Evaluation of a Protein Chip System for Multi-tumor Marker Detection in Clinical Application

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1 Evaluation of a Protein Chip System for Multi-tumor Marker Detection in Clinical Application Nin-he Li, Ning-ning Shan, Yang Gao,Hui-ming Wang, Jian-hong Liu, Hong-yin Xia and Xiong Zou Central Laboratory, Department of Laboratory Medicine, Qilu Hospital, Shandong University, Jinan, Shandong, P.R. China Abstract Objective:To evaluate the clinical applications to cancer diagnosis of the C-12 protein chip system for multi-tumor marker detection and its specificity and sensitivity in detection of non-cancer subjects and healthy controls. Methods: Serum levels of 12 tumor markers, including CA199, NSE,CEA,CA242,CA125, CA153,AFP,FER,f-PSA,PSA,β-HCG and HGH were measured with the protein chip system in 8,330 malignant tumor patients, 9,657 patients with benign diseases and 23,531 healthy individuals. Results: The positive rates were 85.6% for malignant tumor patients, 41.2% for the group with benign diseases and 7.03% for the healthy group, respectively. The malignant tumor group had significantly higher positive rate than that of the controls. Conclusion: Parallel measurement of multiple tumor markers with biochip technique can significantly increase the diagnostic sensitivity for malignant tumors and can be used as an earlier tumor-screening tool for asymptomatic population, especially for those of high cancer risks. Key Words tumor markers; protein biochip; chemiluminescence assay Malignant tumors are a group of devastating diseases, with the incidence increasing in recent years (1). The key to improving the survival of cancer patients lies in early detection, diagnosis and treatment. Tumor markers have important clinical significance. Many researches have shown that combined measurement of multiple tumor markers is an important approach to improve the tumor detection rate of single tumor markers (2). The C-12 protein chip system for multi-tumor marker detection (the C-12 protein chip) developed by Shanghai HealthDigit Co. Ltd. is a solid surface-based antibody microarray analytical system. The C-12 protein chip can simultaneously measure 12 tumor markers and generate test results with fast speed and high throughput (3). We have applied the C-2 protein chip in clinical practice since During this period, a total of 8,330 samples from various cancer patients, 9,657 samples from non-malignant tumor patients, and 23,531 samples from apparently healthy individuals were tested using this product for the measurement of serum levels of the tumor markers AFP, CEA, CA19-9, CA242, CA 125, CA153, NSE, PSA, f-psa, Ferritin,β-HCG and HGH. The clinical value of this system is discussed. Materials and Methods Subjects: A total of 8,330 blood samples of the disease group were all from cancer patients of the Qilu Hospital from January 2003 to April The ages of the patients ranged from 15 years old to 89 years old, with the average age of 53 years old. Among them 4,693 were male and 3637 were females. All patients were diagnosed with surgery and pathology. One hundred sixty-three (163) subjects, 91 males and 72 female with averaged age of 46 years old and age range between 16 years old and 79 years old, were used for posttreatment monitoring study. The 9,657 blood samples of the disease control group were from non-cancer patients from the Qilu Hospital, 4,569 males and 5088 female with averaged age of 49 years old and age range between 17 years old and 81 years old. The 23,531 blood samples of the healthy control group were from health check individuals with averaged age of 51 years old and age range between 8 years old and 92 years old. Measurement of serum tumor markers is according to the product instruction. Reference ranges (cutoff values) of the tumor markers are according to the product instruction (See Table 1). Evaluation method is as follows. Samples with serum level of at least one of the 12 tumor markers above cutoff values is classified as positives (with the exception of the tumor marker ferritin. A value above cutoff for ferritin alone is not considered a positive result). Statistics analysis is done with the SPSS11.0 software. All statistics indicators were expressed as Average+SD (Standard Deviation).

2 Results 1 The positive rates of the malignant tumor group, the non-cancer disease control group, and the health checkup control group were 81.4%, 38%, and 7.5%, respectively (Table 2). Therefore, the disease (malignant tumor) group has a positive rate significantly higher than that from the control groups (P<0.05). One hundred fifty-three samples with negative results were randomly taken and tested with the single marker testing kit using the E170 system (Roche). No significant differences were found between the two systems. All the 153 C-12 negative samples were also tested negative with the E170 kit. 2 In the malignant tumor group, the positive rates as detected by the C-12 protein chip are high for liver cancer, pancreas cancer, ovarian cancer, lung cancer, gastric cancer and colorectal cancer at 85.6% 88.7%, 79.4%, 69.9%, 61.9%, and 76.4%, respectively (See Tables 3, 4, Figure 1). 3 Follow up testing after treatment: Forty-nine C-12 protein chip positive lung cancer patients, 52 liver cancer patients, 14 ovarian cancer patients and 48 colorectal cancer patients were tested with the C-12 protein chip at 3-4 weeks, 6-9 weeks, 3 months, 6 months, and 12 months after surgery. The serum levels of tumor markers decreased significantly as compared to the levels before surgery (P<0.05). However, no significant difference was found between the levels of the tumor markers at the time of 3 months, 6 months, and 12 months after surgery (Table 5). In the follow up, 5 lung cancer patients, 6 liver cancer patients, 1 ovarian cancer patients, and 4 colorectal cancer patients were found to have positive serum levels of tumor marker with the trend of continuing increasing. Those patients were later confirmed to have cancer recurrence or metastasis as examined by CT, ultrasound and biopsy pathology. 4 The serum tumor marker levels of the positive samples from the non-cancer disease control group and the health checkup control group showed moderate elevations compared to the malignant tumor group, and the number of the tumor markers showing positives were mostly single marker or 2 markers, but not multiple markers. Among the 23,531 samples from the health checkup group, there were 1,656 positives with a positive rate of 7.0%. After further diagnosis, 1,489 of them had benign diseases such as chronic inflammation, polypus, drug damage, endometriosis, and pregnancy, 98 of them were diagnosed pathologically as having malignant tumors, and 69 of them showed no apparent cause for the elevation of the tumor markers and are still under monitoring. Discussion The death incidence of malignant tumors in China is /100,000, accounting for 17.9% of all death, and ranks as the second major disease cause of death. Early detection, diagnosis, and treatment are critical for cancer patients. Measurement of serum tumor markers is one of the important means for cancer diagnosis and has broad clinical applications. However, individual tumor markers often have low sensitivity and specificity, especially with low detection rate for early stage tumors. Combined detection of multiple tumor markers can compensate for the low sensitivity and specificity of single marker detection, and thus is getting increased application in clinical practice. Biochip is a technology developed in recent years that is essentially a micro system for chemical analysis based on a microarray on a solid face. Biochip has the capacity of obtaining large quantity of information fast and accurately with small amount of reagents and samples, enabling efficient analysis of proteins, DNA etc. There are three major types of biochip, i.e., gene chip, protein chip and chip-on-a-lab. Protein chip employs probes (mostly antibodies) immobilized on the chip matrix to capture and analyze the protein targets (4). Protein chip has gained broad use in the detection of tumor markers in recent years (5, 6, 7). The protein chip system has the advantages over traditional analytical methods for proteins that it can analyze multiple protein targets in parallel from different samples including tissues and serum and thus provides far more information in one single analysis. Our results show that among the health checkup individuals with no apparent clinical symptoms, the C-12 protein chip detected 98 cases of malignant tumors, a detection rate of 0.2%. Among the 98 cases, 5 lung cancer patients, 6 liver cancer patients, 1 ovarian cancer patients, 4 colorectal cancer patients showed increased serum levels of tumor markers in scheduled monitoring after surgery. Those patients were later confirmed by imaging scanning and pathology as having recurrence or metastasis. Our results showed that the C-12 protein chip has clinical values in early detection of the cancer as well as in post treatment monitoring and prognostic prediction. Our results also showed that multiple tumor markers were positive for many cancer patients, and this phenomenon is caused by the fact that most tumor markers do not have high specificity. Therefore, many clinical laboratories are using combined testing of multiple tumor markers to improve sensitivity and specificity. Our data showed that combined testing of CA199, AFP, CEA and ferritin had high value for the diagnosis of liver cancer, combined testing of CA199, CA125, CEA and ferritin had better detection rate for the cancer types of the digestive

3 system, and combined testing of CA199, CA125, CEA and ferritin was useful for the early diagnosis of lung cancer. The overall positive rate of the C-12 protein chip for the detection of malignant tumor was 85.6%, which is comparable to combined sensitivity of those tumor markers using other single marker detection methods, suggesting the basic clinical value of the C-12 protein chip for clinical diagnosis is significant. The ability of the C-12 protein chip to detect 12 tumor markers not only improves the clinical diagnosis of cancer types such as liver cancer, which has a relatively sensitive and specific marker in AFP, by complementing with positive detections from non-specific markers in CA199, CEA and ferritin, but is also important for cancer types such as lung cancer, breast cancer and gastric cancer, which do not have a sensitive or specific tumor marker, but still have reasonable detection rate because of the contributions from multiple markers. It must be pointed out that the C-12 protein chip not only improves the detection sensitivity for cancer types such as lung cancer, breast cancer and gastric cancer, it also helps find the proper tumor marker for effective monitoring for treatment. The overall positive rate for the health checkup group was relatively high at 7.03%, with ferritin being the highest marker at 4.11%,followed by CEA at 1.41%,CA199 at 0.69%, and CA125 at 0.36%. Among the positive cases, 98 showed having at least three tumor markers positive simultaneously and were confirmed as having malignant tumors further by imaging scanning and pathology The establishment of the C-12 protein chip technology provides a fast and direct means to obtain large amount of information in tumor marker detection. However, because of the complexity of the malignant tumors, there is presently no ideal tumor marker that has 100% sensitivity or specificity. Even with combined detection of multiple tumor markers as with the C-12 protein chip, our results achieved an overall detection rate of only 85.6% for malignant tumors, with still significant number of cancer patients undetected. There is still plenty of work to be done in the future in using tumor markers to diagnose and screen for malignant tumors. References [1] Zou, X. and Zhang, LN. Editors, Molecular immunology and clinical practice (in Chinese):283~300, Shandong Science and Technology Press, Jinan, China. [2] Zou, X. Study of tumor markers in early diagnosis of early tumors and application progress (in Chinese). Chinese Journal of Laboratory Medicine 25(2):71-72, [3] Weinberger, SR, Morris, TS, Pawlak, M. Recent trends in protein biochip technology. Pharmacogenomics 1(4): , [4] Chapman, K. The protein chip biomarker system from Ciphergen Biosystems: a novel proteomics platform for rapid biomarker discovery and validation. Biochem Soc Trans. 30(2):82, [5] Sun, ZH., Fu, XL., Zhang, L. et al. A protein chip system for parallel analysis of multi-tumor markers and its application in cancer detection. Anticancer Research 24: , [6] Rubin, RB. and Merchant, M. A rapid profiling system that speeds of cancer and other diseases. Am Clin Lab. 19(8):28-29, [7] Qi, J. and Che, YQ. Application of a protein chip system for multi-tumor marker detection in the diagnosis of ovarian cancer (in Chinese). Chinese Journal of Laboratory Medicine 26(6): , Table 1. Cutoff Values for the C-12 Protein Chip Tumor marker Cutoff value Tumor marker Cutoff value CA125 <35 KU/L NSE <13 ng/ml CA199 <35 KU/L PSA <5 ng/ml CA153 <35 KU/L f-psa <1 ng/ml CA242 <20 KU/L β-hcg <3mIU/mL CEA <5 ng/ml HGH <7.5 ng/ml AFP <20 ng/ml FER <322 ng/ml(male);<219 ng/ml(female)

4 Table 2. Positive Rates (%) of Three Groups with the C-12 Protein Chip Group Number of samples Positive samples Negative samples Positive rate Malignant tumor group Benign tumor group Healthy Checkup control group Table 3. Positive Rates (%) of Cancer Types with the C-12 Protein Chip Cancer type Total Positive Positive rate Liver Stomach Esophagus Colon Lung Pancreas Ovary Cervix Prostate Esophagus Testis Figure 1. Positive Rates (%) of Cancer Types with the C-12 Protein Chip Positive Rates Liver Esophagus Lung Ovary Prostate Testis

5 Table 4. Positive Rates of Individual Tumor Markers for Each Cancer Type (N=11,256) Cancer Type Samples CA199 NSE CEA CA242 FER β-hcg AFP F-PSA PSA CA125 HGH CA153 Liver Stomach Esophagus Colon Lung Pancreas Ovary Cervix Prostate Esophagus Testis Tumor marker Table 5. Serum Levels (X±S) of Tumor Markers for Liver Cancer Patients Pre- and Pre-surgery (52 samples) 3-4 weeks (51 samples) 6-9 weeks (49 samples) 3 months (47samples) 6 months (40samples) 12 months (39samples) CA ± ± ± ± ± ±20.63 NSE 3.25± ± ± ± ± ±1.17 CEA 39.47± ± ± ± ± ±7.11 CA ± ± ± ± ± ±7.51 FER ± ± ± ± ± ± β-hcg 0.97± ± ± ± ± ±0.74 AFP ± ± ± ± ± ±30.77 f-psa 0.07± ± ± ± ± ±0.27 PSA 4.63± ± ± ± ± ±2.34 CA ± ± ± ± ± ±23.37 HGH 2.07± ± ± ± ± ±5.27 CA ± ± ± ± ± ±3.45