Abstract
OBJECTIVE To investigate the clinical value of hepatoma-specific alpha-fetoprotein (HS-AFP) and gamma-glutamyltransferase II (GGT II) for judgment of postoperative prognosis of patients with hepatocellular carcinoma (HCC).
METHODS HS-AFP was separated and determined using native polyacrylamide electrophoresis with a discontinuous buffer system and West-ern blots. GGT II was separated with native polyacrylamide electrophoresis with a discontinuous buffer system and detected by enzyme staining. Forty cases with HCC underwent serial determination of HS-AFP and GGT II before and after radical excision. The correlations were analyzed between the two indices and survival time.
RESULTS In the 40 cases with HCC, before radical excision the positive rates of HS-AFP and GGT II were 57.5% and 67.5% respectively, with the positive rate of combined HS-AFP and GGT II reaching 80.0%. After operation, the recurrence and metastasis rate in the groups with positive HS-AFP and GGT II were 90.9% and 58.8% respectively, while in the groups with negative HS-AFP and GGT II the rates were 20.7% and 26.1% respectively. Recurrence and metastasis occurred in all cases with both postoperative positive HS-AFP and GGT II but only in 9.5% of the cases in whom both postoperative HS-AFP and GGT II were negative. Univariate analysis revealed that postoperative HS-AFP and GGT II were related to the prognosis in HCC.
CONCLUSION Postoperative serum HS-AFP and GGT II are very useful in predicting the prognosis of HCC patients.
keywords
Hepatocellular carcinoma (HCC) is a common malignant tumor in China. A radical excision is the most effective means to elevate the 5-year survival rate, but some patients develop recurrence. Early diagnosis of a postoperative recurrence and prompt treatment are important for improving the survival time. Serum alpha-fetoprotein (AFP) is a valuable index for monitoring recurrence after operation, but there is a need to enhance its specificity because in some cases with benign liver diseases AFP may also be elevated. Recently, we have separated and determined hepatoma-specific alpha-fetoprotein. (HS-AFP) using a newly developed native polyacrylamide electrophoresis procedure with a discontinuous buffer system and Western blots. The studies demonstrated that HS-AFP is of great value for the diagnosis and differential diagnosis of HCC, with a higher specificity than AFP.[1] Gamma-glutamyltransferase II (GGT II) is complementary with AFP for the diagnosis of HCC and other than AFP it is considered to be the best HCC marker. In our study, we determined HS-AFP and GGT II in 40 cases with HCC before and after radical excision. The relationships were studied between HS-AFP, GGT II and tumor size, stage, postoperative recurrence as well as survival time. The clinical value of postoperative serum HSAFP and GGT II were evaluated as a means for judgment of prognosis in HCC cases.
MATERIALS AND METHODS
Patients
All cases enrolled in this study were hospitalized HCC cases and undergone radical excision between December of 2003 and May of 2005 in the Nantong Tumor Hospital. The cases included 27 males and 13 females, between ages 20 and 70 years. AFP was positive (> 20 μg/L) in 25 cases, among whom AFP was higher than 400 μg/L in 20 cases. The cases were divided into Stage I (13 cases) and Stage II a+b (27 cases) according to the HCC stage standard drawn up by Liver Cancer Committee of Chinese Anti-cancer Association. Tumor size was defined as the maximal diameter of the tumor measured after operation. Pathological stages were determined based on the Edmondson standard. Ten cases belonged to Stage I and 30 cases to Stage II. HS-AFP and GGT II were measured serially with the first time being at admission and the last time at the third month after operation. The results of the last determination result were used for statistical analysis. Follow-up was conducted for 10 months after operation or until the death of the patient.
Reagents
Acrylamide was purchased from the Fluka Co. (USA) and rabbit anti-human AFP-IgG was the product of the DAKO Co. (Denmark). Horseradish peroxidase-conjugated goat anti-rabbit IgG was bought from the Xi'an Huamei Co.(China). DAB, glutamyl-p-nitroaniline and glycylglycine were purchased from the Shanghai Chemical Reagent Company of the China Medicine Group.
Determination of HS-AFP
HS-AFP was separated and determined using native polyacrylamide electrophoresis with a discontinuous buffer system and Western blots as previously reported.121 Briefly, a vertical slab gel was made and inserted into the electrophoresis apparatus. Electrophoresis was run for 4.5 h and then proteins were transferred to a nitrocellulose membrane for 3.0-3.5 h with a current of 1 mA per cm2. The nitrocellulose membrane was blocked with 50 g/L non-fat milk and then reacted with 1:100 rabbit anti-human AFP-IgG overnight at 4°C. The membrane was washed 3 times the next day and then reacted with horseradish peroxidase-conjugated goat anti-rabbit IgG at 37 °C for 1 h. The membrane was washed and put into a DAB solution in the dark for 3 min. The AFP bands were visualized without optical enhancement.
Determination of GGT II
GGT II was separated employing native polyacrylamide electrophoresis with a discontinuous buffer system and detected by enzyme staining as described previously.131 After electrophoresis, the gel was placed between two acetate cellulose membranes which were soaked with a GGT substrate solution (glutamyl-p-nitroaniline and glycylglycine) and then incubated in a humid box for 1 h at 37 °C. At last the acetate membrane was stained by immersion in a solution of acetate trichloride-glycerol. The GGT isoenzyme bands were clearly observed without optical enhancement.
Statistical analysis
The Chi-square test was used and P<0.05 was considered as statistically significant. The Log-rank test was performed to study the correlation between HS-AFP, GGT II and postoperative prognosis for the HCC patients.
RESULTS
HS-AFP and GGT II in 40 patients with HCC before and after radical excision
HS-AFP and GGT II levels were determined before and after radical excision in 40 patients with HCC. It was shown that HS-AFP and GGT II were positive in most cases and the two markers were complementary for diagnosis of HCC. The diagnostic sensitivity for HCC was 80% when HS-AFP and GGT II were simultaneously determined. After operation HS-AFP and GGT II changed to negative in some cases, but remained positive in the other casesTable 1).
HS-AFP and GGTH in 40 HCC patients before and after radical excision
Relationship between HS-AFP, GGT II and recurrence and/or metastasis after radical excision in HCC patients
Before operation, no métastasés were found in the lymph nodes of the peritoneal cavity or remote organs in all of the HCC cases. HS-AFP and GGT II were monitored within 3 months after operation and followed-up for 10 months. The results showed that the rates of metastasis and recurrence were higher in the positive HS-AFP and GGT II groups compared to those in the negative groups. The rates of metastasis and recurrence in the positive HS-AFP group (90.9%) were higher compared to those in the positive GGT II group (58.8%). Metastasis and recurrence occurred in all of the patients who were positive for both HS-AFP and GGT II after operation, but in only 9.5% of the cases who were negative for both postoperative HSAFP and GGT II Table 2).
HS-AFP and GGTH in 40 HCC patients before and after radical excision
Relationship belween clinical parameters and prognosis after operation in HCC cases
As shown in Table 3), univariate analysis indicated that the prognosis was poorer in HCC cases after operation if the tumor size was larger than 5 cm, pathological grade II~III and postoperative positive for HS-AFP or GGT II (P<0.01). There was no correlation between prognosis and the following indices: ALT, TBil, ALB, preoperative AFP, HS-AFP and GGT II (P>0.05 ). Postoperative AFP was not closely associated with prognosis (jP=0.056).
Univariate analysis of correlation between clinical parameters and prognosis after operation
DISCUSSION
AFP is now the most widely used serum HCC marker. However, AFP is not very specific for diagnosing HCC because AFP may also increase in some benign liver diseases. It has been found that there are differences TJTJW between the sugar chains of AFP molecules produced by liver cancer cells and those produced by hépatocytes. The AFP derived from liver cancer cells can be separated from that produced by hepatocytes with Lens culinaris agglutinin. The former is called AFP-L3.[4] Because Lens culinaris agglutinin is very expensive, AFP-L3 has not been widely used in clinical practice. We have previously reported that HS-AFP can be separated by native polyacrylamide electrophoresis and detected with Western blots.[1] Because of the low cost and high specificity, HS-AFP is suitable for clinical use. GGT II is an isoenzyme predominantly produced by liver cancer cells and is complementary to AFP for diagnosis of HCC.[3] Therefore, GGT II is an important HCC marker especially in the cases with negative AFP. The present study demonstrated that HS-AFP and GGT II were positive in the majority of HCC cases and that HS-AFP and GGT II were complementary for HCC. Combined determination of HS-AFP and GGTII may increase the diagnostic sensitivity to 80%, indicating that HS-AFP and GGT II are valuable for HCC diagnosis.
The factors governing the synthesis of HS-AFP and GGT II have not been completely elucidated. Some studies have shown that the malignancy, tumor size and staging are related to serum AFP-L3 and GGT II. [5-7] After resection of a liver tumor, HS-AFP and GGT II decrease due to the removal of the liver cancer cells. It has been reported that AFP production was not induced by partial hepatectomy.[8] After hepatoma resection, the serum AFP level depends on the AFP level before operation and its half time. Generally, it falls to negative within 2 months after operation.
AFP-L3 is superior to AFP for monitoring HCC. Okuda et al.[9] found that regardless of the preoperative serum AFP-L3 level, the postoperative AFP-L3 positive patients had a poorer recurrence-free rate. AFP-L3 was considered as a valuable marker for evaluation of curability by surgical treatment and for improving the accuracy of prognosis. The study by Hayashi et al.[10] concluded that measurement of AFP-L3 after operation is useful for understanding prognosis and recurrence of HCC.
In the present study, we conducted serial measurements of HS-APP and GGT II in 40 HCC cases after radical excision. Within 10 months after operation, recurrence and/or metastasis were found in 90% of the HCC cases with the HS-AFP positive group, while in only 20.7% of the negative group. However, serum AFP is not closely correlated with the postoperative prognosis for HCC patients. Similar to HS-AFP, the postoperative GGT II positive group had a higher rate of recurrence and/or metastasis compared to the GGT II negative group. Recurrence and/or metastasis occurred within 10 months after operation in all cases who were positive for both HS-AFP and GGT II, but in only 9.5% of the cases in whom both HS-AFP and GGT II were negative. These results indicate that HSAFP and GGT II are of value to predict postoperative prognosis of HCC cases.
Conclusively, measurements of both preoperative HS-AFP and GGT II are helpful for the diagnosis of HCC. postoperative HS-AFP and GGT II are of great value in evaluation of the prognosis.
Footnotes
This work was supported by grants from the Jiangsu Science and Technology Department (BS2004528) and Jiangsu Health Department (H200521).
- Received May 13, 2006.
- Accepted August 29, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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