Epidemiological characteristics of peripheral circulating tumor cells and their relationship with the morbid state in gastric polyps and non-metastatic gastric cancer

Circulating cells (CTCs) can in of many but low numbers of can be in of as well as with benign It is not known whether peripheral CTC counts differ between patients with benign gastric diseases and those with gastric cancer. It is still unknown whether peripheral CTC counts can act as a superior factor to assess the stage in gastric Comparative analysis of preoperative peripheral CTC counts was completed in patients with gastric polyps and non-metastatic gastric cancer. Correlation analysis was carried out between peripheral CTC counts and histological differentiation types, histopathological types, depth of tumor inltration, positive lymph node metastasis rate and tumor markers (CEA, CA19-9 and CA72-4). Cancer Antigen 72 − 4. Positive = Serum test results higher than the clinical reference range (CEA: 0 (cid:0) 10 ng/ml, CA19-9: 0 (cid:0) 34 U/ml, CA72-4: 0 (cid:0) 8.2 U/ml); Negative = Serum test results within the clinical reference range (CEA: 0 (cid:0) 10 ng/ml, CA19-9: 0 (cid:0) 34 U/ml, CA72-4: 0 (cid:0) 8.2 U/ml).


Introduction
Gastric cancer, although the incidence of it varies around the world, is still a very common cancer worldwide [1][2][3], consequently radical surgery is still the preferred therapy method for patients with resectable gastric tumors. Although the current integrated therapies for gastric cancer have made a signi cant progress, there are still postoperative recurrence cases, many patients with stage II and III gastric cancers still exhibit tumor recurrence within one year following integrated therapies [4], so early detection and treatment is in highly demand. Circulating tumor cells (CTCs) can be detected at the preoperation and post-operation in peripheral blood in gastric cancer patients, the presence of post-operative peripheral blood CTCs may indicate the potential for tumor recurrence and metastasis [5][6][7].
The presence of CTCs suggests that a tumor may be present in vivo, and preoperative detection of peripheral blood CTCs can be used to assess early stage epidemiological characteristics and predictive values of circulating tumor cells in these patients with tumor [8][9][10]. When combining the clinicopathologic staging of the tumor, surgeons can comprehensively assess the necessity of subsequent treatments, such as chemotherapy and radiation.
However, a low number of CTCs can also be detected in the peripheral blood of some healthy individuals as well as patients with benign tumors [11][12][13]. Therefore, further studies are required to determine whether peripheral CTCs are differentially expressed in benign gastric disease and non-metastatic gastric cancer, and whether detection of CTCs accurately re ects the clinicopathologic characteristics of the primary tumor in benign and malignant gastric diseases. To address these questions, we carried out the comparative analysis of peripheral CTC counts in patients with gastric polyps and gastric cancer.

Patients And Methods
A total of 13 patients (5 males, 8 females) with gastric polyps, and 58 patients (40 males, 18 females) with gastric carcinoma were retrospectively studied at the department of surgery of Shanghai Tongren Hospital (Shanghai, China) between December 2014 and August 2016. After received patient consent and ethics committee recognition, peripheral CTCs quanti cation was performed the day before operation.
Patient general information and pathological tumor characteristics are listed in Table 1. All tumor samples were identi ed as malignant epithelial tumors using Cytokeratin (AE1/AE3), carcinoembryonic antigen, Cytokeratin 7, and Cytokeratin 20. For carcinomas, the degree of tissue differentiation was classi ed as high, moderate, or poor differentiation, and tissue pathology was classi ed as papillary adenocarcinoma, tubular adenocarcinoma, papillary tubular adenocarcinoma, signet-ring cell carcinoma, or mucous adenocarcinoma, patients with tumor cells invasive depth from T 0 to T 4a , lymph node positive from N 0 to N 3b and no tumor cells distant metastases (M 0 ) were taken into accounted and subsequently observed. For polyps, degree oftissue differentiation was classi ed as low-grade intraepithelial neoplasia or high-grade intraepithelial neoplasia, and tissue pathology was classi ed as adenomatous polyp and proliferative polyps. Pathological consultations were completed by three experienced pathology specialists.

Circulating Tumor Cells
CTCs were enriched by negative separation as described in previous studies [13,14]. CD45 antibody beads were used to bind white blood cells, and all CTCs were enriched in precipitation. A total of 9.6 ml of peripheral blood was obtained peripherally from each patient, across three collections (3.2 ml per collection) at three time points(8am, 2 pm, and 8 pm), using an indwelling sheathe syringe needle on the day before the operation. CTC counts were measured for each blood sample using uorescence in situ hybridization in triplicate and mean values were adopted. The following criteria were used to identify CTCs: nuclear signal greater or equal to the triploid, positive nuclear DAPI staining, and negative CD45 staining [13]. Staining of cells was observed and counted using a uorescence microscope (Nikon CI-S, Tokyo, Japan). Peripheral blood CTC counts were measured for each 3.2 ml blood sample using uorescence in situ hybridization.

Pathological Specimens
Tumor tissues, lymph node and polyp samples were immediately xed in formalin for 24 hand para n embedded, tissue sections were prepared as described in our previous study [13]. Primary antibodies (mouse anti human, 1:200) were added and incubated at 4 °C for one night. HRP-labeled secondary antibodies (goat anti mouse, 1:500) were added and incubated for 1 h. Sections were sealed with neutral resin and pictures were taken using a microscope.

Statistical analysis.
Values for all continuous variables are expressed as the mean ± standard error (SE) of the mean. Data were analyzed with Origin 8.0 software (Origin Lab Corp. Northampton, MA, USA

CTC counts and tissue invasive depth in gastric carcinoma
In gastric carcinoma patients, CTC counts was higher in mucous membrane layer (3.0 ± 0.33/3.2 ml) and serous layer (2.56 ± 0.39/3.2 ml), lower CTC count was observed in muscular layer (1.4 ± 0.4/3.2 ml), signi cant difference was observed between mucous membrane layer and muscular layer (t = 2.89, P = 0.012, Fig. 2.), whereas signi cant difference was observed between serous layer and muscular layer(t = 0.97, P = 0.333). Also, no signi cant difference was observed between serous layer and mucous membrane layer (t = 0.19, P = 0.849). These results suggested that CTC counts were higher in early gastric carcinoma (mucous membrane layer invasive), and CTC were detached easily in early gastric carcinoma other than advanced gastric carcinoma.

Comparison of positive rate of CTC counts and tumor markers in gastric carcinoma
Tumor markers CEA, CA19-9, CA72-4 were detected in non-metastatic gastric cancer in this study, only 13.79% positive rate was shown in gastric carcinoma patients when CEA acted as an index to re ect the existence of gastric carcinoma, and 12.07% positive rate with CA19-9, 17.24% positive rate with CA72-4, respectively (Table 6.). Even the joint detection of CEA, CA19-9 and CA72-4, the positive rate was reached at merely 29.31% (Table 6.), which was far below the positive rate of CTCs > 1/3.2 ml criterion (67.24%) and CTC 0-1/3.2 ml criterion (79.31%). CTC criterion possesses higher positive rate than CEA, CA19-9 or CA72-4 when they acted as an sole index to re ect the existence of gastric carcinoma, signi cant differences in positive rate were observed between CTC > 1/3.2 ml criterion and CEA, CA19-9 and CA72-4 (X 2 = 34.37, X 2 = 36.89, X 2 = 29.72, respectively, P<0.001,

Discussion
The detection of CTCs has been widely reported for the diagnostic and predictive properties in numerous types of cancers, such as breast cancer, prostate cancer, colorectal cancer, gastric cancer [5][6][7]. Clinical implications of CTCs, including screening tool for early diagnosis, biomarkers to speci c therapies, monitoring tool to assess the response of chemotherapies. Research focusing on the association of CTCs and postoperative recurrence, as well as response to chemotherapies has been extensively conducted, whereas the application of CTCs in the early diagnosis received limited attention. Moreover, the use of this system for screening, mainly compared the malignant tumor with the healthy control [17] due to the sensitivity and speci city of detection technique. Nevertheless, the differentiation between cancer and benign diseases (e.g. Polyp and cancer) determined by CTCs has been rarely reported, among which the clinical implication of CTCs was associated to clinical symptomatic parameters, such as the tumor stage, histopathological type, degree of differentiation, depth of invasion, and lymph node metastasis. In the current study, aside from the conventional comparison between cancer patients and their healthy controls, the association between the existence of polyps and CTCs was also analyzed, based on tumor biomarkers, which could provide information for the diagnosis of tumor in early and intermediate stage.
CTCs are extensively distributed in the blood of all types of cancer patients, as the development of the research, the CTCs has been detected almost everywhere, even in the blood sample of tumor free individuals and patients bearing benign tumor [11][12][13]17]. If the peripheral CTCs was also detectable in patients with gastric polyps, how about that in those representative benign tumor-like lesions? If it is possible, would the CTC counting interval be different, comparing to healthy controls and malignant patients. The current study was designed to determine those questions by the observation of CTCs between patients with gastric polyps and malignancy tumor. Our results showed that the CTC counting can be identi ed in peripheral circulation of patients with either polyps or gastric carcinoma, however, the polyps group showed a lower CTCs (0.69 ± 0.17/ 3.2 ml), comparing to that of their counterparts (3.05 ± 0.48/3.2 ml). Although there is a certain overlap of the two intervals, signi cant difference was found among the two groups (P < 0.05). The results suggested that the higher CTC counting is associated with higher risk of cancer, therefore CTCs could be adopted as a valuable cancer screening biomarker.
According to the previous publications and our experience, the CTCs counting number were divided into two groups, > 1/3.2 ml and ≤ 1/3.2 ml [10,13,[15][16][17]. Alvarez Cubero [18] reported that the number of CTCs from metastatic tumor is 1-10/ml, while that of non-metastatic tumor is 0.5-5/ml, whose lowest value (0.5/ml) is close to our criteria (1/3.2 ml). Our study found that the probability of malignant tumor in CTC value over (1/3.2 ml) is 67.24%, meanwhile, the rate for benign tumor in CTC value below (1/3.2 ml) is 84.62%. Additionally, we found that there is a signi cant difference between patients with gastric polyps and gastric carcinoma (X 2 = 9.67, P<0.005). The highest CTC counting is mostly detected in carcinoma patients, indicated that CTCs could be used for the preliminary clarify benign and malignant.
For the early stage resectable gastroenteric cancer, the tumor specimens can be collected to determine the differentiation, invasion depth including peripheral vessel, lymphangion and nerves, as well as the lymphatic metastasis. As for patients with gastric polyps, the pathological type can be identi ed by biopsies after endoscopic polypectomy. In the current study, a re ned clinical correlation analysis was carried out by the clinical pathological stage combined with the CTCs, provided reference for the application of CTCs monitor for early stage.After comparing the gastric carcinoma pathological type of two CTCs groups (> 1/3.2 ml vs. ≤1/3.2 ml), there is no signi cant difference of the distribution of pathological types among two groups (P > 0.05), suggested that CTCs is not in uenced by the pathological types. The gradient analysis based the positive rate of lymphatic metastasis, showed that the higher CTCs group showed a relative low rate of lymphatic metastasis, is negatively correlated, though needs further determination. As for the depth of tumor invasion, lower CTC counting was observed in muscular layer, while higher numbers in mucous membrane layer and serous layer. Signi cant difference was observed in muscular layer, compared with the other two layers (P < 0.05), indicating that the higher CTCs is possible detected in the early gastric carcinoma, which therefore could be used as an indicator for early gastric carcinoma roughly screening.
CEA, CA19-9 and CA72-4 have long been employed for tumor preliminary screening, especially CEA in monitoring the gastroenteric tumor relapse. There is no improved tumor marker has been identi ed, although these biomarkers possess limited value in tumor early screening. Our results suggested that the CTCs counting showed superior value in early monitor comparing to CEA, CA19-9 and CA72-4, which could be developed as one of the most powerful approaches for early gastroenteric tumor regardless of the higher expense [17].

Conclusion
CTCs exist widely in patients either with gastric polyps or gastric carcinoma, however the latter showed a dramatic higher counting number. CTCs could be used as a predictor for the existence of gastroenteric neoplastic disorders, which is helpful to roughly tell the benign and malignant of tumor, furtherly, it showed a better re ection on early tumor development comparing to the conventional tumor marker, such as CEA, CA19-9 and CA72-4.

Declarations
Ethics approval and consent to participate The study was carried on after acquiring patients' consent and Tongren Hospital ethics committee approval and consent.

Consent for publication
Not applicable.

Availability of data and material
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
Competing interests