CA242 as a biomarker for pancreatic cancer and other diseases

CA242 as a biomarker for pancreatic cancer and other diseases

CHAPTER TWELVE CA242 as a biomarker for pancreatic cancer and other diseases Huaiqian Doua,*, Guirong Sunb, Lijuan Zhanga,* a Systems Biology and Me...

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CHAPTER TWELVE

CA242 as a biomarker for pancreatic cancer and other diseases Huaiqian Doua,*, Guirong Sunb, Lijuan Zhanga,*

a Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China b Clinical Laboratory, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. Introduction 2. Serum CA242 measurement 3. Serum CA242 levels in 27 diseases 3.1 Materials and methods 3.2 Results 4. Conclusion and discussion Acknowledgment Conflict of interest References

230 232 232 232 233 235 237 237 237

Abstract CA242 is a sialic acid-containing carbohydrate antigen attached to core proteins/lipids detected on the cell surface or in serum. Increased serum levels of CA242 have been used clinically as diagnostic biomarkers for pancreatic, colorectal, and other cancers. Since CA242 is overexpressed in malignant tumor tissues compared to that in normal tissues, it is believed that serum CA242 is a product of cancer cells. Thus far, the serum CA242 levels in patients suffering from different types of cancer and noncancerous diseases have not been systematically compared. In our current study, we acquired data of serum CA242 levels from 34,680 patients with 27 clinically defined diseases and from healthy controls (1317) in the clinical laboratory of our hospital over the past 5 years. The mean, median, and –log10 p values were calculated. Our data showed that patients with pancreatic cancer, cervical cancer, and lymphoma had the highest median values of serum CA242, which were followed by esophagus cancer, colon cancer, ovarian cancer, type 2 diabetes mellitus, rectal cancer, coronary heart disease, breast cancer, diabetic nephropathy, acute myocardial infarction, and 15 other types of diseases compared to that of healthy controls. In contrast, patients suffering from sequela of brain damage and anemia had statistically lower median values than healthy controls.

Progress in Molecular Biology and Translational Science, Volume 162 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2018.12.007

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2019 Elsevier Inc. All rights reserved.

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Based on the –log10 p values, the increased serum CA242 levels could be used not only for diagnosis of pancreatic and other cancers, but also for diagnosis of type 2 diabetes mellitus and coronary heart disease, suggesting CA242 might be a systemic malfunction biomarker associated with cancers and other chronic diseases.

1. Introduction The uncontrolled growth of cancer cells leads many to believe that cancer cells produce and spread special biomarkers via blood circulation, which can be used for cancer detection. The hybridoma technology established in 1970s revolutionizes how the cancer biomarkers are discovered.1 This technology generates monoclonal antibody against specific antigens on cancer cells, which starts by provoking an immune response by injecting a mouse with cancer cells. The B cells then produce antibodies that bind to the injected cancer cells. The B cells are harvested from the mouse spleen and fused with a myeloma, the immortal cancer cells with a B cell origin, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B cell and the immortality of the myeloma. Each hybridoma clone consists of genetically identical hybridomas which produce one antibody per culture. The cancer cell-associated antigen recognized by the monoclonal antibody produced by a specific hybridoma is then isolated and identified. CA1992–4 and CA505 are the first two cancer biomarkers discovered through the hybridoma technology. Interestingly, both antigens are special carbohydrate structures detected on cancer cells. CA199 and CA50 have a good sensitivity and an acceptable specificity as biomarkers for pancreatic cancer diagnosis.6,7 However, both biomarkers are subject to a great influence from hepatic function and cholestasis, resulting in false-positive values in cases of benign cholestatic icterus and diseases of the liver parenchyma.8 Moreover, only a small portion of the patients with early-stage pancreatic cancer have detectable CA199 and CA50 levels. Under such a background, CA242 is isolated by Lindholm et al. in 1985.9 Subsequently, it was revealed that CA242 epitope is a novel sialic acid-containing carbohydrate structure that is present as glycoproteins/glycolipids on the cell surface or as o-linked enriched glycoprotein (mucin) in serum.10 Kuusela et al. reported first the increased levels of serum CA242 in patients with pancreatic cancer in 1991,11 which is supported by followup studies.12,13 Further studies showed that CA242 has similar sensitivities and specificities to those of CA19-9 and CA50 in the diagnosis of pancreatic

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cancer. In general, the sensitivity of CA242 is lower than that of CA50 and CEA, but CA242 has higher specificity when used for the diagnosis of pancreatic cancer. The sensitivity of CA242 can be improved by combining it with CEA or CA50.14 Another study agrees with this notion, which showed that the sensitivities of CA242, CA19.9, and CA50 for pancreatic cancer diagnosis are 66.2%, 70.6%, and 70.6%, respectively. A sensitivity of 75.0% is achieved with CA242 and CA50 combined as diagnostic biomarkers. When CA242 is combined with CA19.9 and CA50, both sensitivity and specificity are improved significantly.15 CA19-9, CA50, and CEA are the commonly used biomarkers for the diagnosis of pancreatic cancer. A series of studies have been conducted in 1980–1990s to compare different cancer biomarkers in serum for the diagnosis of pancreatic and other different types of cancers. Numerous studies showed that the serum levels of CA242 are correlated well with those of CA19-9 and CA50 as biomarkers for digestive tract malignancies.3,7,16–23 These and subsequent studies confirmed that increased serum level of CA242 can be used as biomarkers not only for diagnosis of pancreatic cancer but also for diagnosis of colorectal and other cancers.14,24–28 Immunohistochemical studies demonstrated that CA242 is always coexpressed with CA50, but CA50 and CA242 are recognized by different monoclonal antibodies with completely different carbohydrate epitopes situated on the same macromolecule.29 Another study also showed that CA242 epitope is coexpressed with CA50 and CA199 epitopes on the same macromolecular complex.30 Other immunohistochemical study showed that stage IV pancreatic cancer patients with well- to moderately differentiated carcinomas have significantly higher CA242 levels than patients with poorly differentiated carcinomas.31 This result is in concordance with immunohistochemical findings showing clearly stronger expression of CA242 in well- to moderately differentiated tumors than in poorly differentiated or anaplastic carcinomas.32 In stage I and II–III pancreatic cancer patients, poorly differentiated tumors are associated with slightly higher levels of CA242 expression than well- to moderately differentiated tumors.31 Another study showed that serum CA242 level in patients with advanced pancreatic cancer (stage III) is elevated and implied that high preoperative CA242 levels are related to distant metastasis or a poor prognosis of the patients with pancreatic cancer.33 Both biochemical studies of serum CA242 levels and immunohistochemical studies of tumor tissues suggested that serum CA242 might be a product of cancer cells. However, high frequency of false-positive and false-negative rates during cancer diagnosis when CA242 is used as

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biomarker questions such assumptions. Thus far, the serum CA242 levels in patients suffering from different types of cancers and noncancer diseases have not been systematically compared. Therefore, a retrospective study of serum CA242 levels from 34,680 patients with 27 clinically defined diseases and from healthy controls (1317) in the clinical laboratory of our hospital over the past 5 years was conducted and presented in this chapter.

2. Serum CA242 measurement Immunoradiometric assay (IRMA),34 enzyme-linked immunosorbent assay (ELISA),35–37 and chemiluminescence immunoassay (CLIA)38 are three commonly used methods for determining the levels of serum CA242. IRMA is a noncompetitive RIA and its main characteristic is using radiolabeled antibody. IRMA has the advantage of high sensitivity, but radioactive contamination is its major shortcoming. ELISA is a detection method that measures antigen–antibody binding specificity. It can have high sensitivity and short detection time without using radioactive materials. It is widely used in clinical and other field of studies. CLIA is a mature and advanced technology developed rapidly in the past 10 years with a wide range of applications by using fluorescent labeling. It is more sensitive and accurate. It is expected that CLIA is replacing most of the IRMA and ELISA methods in clinical settings. Based on the advantages mentioned above, CLIA, in the form of a kit, has been widely used in the detection of serum CA242 levels in most of the hospitals.

3. Serum CA242 levels in 27 diseases 3.1 Materials and methods All data for the detection of serum CA242 in the past 5 years were obtained from the Laboratory of Affiliated Hospital of Qingdao University. Serum levels of CA242 from 34,680 patients suffering from 27 clinically defined diseases were measured and analyzed. Each disease had at least 30 independent sets of test data. At the same time, serum levels of CA242 from 1317 healthy controls during their annual health checkup were also obtained and analyzed without exclusion. Serum CA242 levels were quantified by the carbohydrate antigen 242 assay kit (CLIA, Shenzhen New Industries Biomedical Engineering Co., Shenzhen, China) according to manufacturer’s instructions.

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CA242 as a biomarker for pancreatic cancer and other diseases

All data were processed with Microsoft Excel (Microsoft, Inc., USA) and GraphPad Prism 5 (GraphPad Software, Inc., USA). Statistical analysis was performed by using SPSS version 19. Due to a nonnormal distribution, Mann–Whitney test was used for statistical analysis. Two-sided t-test with a 5% significant level (p < 0.05) was considered as statistically significant.

3.2 Results Based on the clinical test data obtained, we calculated and listed the mean, median, and –log10 p values of serum CA242 levels for 27 diseases in Table 1. To visualize the results, the median values for the 27 diseases were plotted. As shown in Fig. 1, the median values for 25/27 diseases were higher than that of healthy controls. Patients suffering from pancreatic cancer, cervical cancer, lymphoma, esophagus cancer, colon cancer, ovarian cancer, type 2 diabetes mellitus, rectum cancer, coronary heart disease, breast cancer, diabetic nephropathy, and acute myocardial infarction had highest levels of serum CA242 compared to other diseases, healthy controls, Table 1 Serum CA242 levels in patients with 27 different types of clinically defined human diseases and in healthy controls. –Log10 CA242 # of cases Mean (SD) Median p value

Pancreatic cancer

209

44.49 (49.28)

28.01

56.28

Cervical cancer

38

11.44 (11.11)

9.29

6.64

Lymphoma

62

26.43 (43.64)

9.15

12.68

177

7.43 (5.94)

5.81

6.91

3736

9.71 (13.36)

5.61

79.83

44

11.40 (16.88)

5.49

1.75

Esophagus cancer Colon cancer Ovarian cancer Type 2 diabetes mellitus

2395

6.89 (5.21)

5.43

46.43

Rectum cancer

4548

9.17 (12.45)

5.40

90.97

Coronary heart disease

673

6.62 (5.07)

5.26

9.55

Breast cancer

186

7.23 (7.84)

5.10

2.18

Diabetic nephropathy

37

10.94 (12.98)

5.09

1.24

Acute myocardial infarction

42

6.70 (4.92)

5.09

1.61 Continued

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Table 1 Serum CA242 levels in patients with 27 different types of clinically defined human diseases and in healthy controls.—cont’d –Log10 CA242 # of cases Mean (SD) Median p value

Cerebral ischemia

145

5.76 (3.88)

4.99

1.40

Lung cancer

307

9.34 (13.67)

4.96

5.80

5708

8.62 (12.60)

4.86

44.13

47

13.59 (24.39)

4.85

2.07

115

6.68 (5.24)

4.81

2.62

Intracranial hemorrhage

41

6.49 (7.75)

4.79

0.31

Gastric ulcer

70

9.09 (13.03)

4.70

2.20

367

6.35 (5.60)

4.57

2.63

Osteoporosis

43

4.84 (3.60)

4.53

0.75

Chronic obstructive PD

39

6.17 (5.63)

4.40

0.32

122

6.26 (6.44)

4.39

0.32

Gout

54

4.45 (2.97)

4.38

1.07

Cerebral arteriosclerosis

39

5.13 (4.09)

4.30

0.56

14,185

5.11 (3.34)

4.20

0.00

302

5.20 (3.90)

3.79

0.67

1317

4.66 (3.12)

3.79

6.54

74

5.00 (5.28)

3.52

1.59

Gastric cancer Pancreatitis Gastritis

Acute cerebral infarction

Cirrhosis

Healthy controls Cerebrovascular disease Healthy controls >65 years old Anemia

Fig. 1 Serum CA242 levels in 27 different types of clinical diseases. The data were sorted in a descending order of the median values. CA242 concentrations for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges were marked in red.

CA242 as a biomarker for pancreatic cancer and other diseases

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Fig. 2 –Log10 p values for CA242 in 27 different types of diseases compared to that of healthy controls. When the serum CA242 levels were lower than that of healthy controls, blue color was used in the plot whereas when the serum CA242 levels were higher than that of healthy controls, red color was used in the plot.

and 65 years and older healthy controls. Patients after gastric surgery, with cerebrovascular disease, suffering from sequela of brain damage, or with anemia, had lower median values compared to that of healthy controls. Interestingly, 65 years and older healthy controls also had lower median values compared to that of healthy controls. The –log10 p values are used in the context of null hypothesis testing in order to quantify if the evidence provided has statistical significance (p < 0.05, log10 p > 1.30). To test this, we used the median values to calculate the –log10 p values. The –log10 p values in different diseases were plotted in both ascending (red) and descending (blue) order relative to the healthy controls, which is shown as heat map in Fig. 2. All 27 diseases had the –log10 p > 1.30, which indicated that increased serum CA242 levels could serve as biomarkers for rectum cancer, colon cancer, pancreatic cancer, type 2 diabetes mellitus, gastric cancer, and many other diseases (Fig. 2) and CA242 was an outstanding biomarker for both cancers and noncancer diseases.

4. Conclusion and discussion Serum CA242 is an important clinical biomarker for the detection of digestive tract cancers, which include pancreatic, colon, rectum, and esophageal cancers. These findings are consistent with the published

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reports.4,6–9,15,39 With 27 clinically defined diseases of 34,680 patients and 1317 of healthy controls, our study also revealed that it could also serve as biomarkers for other human diseases such as type 2 diabetes and lymphoma, based on the data shown in Table 1 and Figs. 1 and 2. Pancreatic cancer is a very aggressive and lethal disease with dismal prognosis. The 5-year relative survival rate of pancreatic cancer, including both exocrine and endocrine malignancies, is only 4% or less.40,41 One of the factors predetermining the poor prognosis of pancreatic cancer is its late diagnosis because of unspecific early symptoms.15 In addition, the lack of an adequate screening method for pancreatic cancer hampers early detection of this devastating disease. Specific biomarkers in the sera of pancreatic cancer patients have been identified by antibody-based detection of cell surface antigen released from cancer cells, such as CA19-9, CA50, CA242.42 CA242, a relatively new biomarker for pancreatic cancer, has contributed to the management of pancreatic cancer treatment as clinical indicators of disease progression during chemotherapy or recurrence after surgery. The sensitivity of CA242 was higher than that of CA50 and CEA at all specificity levels.31 Despite its clinical usefulness, CA242 is not effective enough for the early detection of all cancers, such as pancreatic cancers, since elevated classical tumor biomarker levels indicate the presence of a significant number of cancer cells. Lots of work on combination of tumor indicators has been done by researchers to improve cancer diagnosis rates. In 1992, Pasanen et al. combined CA242, CEA, and CA50, whose sensitivities and specificities were raised up to 76.9% and 89.2%. Other research reported similar results, when combined TSGF, CA242, and CA19-9 together, the sensitivity changed to 77.0% and the specificity and the positive predictive value were 100%. Another research combined detection of CEA and CA242, which improved the diagnostic specificity increase obviously to 92%. Therefore, the combination of multiple indicators and CA242 can better predict cancer. However, high levels of serum biomarkers are associated with advanced stages of the disease which means that tumor biomarkers are easily affected by different stages of cancer. This is an important factor that influence the clinical indicators CA242.31,33,43 This study was a retrospective analysis of the data collected during the past 5 years. In general, our results on CA242 are in concordance with previous studies, and future work must be done to promote the diagnosis of malignant diseases.

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Acknowledgment This research was supported by the Natural Science Foundation of China (Grant 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, the “Double First Class fund” of Shandong Province in China to L.Z., and Postdoctoral application research project in Qingdao China to H.D.

Conflict of interest The authors declare no conflict of interest.

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