Objective Cervical cancer is one of the most common cancers worldwide, and immune function may impact disease progression. Serum markers may also be associated with diagnosis and progression. The aim of this study was to explore the clinical usefulness of determining the levels of peripheral blood immune cells and serum tumor markers in predicting diagnosis and prognosis of patients with cervical cancer.
Methods 82 patients with cervical cancer (early stage group: IA–IB1 and IIA1; locally advanced group: IB2 and IIA2), 54 patients with cervical intra-epithelial neoplasia (CIN), and 54 healthy women (control group) were recruited. Inclusion criteria were: (1) patients whose cervical lesions were determined based on biopsy; and (2) patients who had not undergone immunotherapy, chemotherapy, or radiotherapy. The exclusion criteria were as follows: (1) patients with a history of other malignant tumors; (2) patients with heart, kidney, and other organ failure; (3) patients with immune diseases; and (4) pregnant or lactating women. The levels of immunocytes and tumor markers were assayed. The relationships among histopathologic factors were analyzed. The correlation between the levels of immunocytes and tumor markers in patients with different degrees of cervical lesions (pre-invasive or cancer) and healthy women was evaluated.
Results The squamous cell carcinoma antigen and carcinoembryonic antigen levels in the control group and the CIN group were significantly lower than those in the cervical cancer groups (p<0.01). The incidence of lymph node metastasis in the early stage and locally advanced groups were 22.9% (11/48) and 46.2% (12/26), respectively, and 58.8% (20/34) and 7.5% (3/37) in the positive and negative lymphovascular invasion groups, respectively (p<0.05). The levels of CD8+ and CD8+ CD28+ T cells in the early stage group were markedly lower than those in the CIN group and the control group (p=0.014, p=0.008, respectively). The ratio of CD4+CD25+/CD4+ in the cervical cancer groups was significantly higher than in the control group (p<0.01). The increased serum squamous cell carcinoma and carcinoembryonic antigen levels and CD4+CD25+/CD4+ ratio were risk factors for cervical cancer by logistic regression analysis (p<0.05).
Conclusions In patients with cervical cancer, immune function was impaired compared with that in healthy women and patients with CIN, while squamous cell carcinoma and carcinoembryonic antigen levels were increased. Combined detection of the levels of peripheral blood immune cells and serum tumor markers may be helpful for early detection, diagnosis, and prognosis evaluation of patients with cervical cancer.
- cervical cancer
- lymphatic metastasis
- neoplasm invasiveness
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Lymph node metastasis was closely related to clinical stage and lymphovascular invasion in cervical cancer patients.
Immune function was decreased and the levels of serum squamous cell carcinoma and carcinoembryonic antigen were increased in patients with cervical cancer.
The increased serum squamous cell carcinoma and carcinoembryonic antigen levels and CD4+CD25+/CD4+ T cells ratio were risk factors for cervical cancer.
Cervical cancer is the fourth most commonly diagnosed cancer and the fourth most common cause of cancer related death in women worldwide.1 In the past few decades, the incidence and mortality reported rates of cervical cancer have gradually decreased in many populations worldwide, but it ranks second only to breast cancer in incidence and mortality in areas with a low human development index.1 2 Human papillomavirus (HPV) is a DNA virus that exclusively infects the epithelium and promotes the accumulation of genetic damage and uncontrolled cell division by disrupting the cycle control of normal cells.3 HPV infection is firmly established as a necessary but insufficient cause of cervical cancer. Most HPV infections are cleared spontaneously by the immune response of organisms, but persistent infection with high risk HPV (types 16 and 18) accounts for approximately 70% of all cervical cancers.3 4 The occurrence of cervical cancer is not only related to HPV infection but also to the immune system, which plays an essential role in immunosurveillance and immune clearance. Immune function is closely related to T cells, B cells, and natural killer cells. When the levels of CD8+ and CD4+ T cells are reduced, immune function is impaired so that infected cells cannot be removed effectively and in a timely manner, which may worsen the prognosis of patients with cervical intra-epithelial neoplasia (CIN) and cervical cancer.5–7 Natural killer cells and B cells also play a vital role in immune protection, which can effectively eliminate invading HPV and kill infected cells.8 9 Regulatory T cells can inhibit the antitumor ability of cytotoxic T cells and natural killer cells, thereby promoting the development and progression of tumors.6 7 10 Squamous cell carcinoma antigen, carcinoembryonic antigen, carbohydrate antigen 19-9 (CA19-9) and carbohydrate antigen 125 (CA125) are early stage tumor markers commonly used in cervical cancer, and are closely related to the genesis, development, and prognosis of cervical cancer.11–13 The progression from HPV infection to cervical cancer is a long process that takes over 7 years.14 15 Early cervical cancer can be treated surgically, and its prognosis is significantly better than that of advanced cervical cancer.14 Immune functions and tumor marker levels are altered with the genesis and development of cervical cancer. Therefore, their index levels may contribute to the early detection and prevention of cervical cancer.
In this study, we examined the levels of T cell subsets, natural killer cells, B cells, and tumor markers in patients with different cervical lesions, and provide a theoretical basis for the early diagnosis, prevention, treatment, and prognosis assessment of cervical cancer.
This retrospective study included 82 patients with cervical cancer, 54 patients with CIN, and 54 healthy women diagnosed and treated at Shanghai General Hospital, from November 2016 to December 2018. The inclusion criteria were as follows: (1) patients whose cervical lesions were determined based on pathological biopsy; and (2) patients without a prior history of immunotherapy, chemotherapy, or radiotherapy. The exclusion criteria were as follows: (1) patients with a history of other malignant tumors; (2) patients with heart, kidney, and other organ failure; (3) patients with immune diseases; and (4) pregnant or lactating women. All patients were separated into four groups: (1) control group: 54 healthy women; (2) CIN group: 54 patients with CIN; (3) early stage group: 56 patients with cervical cancer stages IA–IB1 and IIA1; and (4) locally advanced group: 26 patients with cervical cancer stages IB2 and IIA2. The hospital ethics committee approved this study.
Examining Peripheral Blood Immune Cells
To analyze immune cell surface markers, the following fluorochrome labeled antibodies were used: CD3, CD16 (R&D Systems), CD4, CD8, CD28, CD56, CD19, HLA-DR, CD45RO, CD45RA, CD29, and CD25 (Cell Signaling Technology). Fasting venous blood (5 mL) was collected from the patient and anticoagulant was added. Blood to be tested (100 μL) was collected in a test tube and thoroughly mixed with 10 µL of fluorochrome labeled antibodies, and was further incubated in a refrigerator at 4°C for 30 min away from light. Then, red blood cell lysis buffer was added, and the sample was mixed thoroughly and left to stand. After full lysis, the red blood cells were centrifuged and washed three times with phosphate buffered saline, and the remaining cells were resuspended in 500 µL of phosphate buffered saline. Eventually, the cells in the suspension were measured by flow cytometry (BD Biosciences Accuri C6, USA).
Assaying Serum Tumor Markers
The procedure for detecting serum tumor markers was as follows: four serum tumor markers were assayed with human squamous cell carcinoma enzyme linked immunosorbent assay (ELISA), human CA125 ELISA, human CA199 ELISA, and human carcinoembryonic antigen ELISA kits (Roche). Venous blood (5 mL) was collected on the first day of admission and allowed to stand at room temperature for natural coagulation. The supernatant was collected after centrifugation. Samples or standards (50 μL) were added to the appropriate wells in the enzyme coated plate and 50 µL of the sample diluent buffer was added to the blank control well. After 5 min of incubation at 37°C, these wells were washed five times, and 50 µL of the enzyme labeled reagent was placed in each well except the blank control well. The plate was washed five times after incubation at 37°C for 30 min. Then, 50 µL of substrate A and 50 µL of substrate B were placed in each well. After incubation at 37°C for 15 min in the dark, 50 µL of stop solution was added to each well and mixed. The optical density value was measured at a wavelength of 450 nm.
Measured data are expressed as mean±standard deviation, and analyzed using one way analysis of variance (ANOVA) or the Kruskal–Wallis test; count data are presented as cases or percentages, analyzed by χ2test or Fisher’s exact test. Statistical analysis was performed by using the Statistic Package for Social Science 24.0 software with p values <0.05 considered statistically significant.
There were no significant differences in clinical factors such as age, pregnancy number, and previous medical history among the four groups (p=0.06, p=0.18, p=0.99, respectively); a relationship between the general clinical variables and the degree of cervical lesions is presented in Table 1.
Serum Tumor Marker Levels
The normal values were defined using an upper limit of 30.2 U/mL for CA125, 5 ng/mL for carcinoembryonic antigen, 1.5 ng/mL for squamous cell carcinoma, and 37 U/mL for CA19-9. The relationship between the levels of serum tumor markers and the degree of cervical lesions is analyzed in Table 2. The proportions of patients with positive squamous cell carcinoma and carcinoembryonic antigen expression in the control group and the CIN group were markedly lower than those in the cervical cancer groups (p<0.001, p=0.007, respectively), and the locally advanced group showed a significantly higher percentage of patients with positive squamous cell carcinoma expression than the early stage group (p<0.001). Furthermore, there were no statistically significant differences in serum CA125 and CA19-9 levels among patients in each group.
Cervical Cancer Groups: Impact of Lymphovascular Invasion and Lymph Node Metastasis
Postoperative pathology examination revealed that 23 (28%) of 82 patients with cervical cancer had a lymph node metastasis and 51 had no lymph node metastasis; the other 8 were of unknown status and were not included in the statistical analysis. Lymphovascular invasion was found in 36 (44%) of 82 patients, and no lymphovascular invasion was found in 46 patients. The correlation between lymph node metastasis and cervical cancer group or lymphovascular invasion was investigated. As shown in Table 3, there was a significant difference in lymph node metastases based on extent of disease: 46.2% of patients with locally advanced cervical cancer had a lymphatic metastasis compared with 22.9% of patients with early cervical cancer (p=0.04). Lymphatic metastasis occurred in 58.8% of patients with lymphovascular invasion compared with 7.5% of patients without it (p<0.001).
Immune Cells in Peripheral Blood
To analyze whether the levels of peripheral blood immune cell subsets were related to the degree of cervical lesions, we examined different types of immune cells in the peripheral blood of healthy women, in patients with CIN, and in those with cervical cancer (Table 4). The levels of CD8+ T cells and CD8+CD28+ T cells in the control group and the CIN group were markedly higher than those in the early stage group (p=0.014, p=0.008, respectively). As CD25+ expression is thought to be critical for distinguishing whether cells are regulatory CD4+ T cells, we assayed the CD4+CD25+/CD4+ T cell ratio in the peripheral blood and found that the ratio in the control group was significantly lower than that in the cervical cancer groups (p=0.005).The increased CD4+CD25+/CD4+ T cell ratio means that immune function is suppressed in patients with cervical cancer. The ratio of CD4+/CD8+ was markedly higher in the early stage group than in the CIN group and the control group (p=0.006). There were no significant differences in the levels of other T cell subsets, natural killer cells, or B cells in the peripheral blood (p>0.05).
Analysis of Relative Factors Between Healthy Women and Patients with Cervical Cancer
Taking the control group and the cervical cancer group as the dependent variables and the age, pregnancy number, levels of serum tumor markers, and levels of immune cell subsets as independent variables, univariate and multivariate binary logistic regression analysis showed that the increased serum squamous cell carcinoma and carcinoembryonic antigen levels and CD4+CD25+/CD4+ T cells ratio in the peripheral blood were risk factors for cervical cancer (p<0.05) (Table 5).
In this study, we found that serum squamous cell carcinoma and carcinoembryonic antigen levels, CD8+CD28+ T cell level, and CD4+CD25+/CD4+ T cell ratios in patients with cervical cancer were significantly different from those in healthy women.
The development of cervical cancer is a long pathological process and usually evolves from pre-invasive lesions into cervical cancer.15 Squamous cell carcinoma is one of the most frequently used early tumor markers for cervical cancer and is often used for monitoring metastasis, recurrence, and prognosis.11 12 Carcinoembryonic antigen is a broad spectrum tumor marker. Several studies have shown that cervical mucus can produce a large amount of carcinoembryonic antigen in patients with cervical cancer, which could be used in the initial evaluation of cervical cancer.13 CA125 is a tumor marker that may be related to lymph node metastasis and prognosis in patients with cervical cancer.12 13 CA19-9 expression on endothelial cells is usually helpful for the diagnosis of gastrointestinal tumors, but some studies have shown that CA19-9 may be related to the clinical stage of cervical cancer.12 13
HPV triggers specific immunity after invading the body, and cellular immunity plays a key role in eliminating HPV so that most HPV infections may be cleared spontaneously.16 The number and proportion of peripheral blood immune cells of normal organisms are stable, but this balance is disrupted during tumor development. Lymphocytes are divided into T cells, B cells, and natural killer cells, according to their function, surface molecules, and migration. CD4+ T cells can directly clear tumor cells through the cytolytic mechanism, and can also indirectly kill tumor cells by regulating the tumor microenvironment. In addition, CD4+ T cells can promote the immune reaction of CD8+ T cells and B cells in secondary lymphoid organs.17 CD8+ T cells are divided into cytotoxic T cells (CD8+CD28+) and suppressor T cells (CD8+CD28-). The activated cytotoxic T cells kill their target cells using two main pathways: (1) the granule exocytosis pathway in which CD8+CD28+ T cells release granzymes to enter target cells and cleave the intracellular matrix after lysing the plasma membrane of target cells by secreting perforin; and (2) the Fas ligand pathway of apoptosis by which Fas on the surface of the target cells binds to Fas ligands which CD8+ CD28+ T cells express, triggering the intrinsic apoptosis mechanism of the target cells.18 CD4+CD25+ and CD8+CD28- T cells are both regulatory T cells that can secrete cytokines to suppress the antigen presenting capacity of dendritic cells, the killing ability of natural killer cells, and the proliferation and cytotoxic effect of T cells, thereby reducing the antitumor response.6 7 10 19 20 B cells can form a stable relationship with T cells as antigen presenting cells, and can also secrete cytokines to cooperate with other immune cells, especially to enhance the activity of cytotoxic T cells.7 21 Natural killer cells can non-specifically kill virus infected cells and tumor cells without prior sensitization by secreting cytokines and perforins.9 22 The analysis of the proportion of lymphocytes in the peripheral blood helps to understand immune function and provides evidence for clinical treatment.
As shown in our study, the proportions of patients with positive squamous cell carcinoma and carcinoembryonic antigen expression in the control group and the CIN group were significantly lower than those in the cervical cancer groups. Meanwhile, the locally advanced cervical cancer group showed a markedly higher percentage of patients with positive squamous cell carcinoma expression than the early stage group. The results show that serum squamous cell carcinoma and carcinoembryonic antigen levels may be used as auxiliary indicators in the evaluation of cervical cancer, and may have significance for the early detection of cervical cancer. As it pertains to immune modulators, we showed that compared with the control group and the CIN group, the early stage group had significantly lower CD8+CD28+ T cell and CD8+ T cell levels, suggesting that the immune capacity of patients with cervical cancer is weakened. This may be because the patient’s immunity has been reduced over a period of time, leading to ease of infection with HPV, gradually evolving into cervical cancer. This may also be because the mechanism of tumor immune escape inhibits the proliferation of antitumor effector cells. Although the levels of CD8+CD28+ T cells and CD8+ T cells in the locally advanced group were lower than those in the control group, the differences were not statistically significant, which may be related to the small number of cases analyzed in this study. The cervical cancer groups showed a significantly higher level of CD4+CD25+ cells than the control group, indicating that an increased level of cells that negatively regulate immune function can reduce the level and activity of antitumor effector cells, contribute to tumor immunity escape, and may lead to the continuous deterioration of the disease. The ratio of CD4+/CD8+ T cells was higher in the early stage group than that in the CIN group and the control group. This may be related to the decrease in the number and activity of CD8+ T cells in patients with cervical cancer. Further application of univariate and multivariate binary logistic regression analyses showed that the increased serum squamous cell carcinoma and carcinoembryonic antigen levels and CD4+CD25+/CD4+ T cell ratios were risk factors between healthy women and patients with cervical cancer. This result suggests that the levels of serum squamous cell carcinoma and carcinoembryonic antigen and the ratio of CD4+CD25+/CD4+ T cells may be used as an indicator in the assessment of cervical cancer.
In summary, the immune function of patients is impaired in cervical cancer, as shown by the proportion of lymphocytes in the peripheral blood. Combined detection of lymphocytes and serum tumor markers can provide a reference for early diagnosis and prognosis of patients with cervical cancer. However, these two indicators can only be used as auxiliary diagnostic indicators for tumorigenesis and progression, and there are differences in these indicators among patients. Thus diagnosis must be based on histopathological examination. In the future, more cases should be collected and analyzed to evaluate the conclusion of this study.
Contributors YS formulated the research protocol and guided the work. LZ collected and analyzed the data for this study and wrote the article, and is the first author. HZ and YH collected the data for this study. XX guided the work.
Funding This study was funded by the National Natural Science Foundation of China (NSFC) (grant: 81772768). The content only expresses the author's views and is not the responsibility of the NSFC.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval The hospital ethics committee approved this study.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available upon reasonable request. All data, models, or code generated or used during the study are available from the corresponding author by request, such as revalidation of data reliability.