Abstract
OBJECTIVE To conduct a comparative study of the effects of treatment using microwave ablation versus surgical resection on hematogenous dissemination of cancer cells, and on the level of immune cells of the peripheral blood in patients with small primary hepatocellular carcinoma (PHC, ≤5 cm).
METHODS Forty patients with small PHC (maximal diameter ≤5 cm) were divided Into a microwave group (19 cases) and a surgical operation group (21 cases). A real-time (RT) quantitative nested RT-PCR examination was performed for peripheral blood alpha-fetoprotein (AFP) mRNA. Studies were conducted to determine the level of CD3, CD4, CD8 and CD4/CD8 cells and for liver function at 30 min before, and 30 min,1 day and 3 days after the treatment.
RESULTS Compared to the value before ablation, no obvious changes of CD3, CD4, CD8 and CD4/CD8 cells were found in patients of the microwave group within 7 days after ablation, but CD3, CD4 and CD4/CD8 cells in the operation group were lower compared to that before operation. The copy number of AFP mRNA in the peripheral blood samples of the patients of the 2 groups before operation was determined in 67.5% of the patients (27/40). There was an rise in the expression after treatment but no statistical difference was found In comparing the 2 groups. Follow-up of the patients was conducted for 1 to 16 months. For patients with continuous expression of peripheral blood AFP mRNA, the possibility of relapse and metastasis was increased.
CONCLUSION Surgical resection or microwave ablation can cause more exfoliation of hepatoma carcinoma cells in the peripheral blood of patients with small PHC. The immune function of peripheral blood cells decreased in the patients after surgical resection, however, the immune function was better protected following microwave ablation. Microwave ablation causes minor reduction in liver function, and the treatment method presents a definite value for PHC therapy.
keywords
- AFP
- mRNA
- hepatocellular carcinoma
- microwave ablation
- RT-fluorescent quantitative nested RT-PCR
- immune response
At present, surgical resection is still the treatment of choice for patients with primary hepatic carcinoma (PHC, ≤ 5 cm), but the 5-year recurrence rate is up to 43.5%.[1,2] An important feature of liver cancers is that they often metastasize to distant target organs through the blood. It has been reported in past studies that peripheral blood alpha fetoprotein (AFP) mRNA was a sign of the degree of PHC metastasis via the blood, and that exfoliated hepatoma carcinoma cells (HCC) could be assessed by RT-quantitative RT-PCR assay of AFP mRNA in the blood.[1-3]
The research in recent years showed that hepatic resection itself can cause hematogenous spread of HCC.[1-4,5] Clinically, more and more liver cancer patients have received ultrasound-guided microwave ablation. In our study, 19 eligible PHC patients were chosen to investigate whether or not microwave ablation will result in the hematogenous spread of HCC. As controls, 21 PHC patients received surgical resection with estimation of peripheral blood AFP mRNA used as the indicator of the results.
MATERIALS AND METHODS
Clinical data
The liver cancer patients who received microwave ablation or surgical resection in our hospital, from October 2002 to March 2004, met the following requirements, i.e., a full case history and the examinations such as CT and/or MRI, ultrasound and blood serum AFP, etc.; all were diagnosed as small primary liver cancer (diameter ≤ 5 cm), with a single focus and no metastasis; no pretreatment such as transcatheter hepatic arterial chemoembolization or radiofrequency ablation, etc. had been performed; the cases were pathologically confirmed as PHC, and the patients and their relatives had agreed for the patients to be enrolled in the study groups which were made up of 19 cases in the microwave group and 21 in the operation group.
Collection of samples and detection of indexes in the peripheral blood
Blood samples (7 ml) were collected at 30 min before microwave ablation puncturation or laparotomy, and at 30 min, 1 and 7 days after microwave ablation or surgical operations. Five ml of the blood (plus 1 ml of ACD decoagulant) was used within 2 h for extraction of RNA and determination of liver function, and 2 ml was used for CD3, CD4 and CD8 assays within 24 h, after sampling.
Reagents and instruments
HepG2 cells were supplied by the No. 2 Institute of the Academy of Military Medical Science; TRIzol for extraction of total RNA was obtained from the US Invitrogen Co.; the reverse-transcription kit, Taq DNA polymerase, dNTP and the PCR reagents were all from US Promega and the iCycler iQ™ RT quantitative PCR instruments were produced by US BioRad Co.
AFP mRNA and GAPDH mRNA gene primer pairs: Based on design of previous publications,[6] the primer 3 and probe 4 were synthetized by Beijing Sanbo Yuanzhi Biotechnology Co. Ltd., and others by Shanghai Shenggong Bioengineering Technology Service Co. Ltd. PAGE purification was employed for the primers and probes. For the primer and sequences see Table 1.
The designed primer and probe series
The test methods
Extraction of RNA and synthesis of cDNA
Density gradient centrifugation based on lymphocyte isolation was used to separate the kaiyocytes in the peripheral blood.[6] Total RNA of the karyocytes in the peripheral blood was extracted using the TRIzol kit and the reverse-transcription synthesis of cDNA was conducted following the procedure described by the manufacturer.
Construction of the quantitation standard products
RNA was extracted from the HepG2 cells, the targetgene fragment was amplified by RT-PCR, which was connect with pGEM-T and was then introduced into JM109 Escherichia Coli for screening for positive clones. The recombinant plasmid DNA was extracted and after quantitation, diluted to 1x109~1x100 copy/μ1 with sterile water, and stored at -20°C for future use.
The RT fluorescent quantitation PCR reaction
(1) The first amplification: The 20 μ1 of PCR reaction system contained 10 mmol/L TrisCl (pH 8.3), 50 mmol/L KC1, 200 (μmol/L dNTP, 3 mmol/L Mg2+, 0.5 μmol/L primer 1 and 2, 0.2 pmol/L probe 4, 1 U Taq DNA polymerase, and 2 μ1 of each quantitative standard preparations with various dilutions mentioned above and cDNA. After 3 min pre-denaturation at 94 °C on the iCycler iQ™ real-time PCR instrument, a total of 30 cyclings were done for 20 s at 94 °C, 20 s at 56°C and 20 s 72 °C. The results were analyzed by software of the instrument.
(2) The second amplification: The primer 2 was replaced by the primer 3 and the template was the product of the first amplification. The other constituents and conditions for the amplification were the same as that in the first amplification.
The RT quantitative PCR detection of the samples
The gene expression of AFP and GAPDH for each cD-NA were concurrently detected in 2 different tubes, and the quantitative standard curves for the AFP and GAPDH genes were concomitantly set up. The second PCR detection was conducted for the samples and standard preparations not responding to the first PCR assay.
The methods of expression for the AFP mRNA expression level
The absolute value a and b (i.e. the copy number) for the amount of the AFP mRNA and GAPDH mRNA expressions were calculated based on the Ct value and standard curve. The inner level of GAPDH was used to correct for variation of the assay: if the amount of expression for GAPDH is b0 before treatment, the GAPDH is bl at 30 min after treatment and AFP is al at 30 min after treatment. So, at 30 min after treatment the correction value y for the amount of AFP expression will be as follows: y=al×b0/b1.
Statistical methods
The SAS statistical package was used for the unified analysis. The signed-rank test was applied for the comparison between the 2 inter-groups, and intra-group AFP mRNA data before and after treatment; The two-factor ANOVA with a covariant and a repeated measure value was employed for the intergroup comparison after operation and the comparison for the intra-group overall difference. The statistical methods of linear correlation and regression were used for related analysis of the level of peripheral-blood AFP mRNA expression and the serum AFP level.
RESULTS
Comparison between the clinical data of the patients in the microwave and operation groups
There was no statistical significance in comparing the patients in the microwave and operation groups, such as the gender ratio and ages (56.6±9.2 vs. 53.5±9.3), Child stage of liver function, ratio of patients with hepatic cirrhosis (19/19 vs. 18/21), maximum diameter of liver cancer (3.14 ±1.05 cm vs. 3.57 ±1.21 cm) and pathologic cancer grading. There was no significant difference in comparing the inter-groups and intragroup serum AFP before and after treatment (P>0.05) and no correlation between the level of peripheral-blood AFP mRNA expression and serum AFP level (£>0.05).
Comparison between the biochemical indicators of liver function of the patients in the microwave group and operation group
The rise of serum ALT and AST in the patients of the operation group after surgical operation was more significant compared to the patients in the microwave group. In comparison with the same time points between the groups, the ALT value in the patients of the operation group at 30 min and 1 day after operation was higher compared to the microwave group (P< 0.01). Peak value of both ascending ALT indexes occurred 1 day after operation. The levels of both total serum protein and albumin in the two groups at 30 min and 1 day after treatment were lower compared to that before treatment (P<0.01) and there was no statistical significance difference in the overall comparison between the two groups (P>0.05). The postoperative changes in the serum total bilirubin and direct bilirubin of the patients of the two groups was similar. After surgical operation, the rise of both bilirubin values in the patients in the operation group was more significant compared to the patients in the microwave group.
Comparison between the peripheral-blood immune cells of the patients in the microwave and operation groups
Compared to the data before operation, there was no obvious change in the CD3, CD4, CD8 and CD4/CD8 of the immune cells of the peripheral blood in patients of the microwave group within 7 days after operation (P>;0.05). However the immune cell in the peripheral blood of patients in the operation group after their operation decreased compared to that before operation: CD3 was significantly lower at 30 min, 1 and 7 days (P<0.01, P<0.01, P<0.05) and CD4 was significantly lower at 30 min, 1 day, after the surgical operation (P<0.01). These differences showed a statistical significance when compared to the same time points in patients of the microwave group (P<0.01). Preoperative levels were restored 7 days after operation. There was no statistical significance in differences in comparing CD8 between the 2 inter-groups or with the intra-group time points. CD4/CD8 was significantly lower at 30 min, 1 day after the surgical operation compared to that before operation (P<0.05). The preoperative level was restored 7 days after operation.
The RT quantitative PCR assay of peripheral blood samples
Assay of the internal control GAPDH gene
All 40 samples showed a positive response with the level of GAPDH gene expression for most samples found to be between 8×106~3×107 copies/μ1.
Assay of the AFP gene
No fluorescent-response occurred in all the samples at first amplification. After the second amplification, the copies of AFP mRNAs were detected in 67.5%(27/40) of the patients before treatment. There were several regular patterns of dynamic change in the AFP mRNA expression following the treatment: (1) For most patients, there was a definite level of AFP before treatment and the postoperative level increased; (2) For a few patients, the value of AFP mRNA was zero before or after treatment; (3) The expression of AFP was not found before operation but the expression of AFP mRNA could be found after treatment. The peak value of the expression occurred at 30 min after operation; (4) The amount of AFP mRNA expression apparently decreased in patients on the 7th day after operation, compared to that before operation.
Comparison between the changes in the level of AFPmRNA expression in the patients of the two groups
The logarithm of the correction value was used for statistical analysis, and the data were expressed by the value of inter-quartile range (IQR) Table 2. The level of AFP mRNA expression in the peripheral blood in the patients of the two groups at 30 min and 1 day after operation significantly increased compared to that before operation, but there was no statistical significance in the difference between the two groups.
The value of IQR for the expression level of peripheral blood AFP mRNA in different time points
Follow up
Follow-up conducted in the groups by the end of April, 2004 varied greatly, from 1 to 16 months. Loss of a visit occurred in 1 case of the microwave group, multiple bone metastasis in 1 case at 8 months after operation and intra-hepatic relapse in 1 case at 10 months after treatment. Another patient died of other diseases (infection and pulmonary embolism) 10 days after operation. The loss of visits occurred in 3 cases of the operation group and an intra-hepatic recurrence in 2 patients of the same group at 4 and 5 months after the operation. A comparatively high level of the AFP mRNA expression was found before and after treatment in 4 cases of patients with intra-hepatic recurrence or distant metastasis.
Discussion
The RT quantitative nested RT-PCR assay was used for the estimation of AFP mRNA in the peripheral blood samples of patients in both groups. It was found that 67.5% (27/40) of the patients had AFP mRNA expression before treatment, which was in accordance with the reports from the literature.[7] The level of AFP mRNA expression significantly increased in the patients of the two groups at 30 min and 1 day after the operation. On the 7th day, no apparent difference in the expression level was found in comparison with that before treatment. There was no significant difference in various similar time points between the two groups before and after the treatment. No parallel reports from the literature have been found to date. It was noted in the follow-up that there was continued expression of peripheral blood AFP mRNA before and after treatment in patients with intra-hepatic recurrence or distant metastasis, which is in accordance with the litera-ture.[3]
Peripheral blood AFP mRNA is a sign of the presence of hepatic cancer cells in the blood.[7] Because RNase is rich in the blood, the mRNA will be degraded by the RNase once it appears. Since 1994, the findings by many scholars[3,4,5,6] have shown that AFP mRNA has a high tissue specificity related to the liver and that AFP mRNA can be regarded as the sign of circulating liver cancer cells.
The simple identification of AFP mRNA can not be used to differentiate benign and malignant cells. Wu, et al.181 reported a change in the AFP mRNA copy number in the peripheral blood of the patients with PHC and non-PHC before and after operation. After operation, the copy number of AFP mRNA in the PHC group was 40 times that of the non-PHC group (15052/374). It can be seen that there is a minor impact of normal liver cells exfoliated into the blood on the rise of the AFP mRNA, relative to the liver cancer cells exfoliated into the blood. The results of this study shows that the dynamic change of the peripheral-blood AFP mRNA is mainly caused by the hematogenous extension of liver cancer cells.
Whether or not the hematogenous release of cancer cells will cause systemic metastasis, thus resulting in expansion of the tumor, depends on the balance between the selectivity of tumor cells for the metastatic tissues and immunoreactivity of the host.[9] The tumor cells exfoliate from the primary focus, enter the blood, migrate from the blood vessels and result in hyperplasia in suitable tissues. During the course, most of the cells are attacked by immunocytes of the host or sabotaged by mechanical force of the blood flow, and can be killed in the course of platelet aggregation.m Findings from Lennard, et al.[10] have shown that surgical operation and trauma may cause a fall in immunity of the body. The results of animal experiments have demonstrated that operative procedures may result in the hematogenous circulation of tumor cells, which will increase risk of postoperative metastasis.[1
This study has shown that the damage to hepatic function after resection was more severe than that of the microwave ablation group. The patients' cellular immune function in the peripheral blood was significantly decreased within 7 days after operation compared to that before operation, with a minimum at 30 min after operation. The results were in agreement with reports from the literature.[10] The peak value for postoperative exfoliation of tumor cells into the blood occurred 30 min after operation, and microwave ablation resulted in a slight trauma of hepatic function, e.g. the cellular immune function in peripheral blood maintained at a level before treatment within 7 days after operation. This will be of benefit for continuing killing of the tumor cells floating in the blood circulation and preventing the tumors from hematogenous spread and tissular metastasis. The results showed there were favorable characteristics in treatment of liver cancer by microwave ablation.
At this time, it has been generally accepted that surgical resection is the treatment of choice for liver cancer. Although the technological development for microwave ablation and other treatments has been rapid over the past 10 years, this technology is still at the initial stage of investigation. At present, the major indication for application of microwave ablation has been not only small liver cancer, but also for patients with hepatic cancer who can not receive a resection. The main purpose of our clinical research has been to understand the effect of microtraumatic surgery via microwave ablation on hemetogenous dissemination of cancer cells. The clinical efficacy and applied value of this technology still needs validation and evaluation by studying a large number of cases, randomized control and long-term follow-up, based on requirements of the Cochrane Center.
Footnotes
This work was supported by the foundation-aided projects of the 10th Five-year plan for medical research of the PLA (01Z038); the foundation-aided projects of the 10th Five-year plan for medical research of the PLA General Hospital (01YQ07).
- Received July 20, 2006.
- Accepted August 23, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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