Journal of Hainan Medical University. Qing-Hao Gong, Yi-Ting Cai, Hai-Qun Chen, Chao-Feng Zhang, Gang Dai, Song-Ming Zhu. 1.

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1 110 Journal of Hainan Medical University 2016; 22(16): Journal of Hainan Medical University Relationship of serum HMGB1 and sentinel lymph node detection with tumor marker levels and malignant molecule expression levels in tumor tissue of gastric cancer patients Qing-Hao Gong, Yi-Ting Cai, Hai-Qun Chen, Chao-Feng Zhang, Gang Dai, Song-Ming Zhu Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine Chongming Branch, Shanghai, ARTICLE INFO Article history: Received 9 Jul 2016 Received in revised form 19 Jul 2016 Accepted 15 Jul 2016 Available online 24 Jul 2016 Keywords: Ggastric cancer High mobility group box 1 Sentinel lymph node biopsy Tumor marker ABSTRACT Objective: To study the relationship of serum HMGB1 and sentinel lymph node detection with tumor marker levels and malignant molecule expression levels in tumor tissue of gastric cancer patients. Methods: Patients with early gastric cancer were selected as pathology group, healthy volunteers were selected as control group, serum HMGB1, CA72-4, DDK1, TK1, exosome, PG-I and PG-II levels were determined and PGR percentage was calculated, pathology group received intraoperative sentinel lymph node localization and biopsy, tumor tissue was collected and the expression levels of malignant molecules were determined. Results: Serum HMBG1, CA72-4, DDK1, TK1 and exosome levels of pathology group were higher than those of control group, and PGR percentage was lower than that of control group; the higher the serum HMBG1 level in gastric cancer patients, the higher the CA72-4, DDK1, TK1 and exosome levels and the lower the PGR percentage in serum, and the higher the Survivin protein levels and the lower the PTEN, p21, Caspase-3 and Caspase-7 levels in tumor tissue; CA72-4, DDK1, TK1 and exosome levels in serum and Survivin protein level in tumor tissue of patients with SLNS(+) gastric cancer were significantly higher than those of patients with SLNS(-) gastric cancer, and PGR percentage in serum and PTEN, p21, Caspase-3 and Caspase-7 protein levels in tumor tissue were significantly lower than those of patients with SLNS(-) gastric cancer. Conclusion: Serum HMGB1 and sentinel lymph node detection in gastric cancer patients can early assess tumor malignancy and lymph node metastasis. 1. Introduction The global morbidity and mortality of gastric cancer in common malignant tumors rank the fourth and the second respectively, and the main metastatic ways include lymph node metastasis and hematogenous metastasis [1]. Clinical tumor recurrence and metastasis are usually developed from micrometastases. The micrometastases of gastric cancer often exist in the form of single cell or small cell cluster, they can be in G0 phase for a long time or the cell proliferation and apoptosis are in balance for a long time, and only when the body is hit or immunity is weakened, can dormant tumor cells get rid of the inhibited state, continue to proliferate and develop into clinical explicit metastases[2,3]. High mobility group box-1 protein (HMGB1) is a multifunctional cytokine related to cellular movement and tumor infiltration [4]. Sentinel lymph node (SLN) refers to the first (the first stop) lymph node accepting the lymphatic drainage of primary tumor in regional lymph nodes [5]. At present, the change of serum HMGB1 content and the value of sentinel lymph node detection in early gastric cancer patients are not yet clear, and in the following study, the relationship of serum HMGB1 and sentinel lymph node detection with tumor marker levels and malignant molecule expression levels in tumor tissue of gastric cancer patients was analyzed. Corresponding author: Zhu Song-Ming, Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine Chongming Branch, Shanghai, Tel: Fund Project: Funded by Youth Research Project of Shanghai Municipal Health Bureau in 2013 No: 20134Y Subjects and methods

2 Research subjects The following standard was referred to select the research subjects of pathology group, and time range was from January 2014 to December Inclusion criteria: (1) diagnosed with early gastric cancer through histopathology, and after implementing D2 or extended radical surgery, R0 resection (resection-edge tumor cells were negative); (2) without previous history of radiotherapy treatment; (3) without evidence of liver, peritoneum or distant metastasis, without tumor cells in the ascites by cytological analysis and test; (4) without history of upper abdominal surgery; (5) benign gastric disease and healthy subjects were without acute infection or inflammation, and without history of malignant tumor; (6) signed informed consent. Exclusion criteria: (1) with obvious infection or inflammation (temperature of fever more than 38 ); (2) with more than one kind of tumor at the same time, but not including cured carcinoma in situ or skin cancer; (3) in active phase of hepatitis and cirrhosis of the liver; (4) with severe heart disease requiring hospitalization, or with previous history of such heart disease; (5) with severe complications requiring hospitalization (intestinal paralysis, intestinal obstruction, interstitial pneumonia or pulmonary fibrosis, severe diabetes, kidney failure, liver disease or cirrhosis); (6) clinical suspected liver, peritoneum or any distant metastasis; (7) received preoperative chemoradiation therapy; (8) with history of upper abdominal surgery; (9) with other conditions that researchers believed that they should not be tested. 48 patients were included, they were (46.2±7.3) years old, 29 cases were male and 19 cases were female. 50 cases of healthy volunteers who received physical examination in our hospital during the same period were selected as the research subjects of control group, they were (44.3±6.8) years old, 30 cases were male and 20 cases were female Serum index collection and detection 5ml of venous blood was collected from pathology group before operation, 5ml of venous blood was collected from control group during physical examination, both received EDTA anticoagulation and were centrifuged to separate serum, and enzyme-linked immunosorbent kit was used to determine serum high mobility group box-1 protein (HMGB1) levels Gastric cancer tissue collection and detection After intraoperative resection of the tumor tissue, moderate amount of tumor tissue was collected, washed with saline for three times and then stored at -80 ; for test, tumor specimens were taken out and weighed with electronic scales, about mg tissue was collected and added in PBS 300 μl for homogenate, the homogenate was placed in 4 centrifuge and centrifuged for 20 min at a speed of r/min, the sediment was discarded and the supernatant was kept, enzyme-linked immunosorbent kit was used to determine PTEN, Survivin, p21, Caspase-3, and Caspase-7 concentration in homogenate supernatant, BCA kit was used to determine total protein concentration in homogenate supernatant, and the formula, target protein concentration/total protein concentration was referred to calculate specific target protein content per mg total protein Statistical methods SPSS 20.0 software was used to input data, measurement data analysis between two groups was by t test, measurement data analysis among three groups was by variance analysis and P<0.05 indicated statistical significance in differences Sentinel lymph node localization and biopsy Gastric cancer patients received exploratory laparotomy to make sure there was no adjacent organ invasion or distant metastasis, four points were chosen around the primary tumors, 1 ml syringe was used for subserous injection of 1% methylene blue, ml for each point and total amount about ml. After the injection, injection points were pressed for a moment in case that the dye spilled over and polluted the vision. Within 1-4 min, 1 to several blue lymph vessels might extend from local tumor to the distant, and blue lymph nodes could be found following the blue lymph vessels. 1 to several lymph nodes stained at first were regarded as SLN of gastric cancer and removed separately, and then D2 or D3 radical operation for gastric cancer was conducted. Pathologic examination of SLN was conducted through postoperative conventional serial section HE in order to accurately judge the lymph node metastasis and divide it into positive lymph node metastasis [SLN(+)] and negative lymph node metastasis [SLN(-)]. 3. Results 3.1. Serum parameters of pathology group and control group Serum HMBG1 level (29.4±4.1 vs. 5.5±0.8 ng/ml), CA72-4 level (13.1±1.8 vs. 3.2±0.6 U/mL), DDK1 level (78.1±10.2 vs. 24.7±4.2 ng/ml), TK1 level (126.6±18.5 vs. 54.8±7.2 pg/ml) and exosome level (1.52±0.23 vs. 0.67±0.09 pg/ml) of pathology group were significantly higher than those of control group, and PGR percentage (3.61±0.52 vs. 6.85±0.94) was significantly lower than that of control group Serum tumor marker levels in gastric cancer patients with different serum HMGB1 levels Serum CA72-4, DDK1, TK1 and exosome levels and PGR percentage were different among gastric cancer patients with

3 112 Table 1. Comparison of serum parameters between pathology group and control group. group HMGB1(ng/mL) CA72-4(U/mL) DDK1(ng/mL) TK1(pg/mL) Exosome(pg/mL) PGR Pathology 29.4± ± ± ± ± ±0.52 Control 5.5± ± ± ± ± ±0.94 T <0.05 Table 2 Serum tumor marker levels in gastric cancer patients with different serum HMGB1 levels. CA72-4(U/mL) DDK1(ng/mL) TK1(pg/mL) Exosome(pg/mL) PGR Low concentration of HMGB1( ng/ml) 7.3± ± ± ± ±0.81 Median concentration of HMGB1 ( ng/ml) 12.7±1.6a 73.1±9.4a 115.1±15.3a 1.41±0.18a 3.87±0.49a High concentration of HMGB1 ( ng/ml) 17.8±2.4ab 121.3±17.8ab 187.6±25.1ab 2.19±0.30ab 2.21±0.33ab F a: compared with gastric cancer patients with low concentration of HMGB1, P<0.05; b: compared with gastric cancer patients with median concentration of HMGB1, P<0.05. different serum HMGB1 levels, and pair-wise comparison by LSD-t test showed that serum CA72-4, DDK1, TK1 and exosome levels in gastric cancer patients with high and median concentration of HMGB1 were higher than those in gastric cancer patients with low concentration of HMGB1, and PGR percentage was lower than that in gastric cancer patients with low concentration of HMGB1; serum CA72-4, DDK1, TK1 and exosome levels in gastric cancer patients with high concentration of HMGB1 were higher than those in gastric cancer patients with median concentration of HMGB1, and PGR percentage was lower than that in gastric cancer patients with median concentration of HMGB Malignant molecule expression levels in tumor tissue of gastric cancer patients with different serum HMGB1 levels PTEN, Survivin, p21, Caspase-3 and Caspase-7 protein levels in tumor tissue were different among gastric cancer patients with different serum HMGB1 levels, and pair-wise comparison by LSD-t test showed that Survivin protein levels in tumor tissue of gastric cancer patients with high and median concentration of HMGB1 were higher than those of gastric cancer patients with low concentration of HMGB1, and PTEN, p21, Caspase-3 and Caspase-7 protein levels were lower than those of gastric cancer patients with low concentration of HMGB1; Survivin protein level in tumor tissue of gastric cancer patients with high concentration of HMGB1 was higher than that of gastric cancer patients with median concentration Table 3. Malignant molecule expression levels in tumor tissue of gastric cancer patients with different serum HMGB1 levels. PTEN(ng/mg pro) Survivin(ng/mg pro) p21(ng/mg pro) Caspase-3(ng/mg pro) Caspase-7(ng/mg pro) Low concentration of HMGB1 ( ng/ml) 38.5± ± ± ± ±3.5 Median concentration of HMGB1 ( ng/ml) 24.1±4.6 a 45.6±7.3 a 30.4±4.7 a 11.3±1.6 a 16.7±3.0 a High concentration of HMGB1 ( ng/ml) 18.5±2.4 ab 77.5±10.3 ab 17.6±3.2 ab 7.5±0.9 ab 11.4±2.1 ab F a: compared with gastric cancer patients with low concentration of HMGB1, P<0.05; b: compared with gastric cancer patients with median concentration of HMGB1, P<0.05. Table 4. Serum tumor marker levels in tumor tissue of gastric cancer patients with different SLN metastasis. CA72-4(U/mL) DDK1(ng/mL) TK1(pg/mL) Exosome(pg/mL) PGR SLN(+) 20.8± ± ± ± ±0.36 SLN(-) 7.9± ± ± ± ±0.71 F Table 5. Malignant molecule expression levels in tumor tissue of gastric cancer patients with different SLN metastasis. PTEN(ng/mg pro) Survivin(ng/mg pro) p21(ng/mg pro) Caspase-3(ng/mg pro) Caspase-7(ng/mg pro) SLN(+) 16.7± ± ± ± ±1.9 SLN(-) 40.2± ± ± ± ±3.2 F

4 113 of HMGB1, and PTEN, p21, Caspase-3 and Caspase-7 protein levels were lower than those of gastric cancer patients with median concentration of HMGB Serum tumor marker levels and malignant molecule expression levels in tumor tissue of gastric cancer patients with different SLN metastasis CA72-4, DDK1, TK1 and exosome levels in serum of patients with SLNS(+) gastric cancer were significantly higher than those of patients with SLNS(-) gastric cancer, and PGR percentage was significantly lower than that of patients with SLNS(-) gastric cancer, shown in Table 4. Survivin protein level in tumor tissue of patients with SLNS(+) gastric cancer was significantly higher than that of patients with SLNS(-) gastric cancer, and PTEN, p21, Caspase-3 and Caspase-7 protein levels were significantly lower than those of patients with SLNS(-) gastric cancer, shown in Table Discussion Gastric cancer is one of the most common malignant tumors, and its incidence ranks top among the digestive tract tumors. Gastric cancer recurrence and metastasis are the main causes of poor prognosis in patients, and the tumor recurrence and metastasis are mainly developed from micrometastases. There are a variety of metastatic ways of gastric cancer, among which lymph node metastasis and hematogenous metastasis are the most common[6,7]. However, it is very difficult to evaluate whether there has been metastasis or micrometastasis in gastric cancer. Serum tumor markers are the commonly used indicators for clinical evaluation of the tumor malignancy, but the specificity is relatively poor and they are unable to evaluate metastatic conditions. CA72-4, DDK1, TK1 and exosome levels and PGR percentage are common clinical gastric cancer markers [8-10], and in the study, analysis of above markers in patients with early gastric cancer showed that serum CA72-4, DDK1, TK1 and exosome levels in patients with early gastric cancer were significantly higher than those of healthy subjects, and PGR percentage was lower than that of healthy subjects. High mobility group box-1 protein (HMGB1) is a highly conserved nucleoprotein, is a nonhistone chromosomal protein in eukaryotic cell nuclei, is a multifunctional cytokine, plays an important role in maintaining nucleosome stability, DNA recombination, replication and repair as well as gene transcription, and is also related to cell movement and tumor infiltration[11,12]. In the study, analysis of serum HMGB1 levels in patients with early gastric cancer showed that serum HMGB1 level of pathology group was significantly higher than that of control group. This indicated that HMGB1 levels changed in the early stage of gastric cancer development, which might participate in the occurrence and development of gastric cancer. In order to further make clear the relationship between HMGB1 and the malignant degree of gastric cancer, the relationship between different HMGB1 levels and serum tumor marker levels was analyzed in the study, and results showed that the higher the serum HMBG1 levels in patients with early gastric cancer, the higher the serum tumor markers CA72-4, DDK1, TK1 and exosome levels and the lower the PGR percentage. It confirmed that HMGB1 levels could assess the malignant degree of tumor. Sentinel lymph node (SLN) refers to the first (the first stop) lymph node accepting the lymphatic drainage of primary tumor in regional lymph nodes. Sentinel lymph node biopsy (SLNB) technique is a major breakthrough of surgical oncology in recent years, and it obtains development and application in the clinical practice of a variety of tumors[13]. Studies on melanoma and breast cancer prove that the application of sentinel lymph node biopsy technique can predict lymph node metastasis around the tumor, and currently used tracers for sentinel lymph node detection of gastric cancer are mainly divided into three classes: dyes, radionuclide and fluorescence dyes, which color SLN through lymphatic drainage or has high activity of radioactive isotope[14,15]. In the study, methylene blue was used for sentinel lymph node biopsy of gastric cancer sentinel lymph node staining, and analysis of serum tumor marker levels in gastric cancer patients with the different SLN metastasis showed that CA72-4, DDK1, TK1 and exosome levels in serum of patients with SLNS(+) gastric cancer were significantly higher than those of patients with SLNS(-) gastric cancer, and PGR percentage was significantly lower than that of patients with SLNS(-) gastric cancer. This indicated that SLNB could early judge the early lymph node metastasis, and also assess the malignant degree of tumor. According to the above analysis, serum HMGB1 content and sentinel lymph node detection can provide reference and basis for early evaluation of gastric cancer malignancy. The biological behavior directly related to the malignant degree of gastric cancer is cell proliferation and apoptosis, and excessive proliferation and deficient apoptosis can promote the development of gastric cancer and increase the malignant degree. PTEN and Survivin are the important genes regulating gastric cancer cell apoptosis and proliferation. Protein expressed by the PTEN gene can inhibit cell proliferation and induce apoptosis, it is an important tumorsuppressor gene in the body, and deficient expression of the gene can cause enhanced cancer cell proliferation activity; Survivin is the strongest apoptosis-inhibiting protein in the body, can directly inhibit the activation of apoptosis-executing molecules Caspase-3 and Caspase-7 and prevent cell apoptosis, and can also antagonize the blocking effect of p21 protein on the cell cycle and promote the development of the cell cycle, and the excessive expression of the gene will enhance the proliferation activity of cancer cells [16]. In order to define the relationship of serum HMGB1 content and sentinel lymph node detection with gastric cancer cell proliferation and apoptosis, Survivin, PTEN, p21, Caspase-3 and Caspase-7 expression levels in tumor tissue were analyzed in the study, and the results were as follows: the higher the serum HMBG1 levels

5 114 in gastric cancer patients, the higher the Survivin protein level and the lower the PTEN, p21, Caspase-3 and Caspase-7 levels in tumor tissue; Survivin protein level in tumor tissue of patients with SLNS(+) gastric cancer was significantly higher than that of patients with SLNS(-) gastric cancer, and PTEN, p21, Caspase-3 and Caspase-7 protein levels were significantly lower than those of patients with SLNS(-) gastric cancer. To sum up, serum HMGB1 and sentinel lymph node detection in gastric cancer patients can early assess tumor malignancy and lymph node metastasis. References [1] Zhang C, Hu X, Liu XY, Liang P, Zhang J, Cao L, et al. Effect of tumorassociated macrophages on gastric cancer stem cell in omental milky spots and lymph node micrometastasis. Int J Clin Exp Pathol 2015; 8(11): [2] Lee CM, Park SS, Kim JH. Current status and scope of lymph node micrometastasis in gastric cancer. J Gastric Cancer 2015; 15(1): 1-9. [3] Zeng YJ, Zhang CD, Dai DQ. Impact of lymph node micrometastasis on gastric carcinoma prognosis: a meta-analysis. World J Gastroenterol 2015; 21(5): [4] Chung HW, Jang S, Kim H, Lim JB. Combined targeting of highmobility group box-1 and interleukin-8 to control micrometastasis potential in gastric cancer. Int J Cancer 2015; 137(7): [5] Tummers QR, Boogerd LS, de Steur WO, Verbeek FP, Boonstra MC, Handgraaf HJ, et al. Near-infrared fluorescence sentinel lymph node detection in gastric cancer: A pilot study. World J Gastroenterol 2016; 22(13): [6] Zhang C, Hu X, Liu XY, Liang P, Zhang J, Cao L, et al. Effect of tumorassociated macrophages on gastric cancer stem cell in omental milky spots and lymph node micrometastasis. Int J Clin Exp Pathol 2015; 8(11): [7] Lee CM, Cho JM, Jang YJ, Park SS, Park SH, Kim SJ, et al. Should lymph node micrometastasis be considered in node staging for gastric cancer?: the significance of lymph node micrometastasis in gastric cancer. Ann Surg Oncol 2015; 22(3): [8] HUANG Jing. Results of Annual ASCO Gastrointestinal Tumor Conference in Oncol Prog 2014; 12(2): [9] Yin LK, Sun XQ, Mou DZ. Value of Combined Detection of Serum CEA, CA72-4, CA19-9 and TSGF in the Diagnosis of Gastric Cancer. Asian Pac J Cancer Prev 2015; 16(9): [10] SUN Fei, WU Chang-ping, LIU Juan, XU Bin, JIANG Jing-ting. Serum exosome levels in gastric cancer patients and the clinical significance. Chin J Clin Lab Sci 2014; 32(7): [11] Zhang J, Zhang R, Lu WW, Zhu JS, Xia LQ, Lu YM, et al. Clinical significance of hmgb1 expression in human gastric cancer. Int J Immunopathol Pharmacol 2014; 27(4): [12] Abe A, Kuwata T, Yamauchi C, Higuchi Y, Ochiai A. High Mobility Group Box1 (HMGB1) released from cancer cells induces the expression of pro-inflammatory cytokines in peritoneal fibroblasts. Pathol Int 2014; 64(6): [13] Bara T Jr, Gurzu S, Jung I, Kadar Z, Sugimura H, Bara T. Single skip metastasis in sentinel lymph node: In an early gastric cancer. World J Gastroenterol 2015; 21(33): [14] Niihara M, Takeuchi H, Nakahara T, Saikawa Y, Takahashi T, Wada N, et al. Sentinel lymph node mapping for 385 gastric cancer patients. J Surg Res 2016; 200(1): [15] WANG Hui, WANG Cheng-feng, WANG Jing, GAO Ji-dong, Wu Tiecheng, Fang Yi, et al. Imaging methods in the early-stage breast cancer and diagnosis of lymph node metastasis. Oncol Prog 2014; 12(6): [16] WANG Zhi-hong, BAO De-ming, SHI Zhen-wang, ZHAO Min, CHEN Zheng-xu, FANG Dong, et al. PTEN and Survivin expression in gastric carcinoma of ulcerative type and precancerous lesion and the clinical significance. Chin J Clin Exp Pathol 2015; 31(10):