Human Pathology (2011) 42, 1401–1409 www.elsevier.com/locate/humpath Original contribution Evaluation of REG4 for early diagnosis and prognosis of gastric cancer☆ Hou-Quan Tao MD a,⁎, Xu-Jun He MB a, Ying-Yu Ma MS a, Hui-Ju Wang MS a, Ying-Jie Xia MB a, Zai-Yuan Ye MS a, Zhong-Sheng Zhao MS b aKey Laboratory of Gastroenterology of Zhejiang Province, Hangzhou, 310014, China bDepartment of Pathology, Zhejiang Provincial People's Hospital, Hangzhou, 310014, China Received 2 April 2010; revised 30 July 2010; accepted 3 August 2010 Keywords: Summary We explored the correlation between the development of gastric cancer and the concentration REG4; of REG4 and hence the suitability of REG4 as an indicator of the prognosis of patients with GC. Real- Gastric cancer time polymerase chain reaction was conducted to detect REG4 messenger RNA expression. The amount of the REG4 protein was measured by immunohistochemistry staining of tissue and enzyme-linked immunosorbent assay of serum. Serum carcinoembryonic antigen and carbohydrate antigen 19-9 concentrations were measured using a commercial automated immunoassay. Real-time polymerase chain reaction results confirmed that REG4 was significantly up-regulated in gastric cancer compared with paired normal mucosa (P b .001). Immunohistochemistry staining revealed that high expression of REG4 correlated with diffuse type, poor differentiation, lymph node metastasis, distant metastasis, and TNM stage III or IV. The mean survival time for patients in the REG4-positive group was significantly less than that in the REG4-negative group (P = .013). The percentage of serum samples that were REG4 positive was 44.0%, which was higher than that for serum carcinoembryonic antigen (P = .039) or carbohydrate antigen 19-9 (P = .012) in TNM stage I and was significantly higher (P = .031) than that in TNM stage II. Thus, REG4 may be not only a prognostic indicator but also a better serum marker than carcinoembryonic antigen and carbohydrate antigen 19-9 for early diagnosis of gastric cancer. © 2011 Elsevier Inc. All rights reserved. 1. Introduction habits. GC in China constitutes approximately 33% of all worldwide GC cases [1]. As in all cancers, early detection Gastric cancer (GC) is one of the most common human remains the most promising approach to improve the long- cancers and is affected by environmental and behavioral term survival rate. Assessment of tumor markers in serum may be useful for this purpose. There are 2 available tumor markers for GC, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). However, neither is ☆ This work was carried out with the support of grants from Zhejiang an appropriate option for early screening, because preoper- Provincial Program for the Cultivation of High-Level Innovative Health ative positivity for these markers is highly reliant on tumor Talents and the Natural Science Foundation of Zhejiang Province (Grant No. Y207763). stage at the time of detection [2]. Establishment of screening ⁎ Corresponding author. strategies through the development of novel serum markers E-mail address: [email protected] (H. -Q. Tao). that are more specific and more sensitive to GC is thus 0046-8177/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2010.08.023 1402 H. -Q. Tao et al. urgently needed. Genes encoding transmembrane/secretory 2. Materials and methods proteins expressed specifically in cancers may be ideal diagnostic biomarkers [3]. The REG gene family belongs to 2.1. Blood Samples and isolation of serum the calcium-dependent lectin gene superfamily, which is mainly involved in hepatic, pancreatic, gastric, and intestinal Blood samples were obtained from 47 male and 23 female cell proliferation or differentiation. healthy donors (22-68 years old) and 65 male and 27 female The human REG gene family consists of 4 secretory and GC patients (32-86 years old) who were inpatients at the structurally unique proteins that are expressed in different Zhejiang Provincial People's Hospital between January and tissues and play different biological roles [4-6]. REG4,a June 2008. Samples were centrifuged for 10 minutes at member of the REG gene family, which was originally 1000 × g in a swing bucket rotor at 4°C, and the serum was identified by high-throughput sequencing of a complemen- stored in cryovials at −80°C until analysis. Tumor staging tary DNA (cDNA) library derived from a patient with was conducted according to the TNM system [19]. inflammatory bowel disease, maps to chromosome 1 and encodes a 158-amino-acid protein that includes a signal peptide of 22 amino acids and a conserved calcium- 2.2. Tissue samples dependent recognition domain [7]. Although various normal tissues express REG4, the concentrations in normal tissues One hundred ninety-two paraffin specimens of GC (138 are much lower than those in cancerous tissues [8]. In tumor male and 54 female patients aged 29-82 years) were acquired tissues of the gastrointestinal tract and pancreas or in cases in from Zhejiang Provincial People's Hospital, having been which these tissues are inflamed, REG4 can be seen to be collected from January 2000 to December 2001. All cases overexpressed in situ or in ectopias [9]. In colorectal were classified according to the World Health Organization carcinoma (CRC), up-regulation of REG4 was associated Pathological Classification of Tumors. There were 68 cases with tumor differentiation, stage, and lymph node metastasis of well or moderately differentiated tumors and 124 cases of [8,10]. High-throughput analysis showed that REG4 was poor or no differentiation. Histologically, there were 12 cases overexpressed in GC, CRC, pancreatic cancer, and prostate of papillary adenocarcinoma, 112 cases of tubular adeno- cancer but not in lung and breast cancer, suggesting that carcinoma, 58 cases of mucinous adenocarcinoma, and 10 REG4 may be tissue-specific and serve as a marker for cases of signet-ring cell carcinoma. There were 76 cases of tumors of the digestive system and prostate [11-14]. The intestinal histologic type and 116 cases of diffuse histologic REG4 protein is a growth promoter and may function as an type according to the Lauren classification. In 102 cases, antiapoptotic factor early in the development of GC [15,16]. there were lymph node metastases, whereas in 90 cases, no In addition, REG4 is overexpressed in prostate cancer, where such metastases were apparent. Eighteen cases were TNM it is associated with progression and metastasis of hormone- stage I, 45 cases were stage II, 62 cases were stage III, and 67 resistant disease [17]. cases were stage IV. None of the patients had received any Quantitative real-time reverse transcriptase-polymerase radiotherapy or chemotherapy in advance of the operation. chain reaction (PCR) has been used to investigate REG4 Specimens were fixed in formalin and embedded in paraffin. messenger RNA (mRNA) expression in GC cell lines and All patients were followed for more than 5 years. tissues. Results indicate that REG4 is highly overexpressed Thirty-seven fresh specimens from patients with GC were in GC cell lines and tissues established from peritoneal acquired from Zhejiang Provincial People's Hospital from − dissemination, and the quantity of REG4 correlated with wall January 2008 to December 2008 and stored at 80°C until penetration, suggesting that REG4 is involved in the use to detect relative REG4 mRNA expression by real-time peritoneal dissemination of GCs and may be a novel marker PCR. Surrounding normal gastric mucosa also was obtained for such disease [14,18]. and studied. Early detection remains the most promising approach to The project was approved by the ethics committee of improving the long-term survival of patients with GC, and Zhejiang Provincial People's Hospital. serum markers seem to play important roles in its diagnosis. REG4, a secretory protein, could be an alternative serum 2.3. REG4 mRNA expression analysis marker. Few researchers have studied the correlation between REG4 expression and clinicopathologic features Total RNA was isolated with Trizol (Invitrogen, Carls- of GC. In this study, we used immunohistochemical staining bad, CA). A total of 2 μg RNA was reverse transcribed using and enzyme-linked immunosorbent assays (ELISAs) to the SuperScript II RNase-Reverse Transcriptase System determine REG4 expression in sera and tissue and also (Invitrogen). The cDNA was then subjected to real-time PCR investigated the serum concentrations of CEA and CA19-9 with primers specific for REG4. A 0.4-μL aliquot of each using a commercially available automated immunoassay. cDNA and 0.4 μmol/L of forward and reverse primers were The correlation between REG4 expression and tumor added to SYBR Premix ExTaq (Perfect Real Time; TaKaRa invasion and metastasis and the significance of serum Bio Inc, Otsu, Japan) in a total volume of 25 μL. The REG4 concentrations were analyzed. amplification protocol was as follows: denaturation at 95°C REG4 in GC diagnosis and prognosis 1403 for 4 minutes and 40 cycles of 95°C for 10 seconds, 55°C for was added to a 96-well plate that had been coated with REG4 20 seconds, and 72°C for 20 seconds. The sequence of the antibody and incubated at 37°C for 2 hours. Then, 100 μLof forward primer for REG4 was 5′-GCC CTT AGA GTC TTG biotin-antibody working solution was added to each well, and GTT GC-3′ and that of the reverse primer was 5′-GCC ATC the plate was incubated at 37°C for 1 hour, followed by TTC CTC CTA CCC TG-3′. The sequence of the primers addition of 100 μL of horseradish peroxidase–avidin used for the control GAPDH was 5′- TGA AGG TCG GAG working solution. After incubation at 37°C for 1 hour, the TCA ACG G-3′ (forward) and 5′-CTG GAA GAT GGT contents of each well were aspirated and replaced with 90 μL GAT GGG ATT-3′ (reverse; Invitrogen).