and Cochran–Armitage tests. Overall survival (OS) and disease-free survival (DFS) were determined using the Kaplan–Meier method, and the comparison was performed with the log-rank test. Survival was measured from the date of surgery. Nine cases with distant metastasis at the time of surgery were excluded in the analysis of DFS. The Cox proportional hazards model was used for multivariable analysis to evaluate the prognostic value of clinicopathologic factors. The HR and its 95% CI were assessed for each factor. All tests were two-sided, and p≤0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (SPSS, Chicago, Illinois, USA).
Integrin αv expression in colorectal tissue
IHC expression of integrin was mostly membranous, and some of the cases showed membranous and cytoplasmic staining. The IHC scores of integrin αv in normal mucosa, hyperplastic polyp and tubular adenoma with low-grade dysplasia and adenocarcinoma are summarised in figure 1A–E. The median IHC score of adenocarcinoma (6, range 0–12) was higher than that of normal mucosa (0, range 0–4), hyperplastic polyp (3, range 0–4) and tubular adenoma with low-grade dysplasia (2, range 0–8) (figure 1F). High expression of integrin αv expression was observed in 58.1% (115/189) of colorectal adenocarcinoma samples, 11.5% (3/26) of tubular adenoma samples and none of the normal mucosa or hyperplastic polyp samples (table 1).
Integrin αv overexpression is associated with advanced TNM stage in CRC
The associations between integrin αv expression and clinicopathologic variables are summarised in table 2. A high level of integrin αv expression was more frequently found in female patients and less frequently in well differentiated tumours. The proportion of cases with high expression of integrin αv displayed an increasing trend with increases in T stage (p=0.032), N stage (p=0.006) and TNM stage (p=0.001). Tumour recurrence and death were more frequently in the high-expression group. Patients with high expression of integrin αv received adjuvant treatment more frequently, probably reflecting its association with the advanced stage of CRC.
Integrin αv overexpression is associated with shorter survival time in CRC
Kaplan–Meier analysis with log rank ratio revealed significantly shorter OS and DFS for patients expressing high levels of integrin αv as compared with patients with low expression (p=0.001 and p=0.004, respectively; figure 2). The 5-year OS and DFS rates for patients with a high level of integrin αv were 61.7% and 62.3%, respectively, compared with 81.7% and 82.0%, respectively, for patients expressing low integrin αv. In the multivariable analysis regarding patient age, differentiation, TNM stage and integrin αv expression status as covariables, high expression of integrin αv was an independent prognostic factor for OS (HR 2.04, 95% CI 1.16 to 3.56; p=0.013) and DFS (HR 2.19, 95% CI 1.16 to 4.13; p=0.015 (table 3).
In this study, integrin αv expression was highly expressed in 58.1% (115/189) of colorectal adenocarcinoma samples, while only in 11.5% (3/26) of tubular adenoma samples, and in none of the normal mucosa or hyperplastic polyp samples. High expression of integrin αv was associated with advanced T and N stage, and was an unfavourable prognostic factor for CRC in univariate and multivariable survival analyses. Importantly, patients with high expression of integrin αv showed poorer prognosis although they more frequently received adjuvant therapy. As far as the authors are aware, this is the first study to evaluate the prognostic effect of integrin αv expression in CRC patients.
Integrin αv expression has been reported in a variety of human cancers including malignant melanoma,18 ,25 prostate cancer,13 ovary cancer,15 ,21 ,23 lung cancer,14 endometrial cancer,16 renal cell carcinoma,22 ,26 hepatocellular carcinoma,24 laryngeal squamous cell carcinoma,20 and osteosarcoma.17 An association between integrin αv expression and tumour progression and poor survival has been frequently reported.13 ,15 ,17–21 23–26
There have been only a few reports of integrin αv expression in CRCs. Denadai et al27 demonstrated that integrin αv expression was associated with advanced clinical stage and presence of venous or perineural invasion in 114 CRC patients. Bates et al32 reported that αvβ6 integrin expression was markedly increased by inducing epithelial-to-mesenchymal transition in a CRC cell-line model. The latter authors also showed that high expression of β6 integrin, which forms heterodimeric combination with only an αv integrin,5 ,9 was associated with poor prognosis in 488 CRC patients.32 The role of integrin αv in hepatic metastasis of CRC was highlighted concerning tumour cell adhesion to hepatic microvessels or tumour cell migration.28 ,29 The present data support a role of integrin αv in CRC progression. Integrin αv expression was more frequently found in adenocarcinoma than in normal colonic mucosa, hyperplastic polyp, or tubular adenoma with low-grade dysplasia; high expression of integrin αv was associated with advanced T or N stage, and was an independent unfavourable prognostic factor. These results are consistent with those of previous studies.
Integrins could be ideal pharmacological targets because of their location of cell surface, as well as their role in tumour progression.9 Target agents against integrin αv have been investigated in a variety of cancers, and phase I or II clinical trials are in progress.9 ,12 The target agents include MEDI-522 (LM609; a humanised monoclonal antibody against integrin αv β3), CNTO 95, a fully human moncoclonal antibody against integrin αv and Cilengitide, a small cylic RGD peptide against αv β3 and αv β5.9 ,12 CNTO 95 was implicated as a CRC treatment by the demonstration that CNTO 95 plus dasatinib inhibited adhesion, migration and integrin signal transduction in CRC cell lines. The collective data are leading to the validation of integrin αv as a diagnostic and therapeutic target of CRC.
In conclusion, overexpression of integrin αv is associated with advanced T and N stage, and as an independent prognostic factor. These results implicate integrin αv as a potential molecular therapeutic target in CRC.
Take home messages
Integrin αv subunits are involved in tumour angiogenesis and tumour progression in various types of cancers and target agents against integrin αv are on clinical trial. Integrin αv expression was highly expressed in 58.1% of colorectal adenocarcinoma samples and associated with advanced T and N stage, and was an independent unfavourable prognostic factor. These results implicate integrin αv as a potential molecular therapeutic target in colorectal cancers.
Abstract in Korean
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SYH and JS contributed equally.
Contributors S-HK contributed to study design. JS performed the experiments. SYH and S-HK analysed the data. SYH, JS and S-HK wrote the manusript, and all authors revised it critically. All authors read and approved the manuscript.
Funding This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. NRF-2013R1A1A2063277) and by the Samsung Biomedical Research Institute (Grant SS1B30131).
Competing interests None.
Ethics approval Institutional review board of Samsung Medical Center.
Provenance and peer review Not commissioned; externally peer reviewed.