Cell Biochem Biophys DOI 10.1007/s12013-014-0345-2
ORIGINAL PAPER
Serum CA242, CA199, CA125, CEA, and TSGF are Biomarkers for the Efficacy and Prognosis of Cryoablation in Pancreatic Cancer Patients Ye Chen • She-Gan Gao • Jian-Min Chen • Gong-Ping Wang • Zeng-Fang Wang Bo Zhou • Can-Hui Jin • Yan-Tong Yang • Xiao-Shan Feng
•
Ó Springer Science+Business Media New York 2014
Abstract This study aimed to compare the changes and determine the clinical significance of carbohydrate antigens CA242, CA199, CA125, carcinoembryonic antigen (CEA), and tumor-specific growth factor (TSGF) before and after cryoablation by Cryocare system. Thirty-one pancreatic cancer patients were selected to receive cryoablation by Cryocare system. The serum expression levels of CA242, CA199, CA125, CEA, and TSGF before and 1 month after treatment were determined. Meanwhile, the serum level of these factors was also determined in 31 healthy volunteers. The parameter changes were analyzed with the clinical pathological data. The serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). The serum CA199, CEA, and TSGF dramatically decreased 1 month after the treatment, which were statistically different (P \ 0.05). The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). The positive rate of CA199, CEA, and TSGF after the treatment was significantly lower than that before the treatment (P \ 0.05). Serum level of CA242 was correlated with the tumor diameter, clinical staging, tumor differentiation, lymph node, and liver metastasis (P \ 0.05). Except
Y. Chen S.-G. Gao J.-M. Chen G.-P. Wang Z.-F. Wang B. Zhou C.-H. Jin Y.-T. Yang X.-S. Feng (&) Department of Oncology Surgery, The First Affiliated Hospital of Henan University of Science and Technology, 24 Jinghua Road, Jianxi District, Luoyang, Henan Province, China e-mail: [email protected]
gender, CA199 was correlated with all the other clinical pathological parameters (P \ 0.05). The serum levels of CA125 and CEA were correlated with all the other clinical pathological parameters (P \ 0.05). The serum level of TSGF was only correlated with tumor differentiation (P \ 0.05). Cryoablation treatment by Cryocare system can decrease the serum levels of CA199, CEA, TSGF, and the positive rate. Serum CA199, CEA, and TSGF can be important index for pancreatic cancer treatment assessment. Serum levels of CA242, CA199, CA125, and CEA are of great clinical value for metastasis assessment and prognosis in pancreatic cancer patients. Keywords Cryocare cryoablation Carbohydrate antigen Carcinoembryonic antigen Tumor-specific growth factor Pancreatic cancer
Introduction Pancreatic cancer is a common digestive malignancy of very high incidence. Due to its occult nature, diagnostic difficulties, rapid development, and high mortality, it is often beyond surgical removal when the patients are diagnosed [1, 2]. Cryoablation by Cryocare system has been widely used in breast cancer, liver cancer, kidney cancer, etc., which provides a new therapeutic tool for benign and malignant tumors that cannot be surgically removed [3, 4]. Previous studies showed that serum carbohydrate antigens including CA242, CA199, CA125, carcinoembryonic antigen (CEA), and tumor-specific growth factor (TSGF) are important tumor biomarkers for the early diagnosis of pancreatic cancer [5–7]. We determined the serum and tumor expression levels of tumor biomarkers CA242, CA199, CA125, CEA, and TSGF in 31
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selected pancreatic caner patients before and after cryoablation by Cryocare system. We also analyzed the changes of these five biomarkers and evaluated the clinical value of these biomarkers in the diagnosis, prognosis, and assessment of pancreatic cancer.
cryoablation lasts for 10–15 min until freezing area exceeds the tumor border by 0.5 cm. After the argon is stopped, helium is infused under 2,500 psi pressure, which is stopped until the frozen area thaw for 3–5 mins with temperature reaching to 10–20 °C. After the probes are reset, freeze–thaw cycle is repeated. All the patients received either double or multiple cycles of the cycles.
Materials and Methods Sample Collection and Marker Detection Subjects We selected 31 pancreatic patients admitted to our hospital for hospitalization and treatment between February 2013 and February 2014. All the diagnoses were confirmed by emission tomography and X-ray computed tomography and positron diagnosis, and all cases were further histologically confirmed by pathology and received subsequent cryoablation. There were 17 male patients and 14 female patients. The patients are aged between 32 and 75 years old (average 57.2 ± 8.0). We staged the cancer according to the clinical staging of pancreatic cancer (stage I: Tumor confined to the pancreas; Stage II: Tumor invading adjacent tissue only; Stage III: existing regional lymph node metastasis; Stage IV: existing liver and other distant metastasis). There was one stage I case, six Stage II cases, 10 stage III cases, and 10 Stage IV cases. Thirty-one healthy volunteers were randomly selected as the control group. These volunteers (16 males and 15 females aged between 35 and 60 years old, average 56.4 ± 8.2 years old) have no history of cancer, cardiovascular, liver, or kidney diseases. There was no statistical difference (P [ 0.05) between two groups in term of general clinical data, such as gender, age, etc. The expression levels of CA242, CA199, CA125, CEA, and TSGF before and 1 month after the treatment were determined. The results were then analyzed with different parameters and general clinical data. Methods After cryoablation needle, refrigeration efficiency has been checked, and puncture devices will be prepared. With local anesthesia, the selected patient was set at the appropriate position according to the tumor site. Under the guidance of CT, ultrasound probe is inserted through the peritoneum to the tumor site. Considering tumor location, size, and the surrounding structures, cryoablation needle 2 mm in diameter is inserted into the tumors. The number of the probes needed was proportional to the tumor sizes. The general guideline is as follows: 1 probe for tumor \2 cm, 2 probes for tumors between 2 and 4 cm, and 3 or 4 probes for tumors C5 cm. After the probes are set under CT guidance, cryoablation starts with argon infusion under 3,500 psi pressure. The
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Fasting venous blood was collected in the morning in both groups. Patients in the pancreatic cancer group received blood collection before and 1 month after the surgery. Serum was separated by centrifugation and frozen for storage. Serum CA242 was determined by radioimmunoassay. CA radioimmunoassay kits were purchased from China Institute of Oncology Branch of the company. CEA radioimmunoassay kits were purchased from Northern China Institute of Isotopes Company. The c212 counter was purchased from the U.S. DPC Company, and the positive threshold was 20 U/ml. CA199 and CA125 were determined by Access from Kurt company (USA) and Bayer Roche chemiluminescence instrument for quantitative analysis of protein chemistry, and the positive threshold was 35 U/ml. CEA was assayed with radioimmunoassay kit (IRMA) by Beijing Dong Ya biotechnology company, and the positive threshold was 5 ng/ml. TSGF was determined by Hitachi 7060 automatic biochemical analyzer, and the positive threshold was 64 U/ml. All the assays were performed according to manufacturers’ instructions by professionals. Statistical Analysis SPSS20.0 statistical software was used for data entry and analysis. The measurement data were expressed as mean ± standard deviation (x ± s), and counting data were expressed as rates. Measurement data were compared using t test. Counting data were compared with v2 test. P values \0.05 were considered as statistically significant.
Results Comparison of Serum Tumor Biomarkers Before and After Treatment in Control and Pancreatic Cancer Groups The serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). Serum CA199, CEA, and TSGF decreased significantly 1 month after treatment in
Cell Biochem Biophys Table 1 Comparison of serum tumor biomarkers before and after treatment in control and pancreatic cancer groups Group
n
CA242 (U/ml)
CA199 (U/ml)
CA125 (U/ml)
CEA (ng/ml)
TSGF (U/ml)
18.6 ± 3.2*
17.0 ± 1.0*
Pancreatic cancer treatment before
31
98.6 ± 19.7*
1,501.2 ± 764.1*
621.3 ± 206.4*
Pancreatic cancer treatment after
31
85.7 ± 10.6*
1,062.8 ± 631.6*,#
640.7 ± 201.8*
Control
31
11.5 ± 2.3
* Compared with control P \ 0.05;
#
21.3 ± 5.5
22.4 ± 3.1
9.9 ± 2.5*,# 2.5 ± 0.6
14.1 ± 0.9*,# 4.3 ± 1.0
compared with pancreatic cancer group before treatment P \ 0.05
Table 2 Comparison of positive rate for the serum biomarkers before and after the surgery in control and pancreatic groups [n(%)] Group
n
CA242
CA199
CA125
CEA 15 (48.4)*
Pancreatic cancer treatment before
31
23 (74.2)*
29 (93.5)*
23 (74.2)*
Pancreatic cancer treatment after
31
23 (74.2)*
22 (71.0)*,#
23 (74.2)*
31
2 (6.5)
Control * Compared with control P \ 0.05;
#
2 (6.5)
0 (0.0)
TSGF 8 (25.8)*
7 (22.6)*,#
0 (0.0)#
1 (3.2)
0 (0.0)
compared with pancreatic cancer group before treatment P \ 0.05
the pancreatic group compared with pre-surgical results, which were statistically different (P \ 0.05). There was no difference of CA242 and CA125 before and after surgery in the pancreatic cancer group (P [ 0.05, Table 1). Comparison of Positive Rate for the Serum Biomarkers Before and After the Surgery in Control and Pancreatic Groups The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). The positive rate of CA199, CEA, and TSGF after the treatment was significantly lower than that before the treatment (P \ 0.05). There was no difference in the positive rate of serum CA242 and CA125 before and after the treatment in the pancreatic cancer group (P [ 0.05, Table 2). The Correlations of Serum Biomarker Levels and the General Clinical Data in the Pancreatic Cancer Group Before and After the Surgery The serum CA125 and CEA in males were significantly higher than those in females (P \ 0.05, Table 3), and there was no statistical significant for other tumor biomarkers (P [ 0.05). Serum CA199, CA125, and CEA in patients older than 60 were significantly higher than those in patients younger than 60 (P \ 0.05), and the serum levels of CA242 and TSGF were not correlated with age (P [ 0.05). Except TSGF serum level, patients with tumors [4 cm or staged between III and IV exhibited significantly higher serum CA242, CA199, CA125, and CEA than those
with tumors B4 cm or staged between I and II (P \ 0.05). All serum biomarkers were higher in patients with tumor differentiation staged between III and IV than those staged between I and II (P \ 0.05). Except serum TSGF, the serum levels of CA242, CA199, CA125, and CEA in patients with lymph node or liver metastasis were significantly higher than those without (P \ 0.05).
Discussion With features of high degree of malignancy, insidious onset, rapid development, easy metastasis, poor prognosis, and as well as low survival rates, etc. the pancreatic cancer lacks of specific clinical manifestations, which makes difficulties for early diagnosis and treatment [8]. Discovery of tumor biomarker for pancreatic cancer can potentially provide better early diagnosis and prognosis prediction [9]. Currently, there are several tumor biomarkers for the clinical diagnosis and classifications of pancreatic cancer, which increase the general diagnosis and treatment of the disease [10]. At present, there are over 10 tumor biomarkers for clinical diagnosis, but only few has been proven to be of clinical value, among which CA242, CA199, CA125, CEA, and TSGF are included. CA242 is mainly present in the malignant pancreatic and colon cancer cells [11]. It has been shown that CA242 is elevated in pancreatic cancer cells, and the expression level of which is correlated with the development of pancreatic cancer and other GI tract cancers [12]. CA199 is expressed in pancreatic ducts, acinar cells, and salivary gland cells. Because of its high positivity and sensitivity, it is often used for clinical diagnosis, which is of great clinical value for the diagnosis and prognosis of pancreatic cancer [13]. CA125 is a macromolecular glycoprotein, and it has been
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Cell Biochem Biophys Table 3 The correlations of serum biomarker levels and the general clinical data in the pancreatic cancer group before and after the surgery Groups
n
CA242 (U/ml)
Male
17
114.2 ± 22.0
Female
14
98.9 ± 19.9
CA199 (U/ml)
CA125 (U/ml)
CEA (ng/ml)
1,638.5 ± 785.2
698.7 ± 258.6
21.4 ± 4.0
1,456.2 ± 758.5
403.2 ± 198.9*
TSGF (U/ml)
Gender 5.3 ± 2.0*
17.2 ± 0.9 16.6 ± 1.1
Age (years) B60
11
118.3 ± 22.8
1,069.2 ± 587.6
400.5 ± 200.3
6.6 ± 2.9
16.7 ± 1.1
[60
20
103.9 ± 18.4
1,925.6 ± 789.8*
687.6 ± 215.6*
21.5 ± 3.3*
17.3 ± 1.0
Tumor diameter (cm) B4
13
73.6 ± 18.4
1,002.9 ± 587.9
389.6 ± 203.2
10.6 ± 4.8
16.6 ± 1.0
[4
18
142.0 ± 18.0*
1,893.6 ± 885.6*
703.9 ± 241.2*
19.8 ± 4.3*
17.3 ± 1.1
6 25
45.6 ± 16.0 126.0 ± 15.4*
1,023.0 ± 563.2 2,005.6 ± 876.8*
300.4 ± 187.6 721.4 ± 250.0*
4.9 ± 1.2 20.6 ± 4.0*
16.5 ± 0.9 17.2 ± 1.2
Staging I–II III–IV
Tumor differentiation staging I–II
7
92.5 ± 22.5
1,132.3 ± 578.6
312.6 ± 200.2
5.1 ± 1.0
15.5 ± 0.8
III–IV
24
117.3 ± 17.7*
1,987.6 ± 678.9*
714.5 ± 245.3*
21.8 ± 3.9*
17.6 ± 1.2*
Lymph node metastasis No
9
89.0 ± 22.5
1,200.8 ± 670.5
356.6 ± 198.5
4.6 ± 1.0
16.7 ± 1.1
Yes
22
125.9 ± 17.6*
1,987.3 ± 810.2*
698.9 ± 256.3*
21.0 ± 6.3*
17.5 ± 1.1
Liver metastasis No
12
88.0 ± 23.4
1,212.3 ± 663.2
380.2 ± 200.0
12.8 ± 5.0
16.4 ± 1.3
Yes
19
126.4 ± 17.7*
1,988.6 ± 789.8*
701.3 ± 260.0*
17.0 ± 5.2*
17.2 ± 0.9
* Statistically different, P \ 0.05
shown that serum CA125 expression level is elevated in pancreatic cancer patients [14], and thus CA125 has been used for pancreatic cancer diagnosis. CEA was derived from epithelial tissue in endoderm, mostly present in the gastrointestinal tract, urinary tract, respiratory tract, etc., and it is often used clinically for the diagnosis of digestive system tumor [15]. Recently, TSGF has been shown to correlate with the angiogenesis of malignant tumors, which is useful for tumor diagnosis. It has also been shown that TSGF is elevated in pancreatic cancer patients [16]. Clinically, individual sensitivity of specificity of these five biomarkers is not sufficient, but the combination of these markers can significantly increase the sensitivity and specificity. Therefore, we studied the serum levels of CA242, CA199, CA125, CEA, and TSGF before and after cryoablation in pancreatic cancer patients. Our results showed that the serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). These results showed that the expression levels
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of these five biomarkers are correlated with the occurrence and development of pancreatic cancer, which is consistent with previous reports. Recently, cryocare system has been widely used in the treatments of several tumors. As an in situ ablation treatment technique, it provides hope of improvement or even cure for patients who missed the surgical removal opportunities [17]. We showed that serum CA199, CEA, and TSGF levels and positive rates were significantly lower than those before treatment (P \ 0.05), which was consistent with previous reports [18]. It is possible that serum levels of CA199, CEA, and TSGF could help predict the changes of condition or prognosis of pancreatic cancer patients after cryoablation. We also analyzed the correlations of serum biomarker levels and the general clinical data in the pancreatic cancer group before and after the surgery. Our results showed that the serum CA125 and CEA in males were significantly higher than those in females (P \ 0.05). Serum CA199, CA125, and CEA in patients older than 60 were significantly higher than those in patients younger than 60 (P \ 0.05). These results showed that gender and age are correlated with the occurrence and development of pancreatic cancer, which is in agreement with previous reports [19]. The patients with tumors[4 cm or staged between III and IV exhibited significantly higher serum CA242,
Cell Biochem Biophys
CA199, CA125, and CEA than those with tumors B4 cm or staged between I and II (P \ 0.05). We also showed that the invasiveness of pancreatic cancer is correlated with serum levels CA242, CA199, CA125, and CEA. We also showed that the serum levels of CA242, CA199, CA125, and CEA in patients with lymph node or liver metastasis were significantly higher than those without (P \ 0.05). All serum biomarkers were higher in patients with tumor differentiation staged between III and IV than those staged between I and II (P \ 0.05), which also proved these points. These results showed that higher serum levels of CA242, CA199, CA125, and CEA in pancreatic cancer patients were correlated with poor prognosis and distant metastasis. This could be due to adhesion molecule nature of CA242, CA199, CA125, and CEA, which can affect the development and metastasis of pancreatic cancer cells, which is also in agreement with our conclusion [20]. However, there was no correlation of serum TSGF levels in pancreatic patients with gender, age, tumor diameters, clinical staging, lymph node metastasis, and liver metastasis (P [ 0.05), which suggests that TSGF is of little clinical value for predicting the metastasis and prognosis of pancreatic cancer patients. To sum up, the combination detection of CA242, CA199, CA125, CEA, and TSGF is of clinical value for the early diagnosis of pancreatic cancer and predicting the efficacy and prognosis after cryoablation. And CA199, CEA, and TSGF are of great clinical value in evaluating the efficacy after cryoablation. Monitoring the serum levels of CA242, CA199, CA125, and CEA can be valuable for monitoring the distant metastasis and prognosis of pancreatic cancer.
References 1. Jelski, W., Kutylowska, E., Laniewska-Dunaj, M., et al. (2011). Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) as candidates for tumor markers in patients with pancreatic cancer. Journal of Gastrointestinal and Liver Diseases, 20(3), 255–259. 2. Gui, J. C., Yan, W. L., & Liu, X. D. (2014). CA19-9 and CA242 as tumor markers for the diagnosis of pancreatic cancer: A metaanalysis. Clinical and Experimental Medicine, 14(2), 225–233. 3. Liu, H. M., Zhao, S. L., Qu, L. X., et al. (2011). Value of contrast-enhanced ultrasound imaging in monitoring malignant tumor during argon–helium cryosurgery. Nan Fang Yi Ke Da Xue Xue Bao, 31(9), 1622–1625. 4. Zhou, G., Niu, L., Chiu, D., et al. (2012). Changes in the expression of serum markers CA242, CA199, CA125, CEA, TNF-alpha and TSGF after cryosurgery in pancreatic cancer patients. Biotechnology Letters, 34(7), 1235–1241. 5. Wang, Z., & Tian, Y. P. (2014). Clinical value of serum tumor markers CA19-9, CA125 and CA72-4 in the diagnosis of pancreatic carcinoma. Molecular and Clinical Oncology, 2(2), 265–268.
6. Huang, Z., & Liu, F. (2014). Diagnostic value of serum carbohydrate antigen 19-9 in pancreatic cancer: A meta-analysis. Tumour Biology, 35(8), 7459–7465. 7. Bao, B., Ali, S., Ahmad, A., et al. (2014). Differentially expressed miRNAs in cancer-stem-like cells: Markers for tumor cell aggressiveness of pancreatic cancer. Stem Cells and Development, 23(16), 1947–1958. 8. Yoo, T., Lee, W. J., Woo, S. M., et al. (2011). Pretreatment carbohydrate antigen 19-9 level indicates tumor response, early distant metastasis, overall survival, and therapeutic selection in localized and unresectable pancreatic cancer. International Journal of Radiation Oncology Biology Physics, 81(4), e623– e630. 9. Vainshtein, J. M., Schipper, M., Zalupski, M. M., et al. (2013). Prognostic significance of carbohydrate antigen 19-9 in unresectable locally advanced pancreatic cancer treated with doseescalated intensity modulated radiation therapy and concurrent full-dose gemcitabine: Analysis of a prospective phase 1/2 dose escalation study. International Journal of Radiation Oncology Biology Physics, 86(1), 96–101. 10. Zhao, J. G., Hu, Y., Liao, Q., et al. (2014). Prognostic significance of SUVmax and serum carbohydrate antigen 19-9 in pancreatic cancer. World Journal of Gastroenterology, 20(19), 5875–5880. 11. Satoi, S., Yanagimoto, H., Toyokawa, H., et al. (2011). Selective use of staging laparoscopy based on carbohydrate antigen 19-9 level and tumor size in patients with radiographically defined potentially or borderline resectable pancreatic cancer. Pancreas, 40(3), 426–432. 12. Kanda, M., Fujii, T., Takami, H., et al. (2014). The combination of the serum carbohydrate antigen 19-9 and carcinoembryonic antigen is a simple and accurate predictor of mortality in pancreatic cancer patients. Surgery Today, 44(9), 1692–1701. 13. Tzeng, C. W., Balachandran, A., Ahmad, M., et al. (2014). Serum carbohydrate antigen 19-9 represents a marker of response to neoadjuvant therapy in patients with borderline resectable pancreatic cancer. HPB (Oxford), 16(5), 430–438. 14. Li, X., Guo, X., Li, H., et al. (2014). Serum carbohydrate antigen 242 expression exerts crucial function in the diagnosis of pancreatic cancer. Tumour Biology, 35(6), 5281–5286. 15. Singh, S., Tang, S. J., Sreenarasimhaiah, J., et al. (2011). The clinical utility and limitations of serum carbohydrate antigen (CA19-9) as a diagnostic tool for pancreatic cancer and cholangiocarcinoma. Digestive Diseases and Sciences, 56(8), 2491–2496. 16. Singh, S., Tang, S. J., Sreenarasimhaiah, J., et al. (2011). The clinical utility and limitations of serum carbohydrate antigen (CA19-9) as a diagnostic tool for pancreatic cancer and cholangiocarcinoma. Digestive Diseases and Sciences, 56(8), 2491–2496. 17. Liu, H. M., Zhao, S. L., Qu, L. X., et al. (2011). Value of contrast-enhanced ultrasound imaging in monitoring malignant tumor during argon–helium cryosurgery. Nan Fang Yi Ke Da Xue Xue Bao, 31(9), 1622–1625. 18. Bauer, T. M., El-Rayes, B. F., Li, X., et al. (2013). Carbohydrate antigen 19-9 is a prognostic and predictive biomarker in patients with advanced pancreatic cancer who receive gemcitabine-containing chemotherapy: A pooled analysis of 6 prospective trials. Cancer, 119(2), 285–292. 19. Pelzer, U., Hilbig, A., Sinn, M., et al. (2013). Value of carbohydrate antigen 19-9 in predicting response and therapy control in patients with metastatic pancreatic cancer undergoing first-line therapy. Frontiers in Oncology, 3, 155. 20. Kaur, S., Baine, M. J., Guha, S., et al. (2013). Neutrophil gelatinase-associated lipocalin, macrophage inhibitory cytokine 1, and carbohydrate antigen 19-9 in pancreatic juice: Pathobiologic implications in diagnosing benign and malignant disease of the pancreas. Pancreas, 42(3), 494–501.
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ORIGINAL PAPER
Serum CA242, CA199, CA125, CEA, and TSGF are Biomarkers for the Efficacy and Prognosis of Cryoablation in Pancreatic Cancer Patients Ye Chen • She-Gan Gao • Jian-Min Chen • Gong-Ping Wang • Zeng-Fang Wang Bo Zhou • Can-Hui Jin • Yan-Tong Yang • Xiao-Shan Feng
•
Ó Springer Science+Business Media New York 2014
Abstract This study aimed to compare the changes and determine the clinical significance of carbohydrate antigens CA242, CA199, CA125, carcinoembryonic antigen (CEA), and tumor-specific growth factor (TSGF) before and after cryoablation by Cryocare system. Thirty-one pancreatic cancer patients were selected to receive cryoablation by Cryocare system. The serum expression levels of CA242, CA199, CA125, CEA, and TSGF before and 1 month after treatment were determined. Meanwhile, the serum level of these factors was also determined in 31 healthy volunteers. The parameter changes were analyzed with the clinical pathological data. The serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). The serum CA199, CEA, and TSGF dramatically decreased 1 month after the treatment, which were statistically different (P \ 0.05). The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). The positive rate of CA199, CEA, and TSGF after the treatment was significantly lower than that before the treatment (P \ 0.05). Serum level of CA242 was correlated with the tumor diameter, clinical staging, tumor differentiation, lymph node, and liver metastasis (P \ 0.05). Except
Y. Chen S.-G. Gao J.-M. Chen G.-P. Wang Z.-F. Wang B. Zhou C.-H. Jin Y.-T. Yang X.-S. Feng (&) Department of Oncology Surgery, The First Affiliated Hospital of Henan University of Science and Technology, 24 Jinghua Road, Jianxi District, Luoyang, Henan Province, China e-mail: [email protected]
gender, CA199 was correlated with all the other clinical pathological parameters (P \ 0.05). The serum levels of CA125 and CEA were correlated with all the other clinical pathological parameters (P \ 0.05). The serum level of TSGF was only correlated with tumor differentiation (P \ 0.05). Cryoablation treatment by Cryocare system can decrease the serum levels of CA199, CEA, TSGF, and the positive rate. Serum CA199, CEA, and TSGF can be important index for pancreatic cancer treatment assessment. Serum levels of CA242, CA199, CA125, and CEA are of great clinical value for metastasis assessment and prognosis in pancreatic cancer patients. Keywords Cryocare cryoablation Carbohydrate antigen Carcinoembryonic antigen Tumor-specific growth factor Pancreatic cancer
Introduction Pancreatic cancer is a common digestive malignancy of very high incidence. Due to its occult nature, diagnostic difficulties, rapid development, and high mortality, it is often beyond surgical removal when the patients are diagnosed [1, 2]. Cryoablation by Cryocare system has been widely used in breast cancer, liver cancer, kidney cancer, etc., which provides a new therapeutic tool for benign and malignant tumors that cannot be surgically removed [3, 4]. Previous studies showed that serum carbohydrate antigens including CA242, CA199, CA125, carcinoembryonic antigen (CEA), and tumor-specific growth factor (TSGF) are important tumor biomarkers for the early diagnosis of pancreatic cancer [5–7]. We determined the serum and tumor expression levels of tumor biomarkers CA242, CA199, CA125, CEA, and TSGF in 31
123
Cell Biochem Biophys
selected pancreatic caner patients before and after cryoablation by Cryocare system. We also analyzed the changes of these five biomarkers and evaluated the clinical value of these biomarkers in the diagnosis, prognosis, and assessment of pancreatic cancer.
cryoablation lasts for 10–15 min until freezing area exceeds the tumor border by 0.5 cm. After the argon is stopped, helium is infused under 2,500 psi pressure, which is stopped until the frozen area thaw for 3–5 mins with temperature reaching to 10–20 °C. After the probes are reset, freeze–thaw cycle is repeated. All the patients received either double or multiple cycles of the cycles.
Materials and Methods Sample Collection and Marker Detection Subjects We selected 31 pancreatic patients admitted to our hospital for hospitalization and treatment between February 2013 and February 2014. All the diagnoses were confirmed by emission tomography and X-ray computed tomography and positron diagnosis, and all cases were further histologically confirmed by pathology and received subsequent cryoablation. There were 17 male patients and 14 female patients. The patients are aged between 32 and 75 years old (average 57.2 ± 8.0). We staged the cancer according to the clinical staging of pancreatic cancer (stage I: Tumor confined to the pancreas; Stage II: Tumor invading adjacent tissue only; Stage III: existing regional lymph node metastasis; Stage IV: existing liver and other distant metastasis). There was one stage I case, six Stage II cases, 10 stage III cases, and 10 Stage IV cases. Thirty-one healthy volunteers were randomly selected as the control group. These volunteers (16 males and 15 females aged between 35 and 60 years old, average 56.4 ± 8.2 years old) have no history of cancer, cardiovascular, liver, or kidney diseases. There was no statistical difference (P [ 0.05) between two groups in term of general clinical data, such as gender, age, etc. The expression levels of CA242, CA199, CA125, CEA, and TSGF before and 1 month after the treatment were determined. The results were then analyzed with different parameters and general clinical data. Methods After cryoablation needle, refrigeration efficiency has been checked, and puncture devices will be prepared. With local anesthesia, the selected patient was set at the appropriate position according to the tumor site. Under the guidance of CT, ultrasound probe is inserted through the peritoneum to the tumor site. Considering tumor location, size, and the surrounding structures, cryoablation needle 2 mm in diameter is inserted into the tumors. The number of the probes needed was proportional to the tumor sizes. The general guideline is as follows: 1 probe for tumor \2 cm, 2 probes for tumors between 2 and 4 cm, and 3 or 4 probes for tumors C5 cm. After the probes are set under CT guidance, cryoablation starts with argon infusion under 3,500 psi pressure. The
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Fasting venous blood was collected in the morning in both groups. Patients in the pancreatic cancer group received blood collection before and 1 month after the surgery. Serum was separated by centrifugation and frozen for storage. Serum CA242 was determined by radioimmunoassay. CA radioimmunoassay kits were purchased from China Institute of Oncology Branch of the company. CEA radioimmunoassay kits were purchased from Northern China Institute of Isotopes Company. The c212 counter was purchased from the U.S. DPC Company, and the positive threshold was 20 U/ml. CA199 and CA125 were determined by Access from Kurt company (USA) and Bayer Roche chemiluminescence instrument for quantitative analysis of protein chemistry, and the positive threshold was 35 U/ml. CEA was assayed with radioimmunoassay kit (IRMA) by Beijing Dong Ya biotechnology company, and the positive threshold was 5 ng/ml. TSGF was determined by Hitachi 7060 automatic biochemical analyzer, and the positive threshold was 64 U/ml. All the assays were performed according to manufacturers’ instructions by professionals. Statistical Analysis SPSS20.0 statistical software was used for data entry and analysis. The measurement data were expressed as mean ± standard deviation (x ± s), and counting data were expressed as rates. Measurement data were compared using t test. Counting data were compared with v2 test. P values \0.05 were considered as statistically significant.
Results Comparison of Serum Tumor Biomarkers Before and After Treatment in Control and Pancreatic Cancer Groups The serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). Serum CA199, CEA, and TSGF decreased significantly 1 month after treatment in
Cell Biochem Biophys Table 1 Comparison of serum tumor biomarkers before and after treatment in control and pancreatic cancer groups Group
n
CA242 (U/ml)
CA199 (U/ml)
CA125 (U/ml)
CEA (ng/ml)
TSGF (U/ml)
18.6 ± 3.2*
17.0 ± 1.0*
Pancreatic cancer treatment before
31
98.6 ± 19.7*
1,501.2 ± 764.1*
621.3 ± 206.4*
Pancreatic cancer treatment after
31
85.7 ± 10.6*
1,062.8 ± 631.6*,#
640.7 ± 201.8*
Control
31
11.5 ± 2.3
* Compared with control P \ 0.05;
#
21.3 ± 5.5
22.4 ± 3.1
9.9 ± 2.5*,# 2.5 ± 0.6
14.1 ± 0.9*,# 4.3 ± 1.0
compared with pancreatic cancer group before treatment P \ 0.05
Table 2 Comparison of positive rate for the serum biomarkers before and after the surgery in control and pancreatic groups [n(%)] Group
n
CA242
CA199
CA125
CEA 15 (48.4)*
Pancreatic cancer treatment before
31
23 (74.2)*
29 (93.5)*
23 (74.2)*
Pancreatic cancer treatment after
31
23 (74.2)*
22 (71.0)*,#
23 (74.2)*
31
2 (6.5)
Control * Compared with control P \ 0.05;
#
2 (6.5)
0 (0.0)
TSGF 8 (25.8)*
7 (22.6)*,#
0 (0.0)#
1 (3.2)
0 (0.0)
compared with pancreatic cancer group before treatment P \ 0.05
the pancreatic group compared with pre-surgical results, which were statistically different (P \ 0.05). There was no difference of CA242 and CA125 before and after surgery in the pancreatic cancer group (P [ 0.05, Table 1). Comparison of Positive Rate for the Serum Biomarkers Before and After the Surgery in Control and Pancreatic Groups The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). The positive rate of CA199, CEA, and TSGF after the treatment was significantly lower than that before the treatment (P \ 0.05). There was no difference in the positive rate of serum CA242 and CA125 before and after the treatment in the pancreatic cancer group (P [ 0.05, Table 2). The Correlations of Serum Biomarker Levels and the General Clinical Data in the Pancreatic Cancer Group Before and After the Surgery The serum CA125 and CEA in males were significantly higher than those in females (P \ 0.05, Table 3), and there was no statistical significant for other tumor biomarkers (P [ 0.05). Serum CA199, CA125, and CEA in patients older than 60 were significantly higher than those in patients younger than 60 (P \ 0.05), and the serum levels of CA242 and TSGF were not correlated with age (P [ 0.05). Except TSGF serum level, patients with tumors [4 cm or staged between III and IV exhibited significantly higher serum CA242, CA199, CA125, and CEA than those
with tumors B4 cm or staged between I and II (P \ 0.05). All serum biomarkers were higher in patients with tumor differentiation staged between III and IV than those staged between I and II (P \ 0.05). Except serum TSGF, the serum levels of CA242, CA199, CA125, and CEA in patients with lymph node or liver metastasis were significantly higher than those without (P \ 0.05).
Discussion With features of high degree of malignancy, insidious onset, rapid development, easy metastasis, poor prognosis, and as well as low survival rates, etc. the pancreatic cancer lacks of specific clinical manifestations, which makes difficulties for early diagnosis and treatment [8]. Discovery of tumor biomarker for pancreatic cancer can potentially provide better early diagnosis and prognosis prediction [9]. Currently, there are several tumor biomarkers for the clinical diagnosis and classifications of pancreatic cancer, which increase the general diagnosis and treatment of the disease [10]. At present, there are over 10 tumor biomarkers for clinical diagnosis, but only few has been proven to be of clinical value, among which CA242, CA199, CA125, CEA, and TSGF are included. CA242 is mainly present in the malignant pancreatic and colon cancer cells [11]. It has been shown that CA242 is elevated in pancreatic cancer cells, and the expression level of which is correlated with the development of pancreatic cancer and other GI tract cancers [12]. CA199 is expressed in pancreatic ducts, acinar cells, and salivary gland cells. Because of its high positivity and sensitivity, it is often used for clinical diagnosis, which is of great clinical value for the diagnosis and prognosis of pancreatic cancer [13]. CA125 is a macromolecular glycoprotein, and it has been
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Cell Biochem Biophys Table 3 The correlations of serum biomarker levels and the general clinical data in the pancreatic cancer group before and after the surgery Groups
n
CA242 (U/ml)
Male
17
114.2 ± 22.0
Female
14
98.9 ± 19.9
CA199 (U/ml)
CA125 (U/ml)
CEA (ng/ml)
1,638.5 ± 785.2
698.7 ± 258.6
21.4 ± 4.0
1,456.2 ± 758.5
403.2 ± 198.9*
TSGF (U/ml)
Gender 5.3 ± 2.0*
17.2 ± 0.9 16.6 ± 1.1
Age (years) B60
11
118.3 ± 22.8
1,069.2 ± 587.6
400.5 ± 200.3
6.6 ± 2.9
16.7 ± 1.1
[60
20
103.9 ± 18.4
1,925.6 ± 789.8*
687.6 ± 215.6*
21.5 ± 3.3*
17.3 ± 1.0
Tumor diameter (cm) B4
13
73.6 ± 18.4
1,002.9 ± 587.9
389.6 ± 203.2
10.6 ± 4.8
16.6 ± 1.0
[4
18
142.0 ± 18.0*
1,893.6 ± 885.6*
703.9 ± 241.2*
19.8 ± 4.3*
17.3 ± 1.1
6 25
45.6 ± 16.0 126.0 ± 15.4*
1,023.0 ± 563.2 2,005.6 ± 876.8*
300.4 ± 187.6 721.4 ± 250.0*
4.9 ± 1.2 20.6 ± 4.0*
16.5 ± 0.9 17.2 ± 1.2
Staging I–II III–IV
Tumor differentiation staging I–II
7
92.5 ± 22.5
1,132.3 ± 578.6
312.6 ± 200.2
5.1 ± 1.0
15.5 ± 0.8
III–IV
24
117.3 ± 17.7*
1,987.6 ± 678.9*
714.5 ± 245.3*
21.8 ± 3.9*
17.6 ± 1.2*
Lymph node metastasis No
9
89.0 ± 22.5
1,200.8 ± 670.5
356.6 ± 198.5
4.6 ± 1.0
16.7 ± 1.1
Yes
22
125.9 ± 17.6*
1,987.3 ± 810.2*
698.9 ± 256.3*
21.0 ± 6.3*
17.5 ± 1.1
Liver metastasis No
12
88.0 ± 23.4
1,212.3 ± 663.2
380.2 ± 200.0
12.8 ± 5.0
16.4 ± 1.3
Yes
19
126.4 ± 17.7*
1,988.6 ± 789.8*
701.3 ± 260.0*
17.0 ± 5.2*
17.2 ± 0.9
* Statistically different, P \ 0.05
shown that serum CA125 expression level is elevated in pancreatic cancer patients [14], and thus CA125 has been used for pancreatic cancer diagnosis. CEA was derived from epithelial tissue in endoderm, mostly present in the gastrointestinal tract, urinary tract, respiratory tract, etc., and it is often used clinically for the diagnosis of digestive system tumor [15]. Recently, TSGF has been shown to correlate with the angiogenesis of malignant tumors, which is useful for tumor diagnosis. It has also been shown that TSGF is elevated in pancreatic cancer patients [16]. Clinically, individual sensitivity of specificity of these five biomarkers is not sufficient, but the combination of these markers can significantly increase the sensitivity and specificity. Therefore, we studied the serum levels of CA242, CA199, CA125, CEA, and TSGF before and after cryoablation in pancreatic cancer patients. Our results showed that the serum levels of CA242, CA199, CA125, CEA, and TSGF in the pancreatic cancer group were significantly higher than those of the control group both before and after the cryoablation treatment (P \ 0.05). The positive rates of serum CA242, CA199, CA125, and CEA in the pancreatic cancer group were much higher than those in the control group both before and after treatment (P \ 0.05), and the positive rate of TSGF was significantly higher than that of the control group before the treatment (P \ 0.05). These results showed that the expression levels
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of these five biomarkers are correlated with the occurrence and development of pancreatic cancer, which is consistent with previous reports. Recently, cryocare system has been widely used in the treatments of several tumors. As an in situ ablation treatment technique, it provides hope of improvement or even cure for patients who missed the surgical removal opportunities [17]. We showed that serum CA199, CEA, and TSGF levels and positive rates were significantly lower than those before treatment (P \ 0.05), which was consistent with previous reports [18]. It is possible that serum levels of CA199, CEA, and TSGF could help predict the changes of condition or prognosis of pancreatic cancer patients after cryoablation. We also analyzed the correlations of serum biomarker levels and the general clinical data in the pancreatic cancer group before and after the surgery. Our results showed that the serum CA125 and CEA in males were significantly higher than those in females (P \ 0.05). Serum CA199, CA125, and CEA in patients older than 60 were significantly higher than those in patients younger than 60 (P \ 0.05). These results showed that gender and age are correlated with the occurrence and development of pancreatic cancer, which is in agreement with previous reports [19]. The patients with tumors[4 cm or staged between III and IV exhibited significantly higher serum CA242,
Cell Biochem Biophys
CA199, CA125, and CEA than those with tumors B4 cm or staged between I and II (P \ 0.05). We also showed that the invasiveness of pancreatic cancer is correlated with serum levels CA242, CA199, CA125, and CEA. We also showed that the serum levels of CA242, CA199, CA125, and CEA in patients with lymph node or liver metastasis were significantly higher than those without (P \ 0.05). All serum biomarkers were higher in patients with tumor differentiation staged between III and IV than those staged between I and II (P \ 0.05), which also proved these points. These results showed that higher serum levels of CA242, CA199, CA125, and CEA in pancreatic cancer patients were correlated with poor prognosis and distant metastasis. This could be due to adhesion molecule nature of CA242, CA199, CA125, and CEA, which can affect the development and metastasis of pancreatic cancer cells, which is also in agreement with our conclusion [20]. However, there was no correlation of serum TSGF levels in pancreatic patients with gender, age, tumor diameters, clinical staging, lymph node metastasis, and liver metastasis (P [ 0.05), which suggests that TSGF is of little clinical value for predicting the metastasis and prognosis of pancreatic cancer patients. To sum up, the combination detection of CA242, CA199, CA125, CEA, and TSGF is of clinical value for the early diagnosis of pancreatic cancer and predicting the efficacy and prognosis after cryoablation. And CA199, CEA, and TSGF are of great clinical value in evaluating the efficacy after cryoablation. Monitoring the serum levels of CA242, CA199, CA125, and CEA can be valuable for monitoring the distant metastasis and prognosis of pancreatic cancer.
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