Overexpression of FIBCD1 Is Predictive of Poor Prognosis in Gastric Cancer

Chunyi Jiang, MA; Jinhong Zhu, MD; Pengcheng Zhou, MA; Huijun Zhu; Wei Wang, MA; Qin Jin, MA; Peng Li, MD

Disclosures

Am J Clin Pathol. 2018;149(6):474-48. 

In This Article

Materials and Methods

Tumor Tissues and Clinical Data

Gastric cancer samples were collected in the biological sample bank of the Affiliated Hospital of Nantong University from June 2004 to July 2009. Tumor tissues obtained from surgery were fixed in formalin and embedded in paraffin and then examined by at least two pathologists (C.J. and W.W.). This study included 520 gastric cancer samples, 24 chronic gastritis tissues, 46 intestinal metaplasia tissues, 33 low-grade intraepithelial neoplasia tissues, 24 high-grade intraepithelial neoplasia tissues, and 59 surgical margins from patients with gastric cancer. The clinical data of all patients were recorded in detail, including age, sex, tumor location, tumor size, preoperative serum carcinoembryonic antigen (CEA) level, preoperative serum CA19-9 level, human epidermal growth factor receptor 2 (HER-2) level, tumor invasion depth, lymph node metastasis, tumor differentiation, and TNM stage. None of the patients received chemotherapy, radiotherapy, or immunotherapy before surgery. Written informed consent for publication of this study and any accompanying images were obtained from the patients. The study protocol was approved by the Ethics Committee of the Human Research Ethics Committee of the Affiliated Hospital of Nantong University, and all experiments were performed in accordance with approved guidelines of the Affiliated Hospital of Nantong University.

qRT-PCR

Total RNA was isolated from 27 pairs of fresh-frozen tissues. The 27 pairs of tumor tissue and benign surgical marginal tissue were diagnosed definitely by frozen section in 2016. Total RNA was isolated and purified from these fresh-frozen tissues using the RNeasy Mini Kit and QiaShredders (Qiagen, Sussex, UK). Complementary DNA was generated from total RNA using a reverse transcription kit (Fermentas, Foster City, CA) according to the manufacturer's instructions. Quantitative polymerase chain reaction (PCR) was performed on a Biorad iCycler (Biorad, Gladesville, Australia). The primers for FIBCD1 were as follows: forward primer (5′-GTGTGGGGTTCCGTTCTC-3′) and reverse primer (5′-CCAGTGGTGCCAAGTCAA-3′). 18S ribosomal RNA (rRNA) (Life, Carlsbad, CA) served as an endogenous control. Amplification conditions were 6 minutes at 95°C for denaturation followed by 35 cycles of 30 seconds at 95°C and 1 minute at 60°C.

Tissue Microarray Construction and Immunohistochemistry Analysis

TMA-IHC was performed to determine FIBCD1 protein expression in 706 archived gastric tissue blocks, including 520 gastric cancer tissues, 24 chronic gastritis tissues, 46 intestinal metaplasia tissues, 33 low-grade intraepithelial neoplasia tissues, 24 high-grade intraepithelial neoplasia tissues, and 59 surgical margins in gastric cancer. Core tissue biopsy specimens (2 mm in diameter) taken from individual paraffin-embedded sections were arrayed in new recipient paraffin blocks by using a tissue microarray system (Quick-Ray, UT06; UNITMA, Seoul, Korea). Sections from arrayed blocks were sliced into 4-μm sections and placed on Superfrost-charged glass microscope slides. Sections were stained with a polyclonal rabbit anti-FIBCD1 antibody (dilution 1:100) (Atlas Antibodies, Bromma, Sweden; HPA053898) at 4°C overnight, followed by incubation with biotinylated anti-rabbit antibody as a secondary antibody at room temperature for 2 hours.

Positive staining of FIBCD1 was localized predominantly in the nuclei of gastric cells Image 1. However, two chronic gastritis tissues and three intestinal metaplasia tissues showed positive staining of FIBCD1, which localized in the cytoplasm and cytomembrane. But this staining did not achieve statistical significance; therefore, we only discussed tissues with positive staining in nuclei. The staining intensity and percentage scores of FIBCD1 for the core on each chip were evaluated by at least two pathologists (C.J. and W.W.). Staining intensity was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The positive rate of FIBCD1 was scored, ranging from 0 to 100. The product of the intensity score and the percentage were calculated, ranging from 0 to 300, and defined as the final immunohistochemistry (IHC) score. The cutoff value for the FIBCD1 expression data was set by the X-tile software program (Rimm Laboratory, Yale University; http://www.tissuearray.org/rimmlab).[14] The optimal cutoff value of FIBCD1 protein expression was identified based on the maximum χ 2 value estimated by log-rank χ 2 statistics with regard to overall survival. We chose 150 as the cutoff point: a score of 0 to 150 was regarded as low expression while 150 to 300 was regarded as high expression.

Image 1.

Expression of fibrinogen C domain containing 1 (FIBCD1) in gastric benign and malignant tissues detected by an immunohistochemical technique. A, B, Negative FIBCD1 immunohistochemical reaction in chronic gastritis tissue. Arrows indicate negative FIBCD1 staining in chronic gastritis. C, D, Weakly positive expression of FIBCD1 in intestinal metaplasia. Arrow indicates weakly positive FIBCD1 staining in the cytoplasm and cytomembrane of intestinal metaplasia cells. E, F, Weakly positive expression of FIBCD1 in dysplasia (staining intensity: 2; percentage scores: 35%). Arrow indicates positive FIBCD1 staining in the nucleus of dysplasia gastric cancer cells. G, H, Positive expression of FIBCD1 in well-differentiated gastric cancer (staining intensity: 3; percentage scores: 75%). Arrow indicates positive FIBCD1 staining in the nucleus of dysplasia gastric cancer cells. I, J, Positive expression of FIBCD1 in poorly differentiated gastric cancer (staining intensity: 3; percentage scores: 90%). Arrow indicates positive FIBCD1 staining in the nucleus of dysplasia gastric cancer cells. Framed material in low-resolution images (×40) is shown in corresponding high-resolution images (×400).

Bioinformatics Analysis

We further explored the effect of FIBCD1 messenger RNA (mRNA) expression on survival of patients with gastric cancer. The prognostic implication of FIBCD1 was estimated using Kaplan-Meier plotter (KM plotter), an online survival analysis tool. The KM plotter includes transcriptomic data of 1,065 patients with gastric cancer (54,675 genes), with a mean follow-up period of 33 months.[15]

Statistical Analysis

qRT-PCR data were compared between two groups using the Student t test. The correlation between the expression of FIBCD1 and clinicopathologic features was calculated with χ 2 tests. We used univariate and multivariate Cox regression models to determine significant prognostic factors. The Kaplan-Meier method was used to perform survival analysis. For all analyses, a P value less than .05 was regarded as significant. Data analysis was performed using SPSS 20 software package and STATA 12.0 (SPSS, Chicago, IL).

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....