Relationship between Hypercoagulable State and Circulating Tumor Cells in Peripheral Blood, Pathological Characteristics, and Prognosis of Lung Cancer Patients.

OBJECTIVE: To analyze the relationship between hypercoagulable state and circulating tumor cells (CTCs) in peripheral blood, pathological characteristics, and prognosis of lung cancer patients.
METHOD: A total of 148 patients with primary lung cancer diagnosed and treated in our hospital from January 2017 to January 2019 were selected as the research objects. According to the CTC test results, the patients were divided into CTC-positive group and CTC-negative group. Also, the coagulation index of patients was tested. According to the blood coagulation index test results, patients were divided into hypercoagulable group and non-hypercoagulable group. The relationship between hypercoagulable state and pathological characteristics of lung cancer patients was analyzed by single factor analysis and multiple logistic regression model. Kaplan-Meier survival curve was applied to analyze the relationship between hypercoagulable state and the prognosis of lung cancer patients.
RESULTS: The platelets (PLTs), fibrinogen (FIB), D-dimer (D-D), and prothrombin time (PT) in CTC-positive group were significantly higher than those in CTC-negative group. There was no significant relationship between the patient's gender, smoking history, pathological type, and the hypercoagulable state of the patients. The proportion of patients aged 60 years or older, with TMN stage III or IV and lymph node metastasis, in the hypercoagulable group was significantly higher than that in the non-hypercoagulable group. Logistic regression analysis showed that there was an independent relationship between the patient's age, lymph node metastasis, and hypercoagulable state. As of January 2020, among the 148 patients with lung cancer follow-up, 5 patients were lost and 52 died. The median survival time of patients in the hypercoagulable group was 82 weeks, which was significantly lower than the 104 weeks in the nonhypercoagulable group.
CONCLUSION: There is a certain relationship between hypercoagulable state and CTC positive in lung cancer patients. There is an independent relationship between the patient's age, lymph node metastasis, and the hypercoagulable state. The median survival time of patients in the hypercoagulable group was significantly lower than that in the non-hypercoagulable group.

1. Introduction

Lung cancer is a common malignant tumor that has the characteristics of high incidence, high mortality, and many complications, which seriously endangers human health and safety. Thrombosis is one of the common complications of lung cancer, and hypercoagulable state is the direct factor of thrombosis [1, 2]. Hypercoagulable state, also known as prethrombotic state, is a pathological state with dysfunction caused by injury of vascular endothelial cells, such as abnormal clotting and fibrinolysis. The cellular active factors within human body cause inflammatory response or platelet (PLT) function change, which in turn activates prothrombins that lead to blood hypercoagulation [3, 4]. The hypercoagulable state not only promotes tumor development but also is an important factor of thromboembolic disease. Circulating tumor cell (CTC) is a general name of various tumor cells existing in peripheral blood. CTC detection can monitor tumor dynamics in real time by capturing the trend of CTC type and quantity changes measured by the presence of CTC in the detected peripheral blood. Positive CTC detection is a prerequisite for tumor development and metastasis [5]. In this study, we analyzed the relationship between hypercoagulable state and CTC in peripheral blood, clinicopathological characteristics, and prognosis, with the hope to provide clinical guidance on the treatment of hypercoagulable state in lung cancer patients. The details of the study are reported as follows.

2. Data and Methods

2.1. General Information

148 patients with primary lung cancer who were treated in our hospital from January 2017 to January 2019 were selected for the study. Among them, 82 cases were males and 66 cases were females. They were aged from 34 to 76 years old. The mean age was 56.23 ± 8.33 years old. The Carlson scores ranged from 61 to 85 points. The mean point was 77.95 ± 7.59 point. There were 21 cases of stage I, 27 cases of stage II, 55 cases of stage III, and 45 cases of stage IV. There were 102 patients before treatment (61 before first-line, 28 before second-line, and 13 before radiotherapy), 34 patients during chemotherapy (22 first-line and 12 during chemotherapy), and 12 patients after chemotherapy. Inclusion criteria were as follows: meeting the diagnostic criteria of lung cancer; lung cancer diagnosed by pathology or cytology; and not receiving other antitumor treatments. Exclusion criteria were as follows: combined thrombotic disease, chronic obstructive pulmonary disease, or autoimmune disease; history of anticoagulation, hemostasis, or antifibrinolytic drugs; cardiac, renal, and other vital organ insufficiency; and psychiatric disease. All patients consented to the use of medical records for this study and signed an authorization consent form.

2.2. Research Methodology

The Beckman 5DIFF automatic blood cell analyzer and its related reagents were applied to detect the patients' platelet (PLT) level, and the Beckman TOP700 automatic hemagglutinator and its related reagents were applied to detect the fibrinogen (FIB), D-dimer (D-D), prothrombin time (PT), thromboplastin time (TT), and activated partial thromboplastin time (APTT) of patients.

Immunomagnetic bead negative isolation and immunocytochemical staining were used to detect the CTC in peripheral blood. Procedure: the blood and lymphocyte separation medium were allowed to stand at room temperature for 30 minutes; 7.5 ml of anticoagulation reagent and PBS buffer were added into the blood and lymphocyte separation medium and mixed thoroughly; and the mixed liquor was centrifuged at 4000 r/min for 30 minutes. The milky white mononuclear cell layer was aspirated from the liquor, with 5 times the volume of PBS added and mixed into the liquor; the liquor was centrifuged at 3000 r/min for 15 minutes. The supernatant was discarded. The operation was repeated twice. The cells were resuspended in the liquor of 80 μl of magnetic bead buffer and 20 μl of CD45 antibody and incubated at 4°C for 15 minutes. 20 times the volume of magnetic bead buffer was added and centrifuged at 3000 r/min for 10 minutes. The supernatant was discarded, and the cells were resuspended. The cells were separated by MS shunt column, and the cell suspension was collected. The cell suspension was centrifuged at 3000 r/min for ten minutes and then separated, and the supernatant was discarded. Then, the precipitate was applied to slides and air-dried at room temperature. 4% paraformaldehyde was used to fix the slides for 10 min and washed with PBS. The mouse anti-broad-spectrum cytokeratin was added into the slides. Then, the slides were incubated at 4°C, and secondary antibody was added for color development. Hematoxylin was used to re-stain the films for 3–5 min, followed by differentiation, dehydration, transparentization, and sealing with neutral gum. Later, hematoxylin was used to re-stain the cells for 3–5 minutes. The slides then were differentiated, dehydrated, transparentized, and sealed with neutral gum. The CTC isolation reagents and immunocytochemistry kits were purchased from Beijing Boaosen Biotechnology, and the experimental operation was carried out strictly according to the kit instructions.

Hypercoagulable state criteria were as follows: the normal ranges of PLT, FIB, D-D, PT, TT, and APTT were (125–350) × 109/L, 1.80–3.50 g/L, 0–0.55 mg/L, 9–12 s, 16–18 s, and 28–41 s. According to the levels of PLT, FIB, and D-D and the length of PT, TT, and APTT, 77 patients were divided into the hypercoagulability group and 72 cases were divided into the non-hypercoagulability group (if any of the PLT, FIB, D-D, PT, TT, and APTT deviated from the normal range, the patient would be identified as hypercoagulable state). CTC-positive criteria were as follows: the CTC-positive cells had blue-black or brown cytoplasm and abnormal nucleoplasmic ratio, with the cell length greater than 10 μm, as observed by two or more pathologists. The patients were divided into 52 cases in the CTC-positive group and 96 cases in the CTC-negative group according to the result of test, and the coagulation indexes of the two groups were compared. Relevant clinical data were collected from patients, including age, gender, smoking history, TMN stage, pathological type, and postoperative lymph node metastasis.

Follow-up: follow-up was practiced by telephone inquiry or outpatient inquiry, and the follow-up date was up to January 2021. The data collection ended with patient's death or the follow-up cutoff date. The total survival time of patients is defined as the time from admission to death for any reason or the end of follow-up.

2.3. Statistical Methods

SPSS 19.0 software was applied for data processing, and the measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for two-way comparison analysis. Count data were expressed as (%), and the χ2 test was used for comparison between groups. Logistic regression model was used for multifactorial analysis. Kaplan–Meier survival curve was used to analyze the relationship between hypercoagulable state and prognosis of lung cancer patients, and log-rank test was used for comparison. The test level was α = 0.05, and p < 0.05 was considered a statistically significant difference.

3. Results

3.1. Analysis of Coagulation Indexes in the CTC-Positive and CTC-Negative Groups

Among the 148 lung cancer patients, 52 were CTC-positive and 96 were CTC-negative. CTC-positive cells were observed under light microscopy. The cells were found to be round, oval, or rectangular in shape, with intact nuclei. But the nucleoplasmic ratio of the cells showed abnormalities, as shown in Figure 1. The PLT, FIB, D-D, and PT levels in the CTC-positive group were significantly higher than those in the CTC-negative group (p < 0.05), as shown in Table 1.

Figure 1

CTC cell morphology in peripheral blood of lung cancer patients (SP method, ×400).

Table 1

Analysis of coagulation indexes in patients in the CTC-positive group and CTC-negative group (n, mean ± SD).

Data	CTC-positive group (n = 52)	CTC-negative group (n = 96)	t	P
PLT (×109/L)	367.42 ± 68.73	237.95 ± 57.22	6.737	0.008
FIB (g/L)	3.94 ± 0.86	3.12 ± 0.92	3.397	0.021
D-D (mg/L)	2.36 ± 0.37	0.62 ± 0.12	8.652	≤0.001
PT (s)	13.28 ± 1.18	10.15 ± 1.06	4.542	0.013
TT (s)	17.55 ± 1.95	16.23 ± 1.73	1.475	0.182
APTT (s)	32.69 ± 3.15	31.44 ± 3.05	1.058	0.479

3.2. Comparison on Clinical Characteristics of Patients in the Hypercoagulability Group and Non-Hypercoagulability Group

The results of the analysis showed that the gender, smoking history, and type of pathology of patients were not significantly related to the patients hypercoagulable state. None of the differences was statistically significant (p > 0.05). The proportion of patients in the hypercoagulability group who were over 60 years old, had TMN stage III or IV, and had lymph node metastasis was significantly higher than that in the non-hypercoagulability group (p < 0.05), as shown in Table 2.

Table 2

Comparison on clinical characteristics of patients in the hypercoagulability group and non-hypercoagulability group (n, %).

Factors	Number of cases	Hypercoagulability group (n = 76)	Non-hypercoagulability group (n = 72)	χ 2	P
Age (years)
≥60	85	52 (61.18)	33 (38.82)	7.716	0.005
<60	63	24 (38.10)	39 (61.90)
Gender
Male	82	40 (48.78)	42 (51.22)	0.487	0.486
Female	66	36 (54.55)	30 (45.45)
Smoking history
Yes	79	45 (56.96)	34 (43.04)	2.135	0.144
No	69	31 (44.93)	38 (55.07)
TMN stage
I, II	58	23 (39.66)	35 (60.34%)	5.223	0.022
III, IV	90	53 (58.89)	37 (41.11)
Pathology type
Squamous carcinoma	52	30 (57.69)	22 (42.31)	3.742	0.291
Adenocarcinoma	48	23 (41.92)	25 (52.08)
Small-cell carcinoma	30	17 (56.67)	13 (43.33)
Other	18	6 (33.33)	12 (66.67)
Lymph node metastasis
Yes	68	42 (61.76)	26 (38.24)	5.461	0.019
No	80	34 (42.50)	46 (57.50)

3.3. Analysis of Multiple Factors That Affect the Hypercoagulable State of Lung Cancer Patients after Operation

Result of the logistic regression analysis showed that patients' age and lymph node metastasis had something to do with the hypercoagulable state (p < 0.05), as shown in Table 3.

Table 3

Analysis of multiple factors that affect the hypercoagulable state of lung cancer patients after operation.

Influencing factors	B	SE	Walds	df	Sig.	Exp(B)
Age	1.359	0.458	7.168	1	0.015	3.287
TMN stage	0.821	0.372	3.669	1	0.261	1.671
Lymph node metastasis	1.896	0.482	8.007	1	0.008	4.129

3.4. Relationship between the Hypercoagulable State and Prognosis of Patient

As of January 2020, of the 148 lung cancer patients followed up, 5 were lost to follow-up and 52 died. The median survival time of patients in the hypercoagulable group was 82 weeks, which was significantly lower than that of patients in the non-hypercoagulable group of 104 weeks. p = 0.005 was obtained by log-rank test, a statistically significant difference (p < 0.05), as shown in Figure 2.

Figure 2

Relationship between hypercoagulable state and prognosis of patient.

4. Discussion

According to statistics, about 90% of patients with malignant tumors have a hypercoagulable state of blood. The hypercoagulable state is mostly caused by procoagulant factors released by tumor cells, an increase in the number of activated platelets, and cytokines released by macrophages [6, 7]. Hypercoagulable state can change the hemodynamics of tumor patients, affect the blood perfusion of tissues, and cause hypoxia and ischemia of tissues [8]. If not diagnosed and treated in time, hypercoagulable state will further lead to the formation of thrombosis and affect the prognosis of patients. Patients with malignant tumors with hypercoagulable blood are more likely to have hemorrhagic complications, venous thrombosis, etc., leading to poor prognosis and reduced survival rates [9, 10]. Malignant tumors can be transferred to other organs of the body through blood transmission, and tumor metastasis is the main cause of death for tumor patients [11]. Tumor cells invade the surrounding tissues of the primary tumor cells, enter the blood and lymphatic system, and form circulating tumor cells (CTC). These cells are transported to remote tissues, then exuded to adapt to the new microenvironment, and the metastases focus is formed through “sowing,” “proliferation,” and “colonization” finally. Therefore, it is generally accepted that positive CTC means poor prognosis of tumor patients. Therefore, a correct understanding of the relationship between blood hypercoagulability and CTC and clinicopathological characteristics in patients with lung cancer will help improve the prognosis of patients and increase the survival time of patients.

The proliferation and development of CTC are often manifested as the deterioration and metastasis of lung cancer, and the hypercoagulable state of blood can promote tumor proliferation and metastasis. Therefore, the joint monitoring of two indicators is of great significance for predicting the development and prognosis of lung cancer. The results of this study showed that the levels of PLT, FIB, D-D, and PT in the CTC-positive group were significantly higher than those in the CTC-negative group, indicating that there is a certain relationship between the blood hypercoagulability state of tumor patients and the positive CTC. PLT level can reflect the dynamic balance between PLT generation and damage and is an important part of CTC transfer [12]. The body's inflammatory response or infection will affect the content of FIB. After FIB is activated, it participates in the body's coagulation process. When the FIB level in the body increases abnormally, the probability of thrombosis increases. In addition, FIB can reduce the sensitivity of tumor cells to chemotherapy and protect tumor cells while promoting their proliferation and metastasis [13]. D-D is the product of the degradation of cross-linked fibrin, whose level reflects the intensity of secondary fiber activity, and can be used to evaluate the progress of lung cancer or other malignant tumors [14].

The proportion of hypercoagulable patients over 60 years old, TMN stage III or IV, and lymph node metastasis were significantly higher than that of non-hypercoagulable patients. Analysis of blood hypercoagulation state and the clinicopathological characteristics of lung cancer patients showed that there was no obvious relationship between patient gender, smoking history, and pathological type and patients' blood hypercoagulation. Logistic regression analysis showed that the patient's age, lymph node metastasis, and the risk of blood hypercoagulation were related. As the patient ages, physical strength declines and mobility weakens, causing changes in the body's hemodynamics and even blood stasis, thereby increasing the risk of hypercoagulable state [15]. The spread of tumor cells and lymph node metastasis accelerate the formation of thrombus. Besides, the damage of tumor cells to the body's vascular endothelial cells or the formation of new blood vessels by tumor cells will aggravate the blood hypercoagulable state [16].

As of January 2020, among 148 patients with lung cancer follow-up, 5 cases were lost to follow-up and 52 cases died. The median survival time of patients in the hypercoagulability group was 82 weeks, which was significantly lower than 104 weeks in the non-hypercoagulability group. The results indicate that the hypercoagulable state of blood can directly affect the survival time of patients and make the prognosis of patients worse. Therefore, strengthening the monitoring and intervention of coagulation function of patients with lung cancer is of great significance for its treatment.

5. Conclusion

There is a certain relationship between the blood hypercoagulability state of lung cancer patients and the positive CTC. There is an independent relationship between the patient's age, lymph node metastasis, and blood hypercoagulability status. The median survival time of patients in the hypercoagulability group was significantly lower than that of patients in the non-hypercoagulability group. Patients should be screened for coagulation function regularly and diagnosed and treated early to avoid thrombosis caused by hypercoagulable state and prolong the survival time of patients.