Subjects and samples
We identified endoscopic biopsy samples from ileocolic lesion of 83 patients with CD and 372 controls by searching pathological report files at Chonnam National University Hospital between Jan 2018 and June 2019. All 372 control group subjects had no significant pathological findings and no clinical history of inflammatory bowel disease or neoplasia. Among 83 CD patients and 372 controls, 43 of each group were selected through propensity score matching to reduce the effects of age and sex.
Histopathologic assessment of inflammatory activities and Immunohistochemistry of Cyr61
To evaluate the histopathologic assessment of inflammatory activities of biopsy specimens in patients with CD, we used the pathologic scoring system suggested by Naini et al [9] including ileitis score (0-10) and colitis score (0-17)
Cyr61 protein expression in mucosal tissues was evaluated by immunohistochemistry (IHC). Briefly, formalin-fixed paraffin-embedded (FFPE) blocks were cut at 3-μm thickness and immunostained with a specific antibody against Cyr61 protein (1:1500 dilution; catalog no. ab10760; Abcam, Cambridge, UK) using an automated immunostainer (Bond-maX DC2002; Leica Biosystems, Bannockburn, IL, USA). For antigen retrieval, programmed heat-induced epitope retrieval was carried out using bond epitope retrieval solution 1 (containing citrate buffer at pH 6.0) for 15 min. IHC slides were assessed by two experienced pathologists (JHL and KHL), who were blinded to the clinical details. Immunohistochemical staining was re-evaluated for cases showing disagreement between observers. Two pathologists reviewed the cases together and then reached an agreement for inconclusive samples.
The intensity of cytoplasmic immunoreactivity was initially classified into four grades: no staining, weak positivity, moderate positivity, and strong positivity. No cases were completely immunonegative for Cyr61. Cases with weak staining intensity were categorized as ‘low-expression’ and those with moderate or strong staining intensity were considered as ‘high-expression’. The proportion of the stained area was estimated by the ratio of positively stained area over the whole area and expressed as a percentage. IHC scores for Cyr61 expression were calculated by multiplying the intensity grade scale by the stained area percentage.[10]
RNA isolation and real-time polymerase chain reaction of pro-inflammatory genes and Cyr61
In our previous study, we compared the mRNA levels of proinflammatory genes in the colonic paired mucosal biopsy specimens (inflamed and noninflamed lesions) from each 32 patient with CD.[11] In this study, we used the RNA remaining from those samples to evaluate the degree of inflammation and Cyr61 expression in paired samples from each patient. As we did not have enough amount of RNA in 21 patients, we could analyze the mRNA levels from only 11 patients with CD. Total RNA was extracted using Trizol (Takara, Tokyo, Japan). Briefly, 1 mL of Trizol solution was added into each well, and then the suspension was collected into a 1.5 mL tube. After adding 200 μL of chloroform (Sigma-Aldrich, St. Louis, MO, USA) and vortexing for 15 secs, the mixture was centrifuged at 20,000 x g for 20 min. The supernatant was then collected and mixed with equal amounts of isopropyl alcohol (MERCK, Kenilworth, NJ, USA) followed by centrifugation at 20,000 x g rpm for 20 min. The pellet was washed with 1 mL of 70% ethyl alcohol (MERCK) and centrifuged at 20,000 x g for 5 min. After removing the remaining ethyl alcohol, the RNA pellet was air dried at room temperature and then suspended in 50 μL of diethyl pyrocarbonate water.
Outcomes
We retrieved the clinical disease activities score such as Crohn’s disease activity index (CDAI), which was calculated and written on the chart by colonoscopists (KHS and KDH) from the collection of daily status for past 7 days around the time of colonoscopy. We also retrieved endoscopic disease activities scores such as simple endoscopic score of Crohn’s disease (SES-CD) scale and Crohn’s disease endoscopic index of severity (CDEIS) from the medical records, which were calculated by experienced colonoscopists (KHS and KDH) around the time of colonoscopy.
We also reviewed the clinical course of patients in medical records after acquiring biopsy specimens. We defined clinical recurrence as a change in prescription, bowel resection, fistulotomy, strictureplasty, stoma formation, CD-related hospitalization, or flare during the follow-up period.[11] CD-related hospitalization was defined as hospitalization because of complications including the following: CD-related surgery, hospitalization for nonsurgical CD-related events such as CD-related flares, hospitalization related to complications/extraintestinal manifestation of CD, and disease flare.
Statistical analysis
Results are expressed as mean (standard deviation, SD) or median value (range). Continuous variables in 2 groups were compared using Student’s t-test or the Mann-Whitney test. IHC scores for Cyr61 expression in patients with CD were divided into tertiles: 1st, <60 (n=27); 2nd, 60–80 (n=30) and 3rd, ≥80 (n=26). We compared the clinical disease activities such as CDAI, SES-CD and CD-EIS according to tertiles of IHC score for Cyr 61 expression using Kruskall Wallis test. The association between Cyr61 expression and clinical recurrence was assessed using univariate and multivariable binary logistic regression. Odd ratio (OR) and P value are presented. Tests for trend were performed using the Cyr61 expression tertiles as ordinal variables in the corresponding logistic regression models. All statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Two-sided P values<0.05 were considered statistically significant.
Ethical consideration
The study protocol was approved by the Ethics Committee of Chonnam National University Hospital (IRB No. CNUH-2020-121) and was conducted according to the Declaration of Helsinki and Good Clinical Practice guidelines.