Clinical use of hepatic carcinoma associated membrane protein antigen (HAg18-1) for detection of primary hepatocellular carcinoma

Abstract

AIM: To evaluate the sensitivity, specificity, and clinical value of hepatic carcinoma associated membrane protein antigen (HAg18a21) as a reagent in a serum quick enzyme linked immunosorbent assay (ELISA) for detection of primary hepatocellular carcinoma (PHCC).

METHODS: A serum quick enzyme linked immunosorbent assay (ELISA) which uses HAg18a21 as a reagent, was prepared from monoclonal antibodies against human hepatic carcinoma. We assayed blood serum obtained from 100 cases of primary hepatocellular carcinoma (PHCC), 5 cases of hepatic biliary carcinoma (HBC), 10 cases of metastatic hepatic carcinoma (MHC), 20 cases of hepatitis B (HB), 20 cases of liver cirrhosis (LC), 20 cases of malignant gastrointestinal tumors, and 20 cases of inflammatory gastrointestinal diseases (including ulcers). Alpha 2 fetoprotein (AFP) was concurrently detected for each case. Twenty samples of blood bank serum were tested as controls.

RESULTS: The respective positive rates for the HAg18a21 ELISA assay and AFP detection assay were 81% and 68% in PHCC, 20% and 40% in HBC, 19% and 20% in MHC, 10% and 20% in BH, 10% and 20% in LC, 10% and 15% in malignant gastrointestinal tumors, and 5% and 10% in inflammatory gastrointestinal diseases. Neither assay showed a positive result for any tested blood bank sample.

CONCLUSION: The HAg18a21 ELISA demonstrated high sensitivity and specificity in the detection of PHCC. HAg18a21 ELISA and AFP detection, if used together, may compliment each other in the diagnosis of PHCC.

Key Words: Liver neoplasms/diagnosis, Carcinoma, Hepatocellular diagnosis, Antigens, Neoplasm

INTRODUCTION

Hepatic carcinoma is the third leading cause of death in China, and accounts for 15.08% of all cancer-related deaths[1]. Currently, hepatocellular carcinoma (PHCC) is primarily diagnosed by imaging studies and serological tests. While alpha-fetoprotein (AFP) is an internationally accepted specific biological marker for PHCC, and can be detected in 68.5%-76.5% of all cases[2], serum AFP levels can vary with the degree of tumor differentiation and the size of the tumor mass[3]. Also, AFP may be absent or present at only low concentrations in a certain proportion of PHCC cases, whereas cases of metastatic hepatic carcinoma and benign hepatic diseases may have elevated AFP levels[4]. Therefore, there is an urgent need to identify more sensitive and specific markers of hepatic carcinoma.

MATERIALS AND METHODS

Source of materials

All 195 serum samples used in this study were collected from January 1990 to December 1993 from patients at the Chinese PLA 165^th Hospital, The First Affiliated Hospital of Hengyang Medical University, Hengyang Municipal Central Hospital, and The 415^th Hospital of the Ministry of Nuclear Industry. The samples were obtained from 100 patients with PHCC, 5 patients with HBC, 10 patients with MHC, 20 patients with HB, 20 patients with LC, 20 patients with gastrointestinal malignant tumors, and 20 patients with various inflammatory gastrointestinal diseases (including ulcers). Twenty samples of blood bank serum served as control samples.

Methods

HAg18-1 qualitative assays were performed using a HAg18-1 serum quick ELISA Kit (Chinese PLA Fourth Medical University and Zhengzhou Bo-sai Biological Reagent Laboratory, China), and methods described in the kit’s instructions. Quantitative HAg18-1 assays were performed as follows: a known Hag18-1-positive sample (blue in color) was diluted with 1 mL of 0.5 N H₂SO₄ to terminate the reaction; after which, the sample’s OD at 450 nm was measured using a STAT FAX 303 plus photometer system (Awareness Technology; Technology, Palm City, FL, United States). A positive assay sample was defined as one having an OD > 0.15. AFP was detected with an AFP radioimmunoassay (RIA) Kit (Shanghai Biological Product Institute; Shanghai, China), using the procedure described in the instruction manual. AFP antibody was supplied by Fuzhou MaiXin Biological Technique Development Co. (China), and the “SP” method was adopted.

RESULTS

Relationship between HAg18- 1 serum assay results and AFP detection in 100 cases of PHCC (Table 1)

Table 1 The results of HAg18-1 Eenzyme linked immunosorbent assays and alpha-fetoprotein radioimmunoassays.

Disease	No. of cases	No. of positive cases		Positive rate (%)
		HAg18-1	AFP	HAg18-1	AFP
PHCC	100	81	68	81	68
HBC	5	1	2	20	40
MHC	10	1	2	10	20
HB	20	2	4	10	20
LC	20	2	4	10	20
GI malignant tumor	20	2	3	10	15
GI inflammatory diseases	20	1	2	5	10
Bank blood1	20	0	0	0	0

¹Controls

HAg18-1 serum assays and AFP detection assays both showed positive results for 61% (61/100) of all the cases. AFP detection assays showed positive results for 86.4% (70/81) of the HAg18-1 positive cases, and among the 19 HAg18-1 negative cases, 7 cases (36.8%) were AFP positive. The HAg18-1 serum assay showed positive results for 88.23% (62/68) of the AFP positive (> 400 ng/mL) cases, and among the 32 AFP negative cases (< 400 ng/mL), 24 cases (75%) were positive with the HAg18-1 serum assay.

Four HAg18-1 serum assay positive cases (2 clinically diagnosed cases of HB and 2 cases of LC) had been followed up for 3 years. One of the LC cases was later proven to be PHCC by autopsy after death.

The AFP test showed 100% positive results when the “SP” method was used to test sections of paraffin-embedded liver tissue from all 100 patients with PHCC.

DISCUSSION

There are numerous biological markers for human hepatic carcinoma, and substantial advances have been made in both the theories and technology involved in this area of research. Several new techniques that were previously used only in experimental studies are now used in the clinic. In summary, four types of assays are now used to detect PHCC, and these involve detecting proteins, enzymes, hormones, and metabolites, respectively[5]. Because these assays vary greatly in their sensitivity and specificity, there remains an urgent need to identify new a biological marker for hepatic carcinoma.

Practical value of the HAg18-1 assay

In this study, the HAg18-1 assay was able to detect 81% of the PHCC cases, which was markedly or slightly higher than the detection rates reported from studies conducted in Nanjing (65.9%)[6], Xi’an (74%)[4], Chengdu (79.63%)[7], and Lanzhou (80%)[8]. The assay showed a < 10% positive rate when used to detect other types of malignant tumors or inflammatory diseases. The reasons that HAg18-1 is present at certain concentrations in different diseases remain unclear. In our study, the HAg18-1 assay was 90% specific when used to detect PHCC. This result was comparable to the 89.52% specificity the same assay showed in a study conducted in Chengdu. In that study, HAg18-1 was shown to be a tumor-associated antigen (TAA) closely linked with hepatic carcinoma. In our study, the HAg18-1 assay displayed higher sensitivity for detecting PHCC, when compared with the AFP assay (68% detection rate). The HAg18-1 assay showed positive results in 75% (24/32) of the 32 PHCC cases with a serum AFP level < 400 ng/mL, suggesting that it may be especially useful for detecting PHCC cases which are AFP negative or in which the patient has a low serum AFP concentration. On the other hand, among the 19 PHCC cases that tested negative with the HAg18-1 assay, 36.8% (7/19) were detected by the AFP assay. These results indicate that the two assays are complementary to each other in their clinical use. Four HAg18-1 assay positive cases (2 clinically diagnosed HB cases and 2 LC cases) had been followed up for 3 years. Among them, one clinically diagnosed LC case was proven to be PHCC by autopsy after death. Therefore, it is reasonable to believe that the HAg18-1 assay might be helpful in the early diagnosis of PHCC. While the HAg18-1 assay showed positive results in a certain proportion of non-PHCC tumors and inflammatory diseases, these types of results occurred far less often than when using the AFP assay, and had little influence on the final diagnosis of PHCC.

In recent years, researchers have suggested that a combined detection protocol with multiple markers would be superior to detecting only a single marker in the diagnosis of PHCC, and especially when attempting to diagnose patients who are AFP negative or have a low serum AFP concentration. The serum quick enzyme-linked immunosorbent assay with HAg18-1 is easy to perform, provides consistent results, and requires no special instruments. It is a simple technique that is especially suitable for surveying high-risk populations, and can be used in general practice. If combined with AFP detection, it should greatly increase the PHCC detection rate, and thus has great potential for widespread clinical use.