Peripheral blood circulating tumor cells and glycan antigen 153 as biomarkers for diagnosis and prediction of prognosis in breast cancer: a retrospective study

doi:10.21203/rs.3.rs-3554632/v1

Background

Our aim was to study the diagnostic and prognostic value of circulating tumor cells (CTC) and glycan antigen 153 (CA153) expression in breast cancer patients.

Methods

171 breast cancer patients were collected as an observation group, and another 126 cases of benign breast lesions in the same period were selected as a control group, to analyze the difference and correlation between the expression of CA153 and CTC in the two groups, and to compare the diagnostic value of CA153, CTC, and the combined detection for breast cancer. Finally, CTC expression was analyzed in relation to different clinicopathological parameters of breast cancer and CA153.

Results

The CTC positivity rate and CA153 expression level of the observation group were significantly higher than those of the control group, and Point-biserial analysis showed that there was a positive correlation between CTC and CA153 (r = 0.53,P༜0.01); the combined detection of CTC and CA153 had a higher sensitivity than that of the independent detection, and a lower specificity than that of the independent detection, and the combined detection of Youden's-index, Kappa-index, and Kappa-index was more effective in diagnosing breast cancer. index) and Kappa value were the highest at 0.8 and 0.79, respectively, which had higher diagnostic value; CTC expression was related to choroidal infiltration, TNM staging, and CA153 expression (P ༜0.05).

Conclusions

The expression of CA153 and CTC in breast cancer was significantly elevated and correlated, and the combined detection of the two had a higher diagnostic value for breast cancer, and CTC detection predicted the prognosis of breast cancer.

Breast cancer

Circulating tumor cells

Carbohydrate antigen 153

Biomarker

The 2022 National Cancer Center report shows that breast cancer is still one of the most common malignant tumors among women in China, which greatly affects women's physical and mental health.Lebron-Zapata et al [1] Numerous scholars have shown that patients with early-stage breast cancer have a good prognosis and a high survival rate, and the prognosis is correlated with the time of diagnosis. As the symptoms of early breast cancer are not obvious, the effect of early diagnosis of breast cancer is still unsatisfactory. Currently, the widely used blood tumor markers, imaging and pathology examinations have certain limitations. The specificity and sensitivity of blood tumor marker screening is poor, imaging examination is lagging behind and has radiation hazards, and pathology examination is invasive and the specimen is not easy to obtain, which makes it difficult to dynamically assess the disease condition. The authors are searching for a method that is more effective and less hazardous for diagnosis, and can be used for multiple screening and dynamic detection. Peripheral blood circulating tumor cell (CTC) is an important marker in the process of tumor metastasis, which can be used to predict the early stage of breast cancer, and also used in the late stage of treatment decision-making and prognostic assessment [2, 3], the 8th edition of AJCC Breast Cancer specifies and recommends the detection of CTC in breast cancer and its clinical value. The diagnostic efficacy of glycan antigen 153 (CA153) in breast cancer serologic screening index is high [4, 5]. In this study, we analyzed the diagnostic and prognostic value of CTC and CA153 expression in breast cancer patients. This study analyzes the diagnostic and prognostic value of CTC and CA153 expression in breast cancer patients, in order to obtain a higher diagnostic and prognostic method for breast cancer, and to improve the therapeutic effect and survival rate of breast cancer patients.

Ethics approval and consent to participate

This study is approved by the Ethics Committee of Foshan Fosun Chancheng Hospital (approval no.CYIRB-LCYJ-2022009-PJ-20220408).Informed consent was obtained from all patients.

Patients

Collect 171 breast cancer patients who were treated in our hospital between May 2022 and July 2023 as the observation group, with an average age of (53.49±9.53) years old. Inclusion criteria: ① pathological diagnosis of breast cancer; ② no combinations of other malignant tumors; ③ did not receive anti-tumor treatment; ④ complete clinicopathological data. Exclusion criteria: ① leukemia, lymphoma, abnormal elevation of white blood cells, bone marrow transplantation, infusion of leukocyte-containing preparations within 1 month;② pregnant women, lactation. Another 126 cases of benign breast lesions in the same period were selected as the control group, with an average age of (52.67±9.76) years old, and histopathological diagnosis of 19 cases of breast cysts, 6 cases of breast adenopathy, 78 cases of breast fibroadenomas, 23 cases of ordinary ductal hyperplasia of the breast, and 11 cases of intraductal papillomas of the breast. The data of the study subjects collected by our information system contained age, pathologic staging, pathohistologic grading, size of the mass, clinical stage, lymph node metastasis, vascular infiltration, nerve bundle invasion, expression of immunohistochemical indexes, molecular typing, CTC results, and CA153 results. The selected study subjects were all females, and there was no statistical difference between the two groups compared to age (P > 0.05).

Instruments and reagents

The CTC was detected in the peripheral blood samples of the enrolled patients by immunomagnetic bead enrichment (negative cell enrichment) combined with immunofluorescence in situ hybridization (imFISH) kit using the patented technology independently innovated by Jiangsu Lail, and the results were interpreted by Zeiss Z1 fluorescence microscope.CA153 was detected by the kit of Shenzhen New Industry Biomedical Company, and the results of serum CA153 were measured by Maglumi 4000 automatic chemiluminescence assay. CA153 was detected using the kit of Shenzhen New Industry Biomedical Company, and the results of serum CA153 were determined by Maglumi 4000 automatic chemiluminescence assay.

Methods

CTC detection method and interpretation of results 5ml of peripheral blood was collected from patients with ACD anticoagulant, transported at room temperature, and peripheral blood CTC was detected by trained laboratory technicians in strict accordance with the instruction manual of the kit within 24 h. Peripheral blood cells should meet the following four conditions (①single cell, homogeneous nucleus, no delamination found; ②three or more signal dots on the nucleus;③no CD45 coloration under the red channel; ④magnetic beads on the cell surface≤1), and were judged to be CTCs. No CD45 coloration in the red channel;④≤1 magnetic bead on the cell surface), it will be judged as CTC.≥CTC/3.2 ml in peripheral blood will be judged as positive, otherwise, it will be judged as negative.

CA153 detection method and interpretation of results 2ml of venous blood was collected from patients and serum was taken, and CA153 was detected in strict accordance with the detection kit of Shenzhen New Industry Biomedical Company, the reference value of which was <30IU/ml, and the results of CA153≥30IU/ml were defined as positive in this study, and vice versa was defined as negative.

Statistical analysis

All data were statistically processed using SPSS 26. Measurement data were expressed as (x±s) when they conformed to normal distribution, and t-test was used for comparison between two groups. Count data were expressed as frequency (%), and comparisons between two groups were performed using theχ²test or the continuous corrected chi-square test, and comparisons of statistical differences in data between multiple groups were performed using the χ²test in a group design row-multiplication table. Point-biserial correlation was used to analyze the correlation between the two categorical and continuous variables. Kappa test was used to analyze the consistency of the diagnosis of breast cancer by CTC, CA153 and combined test, the diagnostic consistency was excellent when Kappa≥0.75, good when 0.75>Kappa≥0.41, and poor when Kappa≤0.4. Factors with P<0.1 in the univariate analysis were included in the multivariate analysis, and the multivariate analysis was analyzed by the binary logistic regression model; the test level of α= 0.05, and the difference was considered statistically significant at P < 0.05 (two-sided).

Difference in the expression of CTC and CA153 in the observation group and the control group

CTC positive expression was detected in 134 cases in the observation group, with a positive rate of 78.4%, and 9 cases in the control group, with a positive rate of 7.1%, and the difference in the expression of CTC between the two groups was statistically significant (P＜0.05).The level of expression of CA153 in the observation group was (63.6±16.3) IU/ml, which was higher than that in the control group (32.4±6.4) IU/ml. higher than the expression level (32.4±6.4) IU/ml in the control group, and the comparison between the two groups was statistically significant (P < 0.05)（Table 3）.

Correlation between CTC and serum CA153 level

Point-biserial correlation analysis Correlation between CTC expression and serum CA15-3 level showed that there was a significant correlation between CTC and serum CA153 (P < 0.01), with a correlation coefficient of r = 0.53, a It was positively correlated.

Comparison of the diagnostic efficacy of CTC, CA15-3, and combination tests versus individual tests for breast cancer diagnosis

Comparing the diagnostic efficacy of CTC, CA15-3 and combined detection with separate detection for breast cancer using pathological diagnostic results as the standard, the sensitivity of combined diagnostic was higher than that of CTC and CA15-3 detection, and the difference with the independent detection methods was statistically different (χ2value was 21.18, 5.95, respectively, P<0.05), the specificity of combined diagnostic was lower than that of CTC and CA15-3 detection, and the difference with the independent detection methods was not statistically significant (P<0.05). The specificity of combined diagnosis was lower than that of CTC and CA15-3, and the difference with independent detection methods was not statistically significant (P<0.05).The Youden's-index of the 3 diagnostic methods was the highest in the combined test at 0.8, and the comparison of Kappa analysis revealed that the combined test was higher than that of CTC and CA153 at 0.79（Table 2）.

Univariate analysis of the relationship between CTC and CA153 and different clinicopathologic features in breast cancer patients

Taking the enrolled breast cancer patients as the study subjects, further analysis of CTC expression in relation to CA153 and different clinicopathological features showed the results. There were statistically significant differences in TNM stage, vascular infiltration, Her2 expression, and CA153 expression ( P<0.05), and the positive CTC detection rate of patients with stage III/IV, vascular infiltration, Her2 positivity, and CA153 positivity was significantly higher than that of patients with stage I-II, uninfiltrated vasculature, Her2-negative, and CA153-negative; the differences were not statistically significant ( P＞0.05) in different age, histopathologic type, nerve bundle invasion, ER expression status, PR expression status, and Ki-67 expression status, the differences were not statistically significant ( P>0.05)（Table 3）.

Multifactorial Logistic Regression Analysis of CTC and Clinicopathologic Characteristics of Breast Cancer

CTC expression status of breast cancer patients was used as the dependent variable, while choroidal infiltration status, clinical TNM staging, HER-2 expression status and CA153 status were included in multifactorial logistic regression analysis with a univariate analysis of P<0.1. The results showed:The risk of CTC positive expression was increased and statistically significant in patients with vascular infiltration compared with patients without infiltration, in patients with clinical TNM stage III/IV compared with stage I/II, and in CA153-positive patients compared with CA153-negative patients (P<0.05); the difference in CTC positive expression was not statistically significant in HER2-positive patients compared with negative patients (P>0.05)（Table 4）.

Early diagnosis of breast cancer has significantly improved the survival rate, but there is still a risk of recurrence and metastasis. Therefore, finding biomarkers with higher diagnostic efficacy is particularly important for breast cancer, and exploring methods to dynamically detect the risk of recurrence and metastasis in the breast cancer population is essential for improving clinical efficacy and prognosis [6].CTC refers to the tumor cells that enter the peripheral bloodstream from the site of origin either spontaneously or as a result of diagnostic and therapeutic operations. As a new tumor liquid biomarker, it has been widely used in the clinic because it is minimally invasive, timely, accurate, and can achieve dynamic multiple detection [7-10] Currently, it is mainly used for early screening of breast cancer, lung cancer, and intestinal cancer, as well as for prognostic assessment of tumors and determination of treatment options. Among the traditional serologic diagnostic markers for breast cancer, the CA153 index has a higher sensitivity [11] and has long been applied to the screening of the physical examination population.

In this study, we collected clinicopathologic parameters of 171 breast cancer patients and 126 patients with benign breast lesions diagnosed by histopathology, as well as CTC expression and CA153 detection results. Analyzing the differences in the expression of CTC and CA153 in the observation group and the control group, it was found that there was a significant difference in the expression of CTC and the expression level of CA153 between the two groups, and the positive expression of CTC and the expression of CA153 in the observation group were significantly higher than those in the control group, and this result was in line with the relevant reports [12,13]. Therefore, the author further used Point-biserial analysis of the correlation between CTC and serum CA153 levels, and the results showed that there was a positive correlation between CTC and serum CA153 (r = 0.53, P < 0.01), and the conclusion also confirmed that CTC and CA153 expression were statistically significant in the observation group and the control group, and therefore, CTC expression could be initially inferred from serum CA153. CTC expression was inferred, but the causal relationship between CTC and CA153 was not analyzed. Comparative analysis of the diagnostic efficacy of CTC, CA15-3 and combined test for breast cancer showed that the sensitivity of combined diagnosis was higher than that of CTC and CA15-3, and the specificity of combined diagnosis was slightly lower than that of CTC and CA15-3, which indicated that the combined test had a higher sensitivity and specificity in the diagnosis of breast cancer. The Jordon's index of the three diagnostic methods was 0.8, indicating that the accuracy of the combined test was higher than that of the two independent tests in identifying patients with true breast cancer and non-breast cancer. Meanwhile, Kappa analysis showed that the combined test was higher than CTC and CA153 at 0.79, indicating that the consistency of the combined test in diagnosing breast cancer was excellent compared with the consistency of the gold-standard pathologic diagnosis of breast cancer. This further confirms the clinical value of the combined test, which is consistent with Jafari et al [14,15], who concluded that the combined test contributes to both diagnostic sensitivity and specificity.

In this study, we also analyzed the relationship between CTC expression and CA153 and different clinicopathological parameters in breast cancer patients, and the results showed that CTC expression was related to choroidal infiltration, TNM stage, CA153 expression, and HER-2 expression, and it was not related to age, histopathological type, nerve bundles invasion, ER expression, PR expression, and Ki-67 expression. The results were partially consistent with related studies [16-18,13,12]. Further multifactorial logistic regression analysis revealed that CTC expression was associated with vascular infiltration, TNM stage, and CA153 expression, and that patients with vascular infiltration had an increased risk of positive CTC expression compared with patients without infiltration, patients with clinical TNM stage III/IV compared with stage I/II, and CA153-positive patients compared with CA153-negative patients. Analysis of the reasons vascular infiltration may be a support for the formation of CTC blood circulation, due to the tumor's ability to produce vascular growth factors, which promote vascular proliferation and provide the nutrients needed for tumor growth. CTC entering the bloodstream self-aggregates or combines with blood-related components, which in turn forms cancer thrombi; TNM stage III/IV indicates advanced breast cancer, which has a higher degree of malignancy, poorer prognosis, and a higher risk of tumor blood shaped metastasis; CA153 is not only being used as an early screening indicator of breast cancer, but also is significantly elevated in recurrent metastasis, which has a close relationship with its prognosis [14].The CTC expression is not correlated with the CTC expression is not related to HER-2 status, which is inconsistent with [19], probably due to the different detection methods used and study populations.

In conclusion, the expression of CA153 and CTC in breast cancer patients is significantly elevated and correlated, and the combined detection of the two has a high diagnostic value for breast cancer, and CTC is related to clinicopathologic parameters and prognosis, so the detection of CTC can help to dynamically assess the prognosis of breast cancer, and improve the therapeutic efficacy and survival rate of breast cancer patients. Limitations of this study include (1) healthy controls were not included, and there was selection bias in the retrospective study; (2) the peripheral blood CTC detection method is still defective, mainly in CTC enrichment and identification. In the future, CTC can be detected by prospective, more sensitive and specific identification methods, and the relationship between CTC and the treatment and prognosis of breast cancer patients can be further analyzed by long-term follow-up.

Acknowledgments The author thanks the hospital's data support and all the researchers who participated in this study for their help. We also thank the reviewers and editors for their hard work and perseverance.

Author contributions All authors contributed to the study conception and design.Material,

preparation,data collection and analysis were performed by Peiwen Ouyang, Shaoxiong Zhou, Zhaoxing Sun, and Kang Yang. The frst draft of the manuscript was written by Peiwen Ouyang and Kang Yang supervised the study and revised the manuscript.All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding This study was supported by the Medical Research Fund of Foshan Municipal Health Bureau (No.20230226) and the Fourteenth Five-Year Medical Key Specialty Program of Foshan City (Foshan Health Letter [2021] No.107).

Data availability The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest The authors declare no competing interests.

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Table 3 Relationship between CTC expression status and CA153 and different clinicopathologic features of breast cancer

Clinicopathologic features

Cases

CTC expression

χ²value

P value

Positive（n）

Negatives（n）

Age

≤45 years

＞45 years

52

119

39

95

13

24

0.948

0.33

Histopathological type Invasive ductal carcinoma

Invasive lobular carcinoma

Hybrid

Other types

105

14

44

8

84

9

35

6

21

5

9

2

1.705

0.636

Vascular infiltration

Yes

No

105

66

97

37

8

29

31.53

＜0.01

Nerve bundle invasion

Yes

No

11

29

56

78

18

19

0.555

0.456

TNM staggered

Ⅰ/Ⅱstage

Ⅲ/Ⅳstage

51

120

19

115

29

5

67.204

＜0.01

ER expressions

Positive

Negatives

126

45

93

38

33

9

0.923

0.337

PR expressions

Positive

Negatives

109

62

82

50

27

12

0.658

0.414

HER2 expressions

Positive

Negatives

43

128

39

95

4

33

5.155

0.023

Ki-67 expressions

Positive

Negatives

119

52

95

41

24

11

0.001

0.979

CA153

Positive

Negatives

107

64

93

41

14

23

12.335

＜0.01

Table 4 Multifactorial Logistic Regression Analysis of CTC Expression and Pathological Characteristics of Breast Cancer

Variant	β	SD	P value	OR value	95%CI
Vascular infiltration	1.623	0.707	0.022	5.067	（1.268,20.245）
Clinical TNM staging	1.611	0.605	0.008	5.009	（1.531,16.39）
HER2 expressions	-1.193	0.846	0.158	0.303	（0.058,1.592）
CA153 expressions	2.613	0.696	＜0.01	13.643	（3.488,53.366）

Peripheral blood circulating tumor cells and glycan antigen 153 as biomarkers for diagnosis and prediction of prognosis in breast cancer: a retrospective study

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

Results

Discussion

Declarations

References

Tables

Status:

Version 1