Impact of Low Versus Conventional Doses of Chemotherapy During Transcatheter Arterial Chemo-embolization on Serum Fibrosis Indicators and Survival of Liver Cancer Patients

Transcatheter arterial chemoembolization (TACE) is one of the main methods in treating hepatocellular cancer (HCC) (Raoul et al., 2011). Repeated TACE improves the overall survival and the overall response rate of HCC (Artinyan et al., 2008; Toyama et al., 2012). Serum albumin level, Child-Pugh class, tumor number, size, alpha-fetoprotein, alanine aminotransferase, des-gamma carboxy-prothrombin, and gamma-glutamyl transferase are correlated to treatment outcomes (Hakamada et al., 2008; Zhang et al., 2011), but no standardized protocol regarding the selection, dosage, concentration, rate of injection of the chemotherapeutic agent, and optimal retreatment strategy (Paul et al., 2011). Upper gastrointestinal hemorrhage, hepatic failure, atrophy or cirrhosis, pulmonary embolism, and cholecystitis are most common side effects (Li et al., 2012). The number of treatment sessions depends on the response of the tumor and whether serious side effects are seen (Marelli et al, 2007). In recent years, efforts have been directed to improve the delivery system of chemotherapeutic and


Introduction
Transcatheter arterial chemoembolization (TACE) is one of the main methods in treating hepatocellular cancer (HCC) (Raoul et al., 2011). Repeated TACE improves the overall survival and the overall response rate of HCC (Artinyan et al., 2008;Toyama et al., 2012). Serum albumin level, Child-Pugh class, tumor number, size, alpha-fetoprotein, alanine aminotransferase, des-gamma carboxy-prothrombin, and gamma-glutamyl transferase are correlated to treatment outcomes (Hakamada et al., 2008;Zhang et al., 2011), but no standardized protocol regarding the selection, dosage, concentration, rate of injection of the chemotherapeutic agent, and optimal retreatment strategy (Paul et al., 2011). Upper gastrointestinal hemorrhage, hepatic failure, atrophy or cirrhosis, pulmonary embolism, and cholecystitis are most common side effects . The number of treatment sessions depends on the response of the tumor and whether serious side effects are seen (Marelli et al, 2007). In recent years, efforts have been directed radiotherapeutic agents to be used in TACE (Hsu et al., 2011;Tam et al., 2011). Drug-eluting beads are designed to localize a drug to the targeted tumor and minimize systemic exposure to the drug, thus decreasing the common postoperative adverse effects associated with chemoembolization with lipiodol. However, one study from a single center showed that lipiodol TACE was not inferior to TACE with drug-eluting beads in terms of response rate and was superior to the latter regarding time to progression. What is more, this study demonstrated that the median overall survival was 46 months for TACE and only 19 months for TACE with drug-eluting beads (Baroni et al., 2010). Other strategy to low the side effects of TACE is to modify the dosage of regimens. We hypothesize that low dose doxorubicin would reduce sides effects and especially the possibility of liver cirrhosis and in the mean time maintain the treatment efficacy. Because little is known how to predict which patients have liver fibrosis or cirrhosis, this study used serum fibrosis markers [hyaluronic acid (HA), human pro-collagen type-Ⅲ (hPC-Ⅲ), laminin (LN), collagen type-Ⅳ (Ⅳ-C)] to demonstrate the impact of chemotherapy on fibrosis.

Patients eligibility
In this study, all patients shoud be admitted to the hospital and treated from August 2004 to September 2006. Other inclusion criteria were: (1) diagnosed and staged according to the Hepatocellular carcinoma diagnosis standard issued from Guangzhou Conference by Chinese Society of Liver Cancer on 2001; (2) karnofsky performance status of 60 or more; (3) age less than 75 years; (4) adequate hematological (white blood cell count > 3.5×10 9 and platelet count > 80×10 9 ), liver (bilirubin and transaminases < 1.5 times the upper normal limit) and renal function (creatinine leval < 1.5 times the upper normal limit); (5) no pre-existing or concomitant malignant neoplasms or pre-operative chemotherapy and radiotherapy. All eligible patients were divide into two groups, group A received low-dose anticancer agents and group B got conventional-dose. All of our patients were first treated with TACE and without surgical operation and signed informed consent before TACE.

Embolism and chemotherapeutic agents
During TACE, a mixture of epirubicin, cisplatin 60 mg, 5-fluorouracil 1g and super emulsified lipiodol (5-15 ml) was infused. Dose of epirubicin administrated to Group A was estimated according to tumor size: 10mg to tumor diameter<5 cm; 20mg to diameter>5 cm. Dose of epirubicin to Group B was set as 50 mg.

TACE and Serological examination
Seldinger technology was used to puncture the femoral artery. TACE procedures were performed under radiographic guidelines following the steps that has been described elsewhere . Venous blood samples of all patients were collected prior to first TACE and 1 week after TACE. Radioimmunoassay was used to examin serum fibrosis index, including HA, PCⅢ, C-Ⅳ and LN. Variation of hepatic function was monitored before and after TACE using serum indicatiors, eg., total bilirubin, albumin and glutamate pyruvate transaminase.

Evaluation of treatment efficacy
Patients take the CT examination 1 month after TACE, and were evaluated the short term effect based on Response criteria for solid tumor. Effectiveness or stabilization ones were proceeded the chemotherapy 2-6 times every 1-3 months, and evaluated the curative effect every time. We use the best curative effect calculate the rate of effectiveness. We observe the survival rate and the median survival time as long-term efficacy. The survival time was defined from final diagnosis to death or lost.
Efficacy endpoints Efficacy variables included response rate was defined according to RECIST criteria (Therasse et al., 2000). If a patient had a CR or PR that occurred before progression, the patient was classified as a responder. Other efficacy analyses included survival.

Safety analyses
Safety parameters included adverse events, serious adverse events, laboratory abnormalities, and deaths. Safety analyses were evaluated for all patients who received at least one TACE procedure. The WHO Toxicity Grading System was used for all adverse events.

Statistical analysis and research experience
Serological testing data were demonstrated as X±s, life span was calculated by month. Chi-square tests were conducted to compare the differences between frequencies, and t test to check the differences between means.The median survival time was estimated by Kaplan-Merier method, and survival rate were compared by Log-rank test. All statistical processing were analysed by SPSS10.0, P≤0.05 was considered statistical significance. We have enough experience in conducting medical researches, and have published some results elsewhere (Huang et al., 2004;Zhou et al., 2009;Jiang et al., 2010;Yan et al., 2010;Gao et al., 2011;Huang et al., 2011;Yan et al., 2011;Zhang et al., 2011;Gong et al., 2012;Yu et al., 2012).

Treatment of TACE
We recruited 50 eligible hospitalized HCC patients, with 25 patients in group A and 25 in group B. Of all 50 patients, 38 were men and 12 were women, age of patients was arranged from 29 to 72 and the average age was 52.8. Our cases include AFP>400 U/ml pre-TACE 35 cases; merging portal vein tumor thrombus 10 cases; Child-pugh A 37 cases and Child-pugh B 13 cases; Clinical stage 1 period 5 cases, 2 period 38 cases, 3 period 7 cases; Giant mass type 42 cases and Multiple nodular type 8 cases. According to the extent of disease, there were right liver lobe 37 cases ,left liver lobe 5 cases and total liver lobes

Liver function tests
Each liver function index of both groups pre-TACE was in the normal range, while it rose up 17 in 25 cases and down to the normal level 8 cases in Group A one week after TACE. In Group B the transaminase of all 25 cases rose 1-5 times post-TACE.

Liver fibrosis index
The serum fibrosis indicators before and after the TACE of patients in both groups had been described particularly in Table 2. The index pre-TACE in both groups have no significant difference (P>0.05). No significant difference were found from cases in Group A either pre-TACE or post-TACE (P>0.05), while the index rose obviously in Group B (P<0.05). Compared the difference before and after TACE of each index, there were significant differences (P<0.01). While the compare of two groups post-TACE shows all index in Group B were higher than which in Group A.

Short term effect
Evaluation of curative effect have been made in all 50 cases. The recent curative effect was as follows: CR 3 cases, PR 8 cases, SD 16 cases, PD 1 case in Group A and CR 1 case, PR 7 cases, SD 10 cases, PD 6 cases in Group B.

The follow-up results and Long-term evaluation
During the follow-up, 1 in 50 cases was lost(lost rate was 2%). The last follow-up was in October 30, 2006 and the follow-up time was 8 to 32 months. At the last follow-up, survival 16 in 25 cases and death 9 in 25 cases in Group A while survival 13 in 25 cases, death 11 in 25 cases and loss 1 in 25 cases in Group B. Cases in Group A had the survival period of 10-31 months, with a 19.2 days mid-survival rate ,had TTP of 9.5 months and survival rates of 1-, 2-years were 60.9%, 40.1%, whereas in Group B the survival time was 8-28mo, the media time was 18.5 months, the TTP was 9.1 months and the 1-, 2-years survival rates were 58.8%, 39.5%. There was no remarkable difference between two groups (P>0.05).

Discussion
The difference between TAE and TACE is focused on the argument over the rationale of chemotherapeutic agents injected into the blood vessels (Marelli et al., 2007;Miraglia et al., 2007;Pleguezuelo et al., 2008). Those who support TACE suggested that TACE appears to be superior to TAE (Artinyan et al., 2008). TACE derives its antitumor effect by two methods. Firstly, chemotherapy allows a higher dose of chemotherapeutic agents to the tissue while systemic exposure is less extensively increased. Thus, the cytotoxic effect on tumor cells is enhanced and systemic side effects are mild. In the mean time, arterial embolization interrupts blood supply of tumors that are mostly supplied by the hepatic artery, and postpones tumor growth. This effect will be potentiated because chemotherapeutic drug will not washed out from tumor bed after embolization (Miraglia et al., 2007). Secondly, during TACE, embolotherapy and regional chemotherapy are synergic since tumor ischemia caused by embolization increases drug concentration compared to sole infusion and prolongs retention of chemotherapeutic drugs, and with repeated TACE, survival time for a patient with unresectable HCC could be rationally extended for 1 to 2 years (Huang et al., 1999). However, the best regimen and best dosage of chemotherapy is not established. In some study, adriamycine was administrated in 50mg but in others in 25 mg (Choi, 2009;Britten, 2012). No comparison regarding treatment efficacy and side effects was conducted between low-and conventional doses. Our study shows: compared with conventionaldose of chemotherapeutic agent, the value of serum fibrosis indicators measured shortly after TACE in lowdose chemotherapeutic agent group did not increase significantly. This could be interpreted as a result that risk of liver fibrosis is decreased. Liver fibrosis implys the ultra-precipitation of hepatic extracellular matrix especially collagen, which is a repair response of injury in liver parenchyma. Now some indicators were considered to have diagnostic significance in detecting liver cirrhosis, including HA, LN, PCⅢ and C-Ⅳ (Collazos et al., 1993;Resino et al., 2010). As HCC mostly occurred during hepatic cirrhosis in China, the extent of hepatic cirrhosis in a clinical setting will greatly influence curative effect, complication and prognosis of treatment for HCC patients. We found in this study: if serum fibrosis indicators elevated markedly after first TACE, it suggests that chronic liver injury may occur, and will aggravate liver cirrhosis; if chronic liver injury has not been well treated, liver cirrhosis may further deteriorate. In Conclusions, low-, compared with conventional-dose chemotherapy impose same impact on the variation of fibrosis related indicators and has no influence on median survival time and survival rate after TACE in HCC patients, suggesting low dose TACE in treating HCC in China is a proper choice, but this conclusion needs to be verified by large scale randozimed clinical trials.