Research and analysis of circulating tumor cell detection in the diagnosis and treatment of gastric cancer

Abstract

BACKGROUND

Circulating tumor cells (CTCs) are crucial for improving our knowledge regarding tumor progress, prognosis, and recurrence possibility.

AIM

To evaluate the role of CTCs in the early diagnosis and treatment of gastric cancer.

METHODS

From June 2020 to December 2021, a randomized study was conducted in our institution involving 80 patients scheduled for surgery for gastric cancer. The patients were divided into two groups: A control group that was tested for traditional serum markers and a study group that was assessed for serum CTCs.

RESULTS

In the study cohort, CTC levels did not correlate significantly with patient age, gender, or degree of tumor differentiation (P > 0.05). However, there was a significant correlation with the tumor-node-metastasis stage of the tumor (P < 0.05). In the study group, the CTC diagnostic positivity rate was 62.50% (25 out of 40 patients), while the positivity rate for conventional serum markers in the control group was 47.50% (19 out of 40 patients). The positive detection rate in the study group was significantly higher than that of the control group (P < 0.05).

CONCLUSION

CTCs have slight invasion and high sensitivity and specificity, presenting great value for early clinical diagnosis of recurrence and metastasis. It will improve the deceleration of disease development and increase the survival rate.

Key Words: Gastric cancer; Circulation; Tumor cells; Early diagnosis; Randomized controlled trial

Core Tip: Investigate the role of circulating tumor cells in the early detection and treatment of stomach cancer. Here, we discovered that circulating tumor cells had minimal damage, high sensitivity, and strong specificity, making them useful for early clinical identification of recurrence or metastasis, slowing the progression of patients’ disease, and improving patient survival rates.

INTRODUCTION

It is expected that more than one million new cases of gastric cancer occur worldwide[1]. Even with the advances in surgical techniques and adjuvant therapies, the prognosis for patients with advanced gastric cancer is generally poor[1]. Early detection and intervention are important in this disease, as the five-year survival rate significantly increases if the disease is caught at an earlier stage[1]. This underscores the need for innovative diagnostic tools.

The development of minimally invasive diagnostic tools is a promising avenue to improve early diagnosis and management of gastric cancer[1]. These conventional examinations are invasive and could cause discomfort or complications for patients. Circulating tumor cells (CTCs) are cancer cells that break away from the primary tumor and enter the bloodstream. The detection of these cells can serve as an early indicator of cancer, often before any symptoms are apparent[2]. This method is less invasive, which might improve patient compliance and allow for the possibility of surveillance more frequently than what is now available, critical to early diagnosis and treatment of gastric cancer[2].

Despite advances, early gastric cancer detection remains low in sensitivity and specificity, and it is occasionally accompanied by serious consequences such as bleeding and perforation[1]. The principal treatment options for gastric cancer remain surgical, radiotherapy, and chemotherapy; the majority of patients are already elderly[1]. Such minimally invasive techniques including CTC detection may provide an attractive alternative in the future and potentially decrease aggressive interventions. This might enable the clinician to offer more effective treatment options, possibly through early identification of CTCs, and closely monitor the disease progression with much higher precision. This could lead to improved outcomes and a better quality of life for patients with gastric cancer[3]. When diagnosed, most cases of gastric cancer are found to be in an advanced stage, resulting in limited treatment options and generally poor prognosis[1]. Consequently, gastric cancer stands as one of the major diseases posing a severe threat to the life and health of Chinese residents. Therefore, gastric cancer diagnosis and treatment are an urgent need from a public health perspective. Therefore, finding an effective detection method that can be used for reference in clinical treatment is necessary[4].

Serum CTCs are characterized as tumor cells that break away from the primary location of solid tumors, migrate into the peripheral blood or lymphatic system, and infiltrate the bloodstream, bone marrow, and lymph nodes through peripheral circulation[5]. Although they are present in tiny numbers in the blood, CTCs could be the root cause of metastasis and recurrence of a tumor. Currently, in cases of metastatic breast cancer, monitoring CTCs can help predict patient prognosis. Compared to histological detection, CTCs’ advantages include being almost non-invasive, dynamic monitoring could be allowed, and the sensitivity is much higher[6]. CTC testing could emerge as a new therapy response evaluation and prognosis-predictive indicator in metastatic breast cancer[7,8]. However, reports are limited on the efficacy and recurrence evaluation of serum CTCs in gastric cancer patients undergoing surgical intervention.

This work advances our understanding and use of CTC identification for gastric cancer care. Unlike traditional blood indicators, which typically lack sensitivity and specificity for detecting early cancer, CTC monitoring offers a non-invasive yet very accurate technique. This study shows that focusing on real cancer cells in the bloodstream improves diagnostic accuracy in the detection and stage of stomach cancer significantly. The findings highlight the clinical utility of CTC enumeration as a predictive tool for early intervention and tailored therapy methods. This unique technique has the potential to shift the paradigm of early detection, minimize the need for invasive treatments, and increase long-term survival rates for patients with stomach cancer. By bridging advanced diagnostics to practical therapeutic applications, our study provides a helpful paradigm for improving patient outcomes in an illness that has traditionally had a dismal prognosis.

MATERIALS AND METHODS

Data method

General information: In this study, 80 patients who underwent gastric cancer surgery and were admitted to our hospital between June 2020 and December 2021 were chosen and divided into two groups: A control group and a study group, each with 40 participants. Participants in the control group were tested using conventional serum indicators, whereas those in the study group were analyzed using serum CTCs. The goal was to investigate the relationship between positive rates and general information features in both patient groups.

Inclusion criteria and exclusion criteria: The inclusion criteria for this study required that participants have a gastric malignant tumor diagnosed by preoperative pathological biopsy through gastroscopy, confirmed according to the “Diagnostic Criteria for Gastric Cancer (WS316-2010)” of the Health Industry Standards of the People’s Republic of China. Additionally, patients needed to have a tumor-node-metastasis (TNM) stage III, have received no adjuvant treatment before enrollment, have indications for preoperative neoadjuvant chemotherapy that was administered in our hospital, and have voluntarily participated with written informed consent after the study received approval from the hospital ethics committee. The exclusion criteria included a pathological diagnosis of advanced gastric cancer, specifically if the infiltration depth of gastric cancer extended beyond the submucosa, as well as individuals with a history of other types of tumors.

Research method

Control group and study group: The control group was tested using standard serum indicators. Five milliliters of fasting patients’ venous blood were drawn upon admission, separated from the serum using a centrifuge, and then placed in a freezer set at -80 °C. An automated electrochemiluminescence immunoassay analyzer (model: ELECSY2010; manufacturer: Roche, Switzerland) was used to measure the levels of carbohydrate antigen 19-9 (CA199), carcinoembryonic antigen, and CA724. The normal ranges for CA199, carcinoembryonic antigen, and CA724 are 0 to 39 U/mL, 0 to 5 μg/L, and 0 to 6 U/mL, respectively. The study group employed the serum CTC assay. We purchased the CellSearch system and related chemicals from Johnson & Johnson. Patients were fasting when 7.5 mL of their venous blood was drawn and put into a CellSave tube. Before this treatment, blood samples were obtained for routine or biochemical analysis to reduce the influence of epithelial cells. The samples were then sent for evaluation right away.

Blood samples preparation: CellTracks AutoPrep tapered tubes were used to prepare the blood samples, and they were then exposed to a magnetic bead tag that included the antibody for epithelial cell adhesion molecule. 6.5 mL of sodium citrate buffer was added, and the samples were gently mixed before being centrifuged for 10 minutes at 2000 rpm. Tumor cells were then identified using fluorescence scanning with the analyzer system and marked with a red marker (CK+) for manual examination. CKDAPICD45 markers were used to confirm CTC-positive cells. Our hospital’s central laboratory performed the CTC testing, and the costs were funded by internally funded scientific research.

Preparation of the experiment: Preparation for the experiment involved removing the test kit from the cold room at 2-8 °C and allowing it to reach room temperature for at least 30 minutes. The instrument status was confirmed before the experiment, ensuring that the UPS power supply was operational. The switch on the lower right side of the instrument was opened, the power was turned on, and the waste liquid bottle was emptied. A 400 mL bleach solution was added to refill the instrument, ensuring enough buffer was loaded. Blood samples were confirmed according to the information in the sample registration form, checking for clotting and hemolysis within 96 hours.

Experimental steps: Turn on the pretreatment device and ensure that the deionized water has been changed. Initiate the daily cleaning routine and wait for an hour. For sample processing, label each 15 mL conical tube for batch processing, with no more than eight samples per batch. Mix the CellSave tubes and transfer 7.5 mL of blood from each into a labeled CellTracks AutoPrep conical tube. Add 6.5 mL of dilution buffer to each sample-containing tube, seal the tube, and invert to mix. Centrifuge the tubes at 800 × g for 10 minutes with the brake engaged, then process them on the CellSearch system within the hour. For quality control, mark each 15 mL tube with an orange barcode, shake the quality control bottle five times in one direction, then reverse and shake five times more. Pour the product into the marked tube, use a pipette to transfer any remaining liquid, and process these along with the samples on the CellSearch system. When loading samples and controls, follow the on-screen prompts to select the CellSearch CTC kit, opt for no tumor marker, decide on adding quality control, and input the total sample count. Add the reagent rack, Magnest, and tubes as indicated, and ensure the buffer has been replaced before starting the process. The samples will take between 2.5 to 4.5 hours to process, depending on their number. Afterward, remove the Magnest, insert the lid diagonally, release any bubbles, close the lid, and place the samples in a cassette for over 20 minutes. For system verification, log into the analyzer, turn on the mercury lamp, place the verification box in position, and follow the prompts to align and start the system check. Once verified, place the Magnest from the quality control and samples that have been in the dark for over 20 minutes into the system and complete the scanning as directed, with each scan taking at least 10 minutes. After scanning, turn off the lamp. Finally, review the analyzer’s results to interpret the CK + DAPI + CD45-blank channel-CTC findings.

Statistical analysis

The statistical software SPSS20.0 was utilized for data analysis. mean ± SD was the format for presenting measurement data. For comparing the average values between the two groups, a t-test was employed, while a χ² test was applied to assess differences in rates. A threshold of P < 0.05 was set to determine statistical significance.

RESULTS

Comprehensive analysis of CTCs with clinicopathological characteristics of gastric cancer patients

Table 1 outlines the detailed correlation between the presence of CTCs and various clinicopathological features of gastric cancer. It covers a broad spectrum of patient attributes such as age, gender, tumor differentiation level, lymph node involvement, TNM stage, and the occurrence of vascular invasion. The table breaks down the data to show the count of patients testing positive or negative for CTCs across these attributes, giving a detailed picture of how CTCs are distributed among different patient groups. It also includes χ² test results and their associated P values to determine whether these correlations are statistically significant. Strikingly, the table demonstrates a robust link between CTC presence and the later stages of TNM staging as well as vascular invasion, with P values of 0.001 and 0.002, respectively. These minimal P values underscore the importance of the connection between CTC detection and these key markers of cancer advancement. This correlation is a critical discovery, implying that identifying CTCs could serve as a significant biomarker for tracking the spread of the disease. On the other hand, the table shows no substantial correlation between CTC presence and factors like age, gender, or tumor differentiation, with P values > 0.05. The insights from Table 1 are crucial for understanding the role of CTCs in gastric cancer, especially their potential to forecast the severity and spreading of tumors.

Table 1 Relationship between circulating tumor cells level and clinicopathological features of gastric cancer, n (%).

Clinicopathological features	n	CTCs positive	CTCs negative	χ2	P value
Age (years)
≥ 60	23	16 (69.57)	7 (30.43)	2.364	0.315
< 60	17	12 (70.58)	5 (29.41)
Sex
Male	22	16 (72.72)	6 (27.28)	0.028	0.874
Female	18	10 (55.55)	8 (44.44)
Degree of differentiation
Low differentiation	22	13 (59.09)	9 (40.90)	0.587	0.632
High differentiation	18	11 (61.11)	7 (38.89)
Lymph node metastasis
Positive	23	15 (65.21)	8 (34.78)	4.369	0.498
Negative	17	12 (70.58)	5 (29.41)
TNM staging
I + II	20	9 (45.00)	11 (24.44)	4.127	0.001
III + IV	20	13 (32.50)	7 (35.00)
Vascular invasion
Yes	17	12 (70.59)	5 (29.41)	8.153	0.002
No	23	13 (56.52)	10 (43.47)

CTCs: Circulating tumor cells; TNM: Tumor-node-metastasis.

Comparative analysis of gastric cancer diagnostic positive rates between the study and control groups

Table 2 offers an examination of how often gastric cancer was correctly identified in two different groups of patients - one group had their blood tested for CTCs, while the other was checked using standard blood markers. The table lays out the total number of patients tested in each group, how many of them tested positive, and what percentage that represents. The group that was tested for CTCs had a higher percentage of positive results (62.50%) than the group tested with traditional methods (47.50%), which suggests that looking for CTCs might be a more effective way to spot gastric cancer. The difference in positive rates between the two groups is underlined by a χ² test value of 12.725 and a P value < 0.05, which strongly points to the statistical relevance of this difference. This data from Table 2 suggests that using CTCs for diagnosis could be more effective than traditional methods, making a strong case for including CTC testing in how we handle gastric cancer. This analysis not only speaks to the accuracy of CTC testing but also moves us towards improving how we diagnose gastric cancer, which could lead to more tailored treatment plans for patients.

Table 2 Comparison of gastric cancer diagnostic positive rates between two groups of patients.

Group	n	Positive diagnosis of stomach cancer (n)	Positive rate of gastric cancer diagnosis
Study group	40	25	62.50%
Control group	40	19	47.50%
χ2			12.725
P value			< 0.05

DISCUSSION

Gastric carcinoma, emerging from cells lining the stomach, is prevalent in our nation, with the highest occurrence rate compared to other cancers[9,10]. In China, it claims about 170000 lives annually, accounting for nearly a quarter of all cancer-related fatalities[11,12]. Over 20000 individuals are newly diagnosed each year. This cancer poses a significant risk to public health, and timely detection is vital for enhancing survival chances. Research suggests that CTCs, as biomarkers, could aid in the early identification of stomach cancer[13]. The examination of CTCs can detect minute tumor cells, around 2 millimeters, possibly pinpointing cancer cells 3 to 6 months sooner[14,15]. Typically, by the time tumor markers are flagged, the cancer is often in advanced stages, making early discovery challenging. The accuracy of CTC detection in gastric cancer patients surpasses that of traditional markers, with a demonstrated higher sensitivity and specificity[16,17]. This is because CTCs are actual circulating cancer cells, not just byproducts like proteins or enzymes emitted by tumors. This direct detection approach can lead to more precise diagnoses and better prognoses through earlier treatment. Our study found no link between CTCs and factors such as age, gender, tumor grade, or lymph node spread in stomach cancer patients. Among our test subjects, 62.50% (25 out of 40) in the CTC group tested positive for stomach cancer, compared to 47.50% (19 out of 40) in the traditional marker group. This significant difference suggests that CTC detection is more sensitive in diagnosing stomach cancer. With a 95% specificity rate, CTC detection accurately differentiates between stomach cancer patients and healthy individuals. Furthermore, our findings indicate that CTC detection closely mirrors the tumor stage and spread, suggesting its utility in assessing cancer stage and prognosis[18]. The presence of CTCs correlates with tumor advancement and spread, acting as a prognostic indicator across various cancers. Our study confirmed a significant link between CTC count and disease stage, aligning with prior studies. However, the molecular events associated with the release of CTCs from the primary tumor and their survival into the circulation are not well documented. Further studies should focus on targeting genetic and epigenetic alterations that allow the ability of CTCs to evade immune surveillance and to adapt to the circulatory system. Gaining insights into these processes could pave the way for therapies that reduce CTC numbers or hinder their metastatic capabilities. Additionally, advancing CTC detection methods could enhance early cancer diagnosis and progression monitoring, potentially enhancing patient outcomes.

CONCLUSION

The use of CTC technology offers considerable benefits for the prompt identification of gastric cancer, holding the potential to enhance both the longevity and daily well-being of those affected. As this technology continues to be refined and its clinical use becomes more widespread, it is anticipated that CTC will become increasingly instrumental in detecting gastric cancer at an early stage and in tracking the effectiveness of treatments.