Original article Risk of HBV reactivation according to viral status and treatment intensity in patients with hepatocellular carcinoma

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1 Antiviral Therapy 11; 16: (doi:.3851/imp18) Original article Risk of HBV reactivation according to viral status and treatment intensity in patients with hepatocellular carcinoma Jeong Won Jang 1 *, Jung Hyun Kwon 1, Chan Ran You 1, Jin Dong Kim 1, Hyun Young Woo 2, Si Hyun Bae 1, Jong Young Choi 1, Seung Kew Yoon 1, Kyu Won Chung 1 1 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Internal Medicine, Pusan National University School of Medicine, Busan, Korea *Corresponding author garden@catholic.ac.kr Background: There are no convincing data supporting the routine use of pre-emptive therapy against HBV reactivation in various loco-regional therapies for hepatocellular carcinoma (HCC). This study investigated the incidence, severity and risk factors of HBV reactivation during locoregional therapies. Methods: A total of 5 prospectively enrolled patients were classified in order of increasing intensity of locoregional therapies: local ablation therapy (LAT; 43 patients), transarterial chemotherapy using adriamycin (TAC-ADR; 93 patients) or combined epirubicin-cisplatin (TAC-EC; 26 patients), and combined chemo-radiotherapy (TAC-EC+RT; 43 patients). Results: During the follow-up, 62 (.2%) patients developed HBV reactivation. Multivariate analysis identified HBV DNA levels > 4 copies/ml (P=.41) and treatment option (P=.1) to be independent predictors of HBV reactivation. There was a significant trend for increasing risk of reactivation with increasing intensity of therapy, with hazard ratios of 1. for LAT, 2.45 for TAC-ADR, 4.19 for TAC-EC and.17 for TAC-EC+RT. The severity of reactivated disease was also increased with increasing treatment intensity (P-value for trend <.5). Only one of the patients with low-level viraemia receiving LAT alone developed reactivation, whereas a substantial number of patients with high-level viraemia eventually developed reactivation. Conclusions: High-level viraemia and high-level treatment intensity are the major risk factors for HBV reactivation during loco-regional therapy. Trends are evident for the increased risk and severity of reactivation with the aggressiveness of treatment. Pre-emptive antiviral therapy should be recommended for all patients with high-level viraemia irrespective of treatment option, or those undergoing any intensive therapy. Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignant tumour and the third most common cause of cancer-related mortality worldwide [1]. In many patients with HCC, the underlying cirrhosis represents a challenge, and often precludes surgical options with curative intent, such as resection and transplantation. Accordingly, the majority of patients are mainly treated with loco-regional therapies on the basis of tumour stage and the status of underlying cirrhosis. HBV reactivation is a well-known and potentially life-threatening complication in chronic HBV carriers receiving systemic chemotherapy for cancer treatment. In theory, the risk of viral reactivation by loco-regional therapy may be negligible or only minimal compared with systemic chemotherapy, because a loco-regional approach is not likely to significantly disturb host immunity. However, previous observations have demonstrated that this phenomenon indeed occurs during the therapy for HCC. The risk for HBV reactivation in transarterial therapy is reportedly 22 24% [2,3], which appears to be lower than the reported rates of % in patients with HCC receiving systemic chemotherapy [4,5]. Such a varying reactivation risk according to treatment option is a consideration in decision making for pre-emptive measures in the treatment of HCC, and raises the need of detailed further evaluation. The current guidelines recommend the use of preemptive antiviral drugs for chronic carriers undergoing 11 International Medical Press (print) -58 (online) 969

2 JW Jang et al. systemic chemotherapy [6 8], but the consensus on the pre-emptive approach in the setting of loco-regional therapy is not well-established. To date, a comparison of viral reactivation between different loco-regional options has not been prospectively studied. Given the current common use of loco-regional therapies for HCC, a good understanding of HBV reactivation and risk factor identification is of great importance, and this information would help to establish future recommendations regarding the optimal pre-emptive strategy against HBV reactivation during loco-regional therapy. Thus, we conducted a prospective study to investigate and compare the incidence and severity of HBV reactivation in patients with HCC undergoing various locoregional therapies. This analysis allowed us to assess the effect of treatment intensity and immune suppression on the risk of HBV reactivation and the role of preemptive antiviral therapy unique to patients with HCC undergoing loco-regional therapies. Methods Patients Between September 5 and December 9, consecutive patients with newly diagnosed HBV-related unresectable HCC at our liver units (Incheon St Mary s Hospital, Seoul St Mary s Hospital, The Catholic University of Korea, Seoul, Korea) were considered for entry into the study. In Korea, antiviral therapy has not generally been carried out for patients with positive HBV DNA levels and mildly increased aminotransferase levels (< IU/l), because the Korean health insurance policy is strictly confined to viraemic carriers with increased aminotransferase levels >2 the upper limit of normal (ULN; IU/l), irrespective of cirrhosis or HCC. Thus, antiviral-naive patients with a baseline serum alanine aminotransferase (ALT) level <1.5 ULN were eligible for enrolment. The diagnosis of HCC was based on histological evidence or increased serum α-fetoprotein (AFP) levels (> ng/ml) with typical radiologic findings (arterial hypervascularization with portal/venous wash-out). Treatment assignment in our institution was made on the basis of tumour characteristics and hepatic reserve function. Briefly, patients were considered to receive either orthotopic liver transplantation or partial hepatectomy for HCC eligible for surgery. Small and/or multifocal ( 3 nodules) diseases not eligible for surgery were subjected to percutaneous ethanol injection therapy (PEIT) or radiofrequency ablation (RFA) when appropriate. The remaining patients underwent transarterial chemotherapy, and local therapies, such as PEIT or RFA, were added to transarterial therapy if necessary. Patients with portal vein thrombosis (PVT) or extrahepatic metastasis were considered to receive radiotherapy (three-dimensional conformal radiotherapy or helical tomotherapy) in addition to transarterial therapy [9,]. Patients who had any of the following criteria were excluded from the study: a previous history of antiviral therapy, baseline serum ALT level 1.5 ULN, serum HBV DNA level > 8 copies/ml, coexisting serious medical diseases, positive tests for antibody to HCV or HIV, altered blood cell counts (white blood cell [WBC]<3,/ mm 3 or platelet count <,/mm 3 ), Child Pugh classification C, or pre-existing evidence of hepatic decompensation including encephalopathy, ascites, prolonged prothrombin time (>3 s) or a bilirubin level >2.5 ULN. Each patient provided informed consent for this study. The study was approved by the Ethics Committee of The Catholic University of Korea. Treatment and follow-up The intensity of loco-regional treatment protocols used was largely based on tumour stage: intra-arterial chemoembolization using mg of adriamycin alone (TAC-ADR) was performed every 2 months for patients who had multifocal tumours cm with or without peripheral PVT; intra-arterial chemo-lipiodolization using a combination of epirubicin ( mg) and cisplatin ( mg; TAC-EC) was performed at 6-week intervals for patients with Child Pugh class A who had large tumours (> cm) or PVT at the first or second branch; patients with a main PVT or extrahepatic metastasis were considered for radiotherapy in addition to TAC- EC regimen (TAC-EC+RT) [9 11]. The transarterial procedure was repeated until radiologic disappearance or complete necrosis of viable tumour was achieved. At baseline, hepatitis B surface antigen (HBsAg), antibody to HBsAg, hepatitis B e antigen (HBeAg), antibody to HBeAg (anti-hbe; Abbott Laboratories, Abbott Park, IL, USA), and anti-hcv were measured by commercial immunoassays (Abbott Diagnostics, Chicago, IL, USA). Serum HBV DNA was quantitatively measured using a Real-Q HBV quantification kit (BioSewoom Inc., Seoul, Korea) with a detection limit of 56 copies/ml. Serum HBV DNA, HBeAg, anti-hbe and AFP levels were measured before each cycle of treatment or more frequently if necessary (at intervals of <6 8 weeks) on treatment, and every 3 months after complete necrosis was achieved. Complete blood cell counts and liver biochemical tests were performed before and 2 weeks after each cycle of therapy, and monthly thereafter. To exclude treatment-related hepatitis, an abrupt elevation in ALT levels within 2 weeks after treatment was not considered to be reactivation hepatitis. For patients who developed HBV reactivation, antiviral treatment using lamivudine ( mg/day) or entecavir (.5 mg/day) was started immediately when ALT levels increased to 2 ULN, or whenever clinically warranted International Medical Press

3 HBV reactivation in HCC patients Definitions and end points The definitions of HBV reactivation and hepatitis were made and modified based on previous reports [4,12,13]. HBV reactivation was defined as a 1 log increase in serum HBV DNA compared with the baseline level [4,12,13]. Hepatitis due to HBV reactivation was defined as a threefold or greater increase in ALT levels if baseline values were normal, or a twofold or greater increase in ALT levels if baseline values were over the ULN in patients with HBV reactivation [4,12,13]. Hepatic decompensation was defined as newly developed encephalopathy, ascites, variceal bleeding, bilirubin level >2.5 ULN or prolongation of prothrombin time by >3 s [12]. Statistical analysis The duration of the patient s follow-up was calculated from the time of initial treatment to the onset of HBV reactivation, the date of death or the last visit. To ascertain the true effect of each therapy on reactivation, the follow-up was censored at the time of use of either another treatment option or a combination therapy that was clinically warranted. The cumulative event of HBV reactivation was estimated using the Kaplan Meier method, and the differences were analysed using the log-rank test. Univariate and multivariate analyses with the Cox proportional hazard model were used to identify risk factors for HBV reactivation. The cutoff value representing the best discrimination derived from the area under the receiver operating characteristic (AUROC) analysis was chosen for categorization of continuous variables. For additional analyses, the dose response relationship between the degree of treatment intensity and the reactivation risk was examined for statistical significance with a test for trend. Two-tailed P-values <.5 were considered significant. Data were analysed using SPSS version 15. (SPSS Inc., Chicago, IL, USA). Results Study population A total of 332 patients with non-surgical HBV-related HCC were prospectively evaluated at Incheon St Mary s Hospital of The Catholic University of Korea. Among the patients, 165 patients were not included in the study because of increased ALT levels >1.5 ULN (85 patients), high HBV DNA levels > 8 copies/ml (7 patients), use of antiviral drugs (29 patients), Child Pugh class C (18 patients), patient refusal (8 patients), poor performance (6 patients), coexisting medical diseases (4 patients) or loss to follow-up (8 patients). Recruitment of 43 patients in the local ablation therapy (LAT) group was carried out at Seoul St Mary s Hospital (an affiliated teaching hospital, The Catholic University of Korea) according to centre expertise, including five patients referred from Incheon St Mary s Hospital. Thus, the data consisted of observations in 5 consecutive patients. According to the aforementioned treatment allocation, patients were divided into four groups: LAT group (43 patients; 7 with PEIT and 36 with RFA), TAC-ADR group (93 patients), TAC-EC group (26 patients) and TAC-EC+RT group (43 patients). Some exceptions to the treatment protocol were made on a case-by-case basis after a full discussion between expert physicians, including patients, due to unfavourable tumour location or inaccessibility for local therapy (7 patients), haematological or renal dysfunction (6 patients), physician or patient preference ( patients), patient refusal of local therapy (3 patients), the high cost of radiotherapy (9 patients) and health insurance restrictions (5 patients). The median number of transarterial therapy cycles was 4 (range 1 12), 3.5 (range 1 11) and 2 (range 1 5) for the TAC-ADR, TAC-EC and TAC-EC+RT groups, respectively. The median radiation dose in the TAC- EC+RT group was 49.8 Gy (range 58). The baseline characteristics of the study subjects are summarized in Table 1. Risk factors During the mean ±sd follow-up period of 11.1 ±9. months, 62 (.2%) patients in the entire study population eventually developed HBV reactivation and 32 (51.6%) of the 62 patients with HBV reactivation experienced hepatitis. The estimated probabilities of HBV reactivation at 6, 12 and 24 months were 29.4%, 39.7% and 49.6%, respectively. To identify predictors of HBV reactivation in patients undergoing loco-regional therapy, 12 potential variables of interest were analysed, as listed in Table 2. Of these, age (P=.21), baseline HBV DNA levels (P=.67), tumour size (P=.61), portal vein thrombosis (P=.11), extrahepatic metastasis (P=.12) and treatment option (P<.1) were marginally or significantly associated with viral reactivation during therapy. The optimum cutoff levels for age and baseline HBV DNA had been established at 51 years (AUROC=.613, 95% CI ) and 8,621 copies/ml (AUROC=.585, 95% CI.2.6), respectively, and cutoff values of years and 4 copies/ml were used for further statistical analysis. With multivariate analysis using a Cox proportional hazard regression model, a baseline HBV DNA level > 4 copies/ml (hazard ratio [HR]=1.81, 95% CI ; P=.41) and treatment option (HR=1.99, 95% CI ; P=.1) were identified as the two independent predictors of HBV reactivation (Table 2). As shown in Figure 1A, patients with a high viraemia (HBV DNA> 4 copies/ml) tended to have more Antiviral Therapy

4 JW Jang et al. Table 1. Baseline characteristics of all 5 patients Characteristic LAT group (n=43) TAC-ADR group (n=93) TAC-EC group (n=26) TAC-EC+RT group (n=43) Age, years 54.5 ± ± ± ±.1 Male:female sex ratio 33: 71:22 21:5 32:11 ALT, IU/l 35.3 ± ± ± ±16.9 Total bilirubin, mg/dl.9 ± ± ± ±1.6 HBeAg seropositivity, n (%) 7 (16.3) 33 (35.9) 7 (26.9) 12 (27.9) HBV DNA, 3 copies/ml 18.9 (.56 21,589) 94.6 (.86 83,426).9 (.56,354) 14.4 ( ,928) AFP, ng/ml.2 (1.6 2,793).5 ( ,7).1 (.8 337,3) 275. ( ,4) Tumour size, cm 2.6 ± ± ± ±3.5 Tumour number Single, n (%) 35 (81.4) 47 (.5) 6 (23.1) 15 (34.9) Multiple, n (%) 8 (18.6) 46 (49.5) (76.9) 28 (65.1) Portal vein thrombosis Present, n (%) () 16 (17.2) (76.9) 27 (62.8) Absent, n (%) 43 () 77 (82.8) 6 (23.1) 13 (37.2) Platelet count, 3 /mm 3 4. ± ± ± ±73.8 Imaging finding of cirrhosis, n (%) a 32 (74.4) 73 (78.5) 18 (69.2) 33 (76.7) Child-Pugh classification A 41 (95.3) 73 (78.5) 21 (.8) 32 (74.4) B 2 (4.7) (21.5) 5 (19.2) 11 (25.6) Data are mean ±sd unless indicated otherwise. a Imaging findings of liver cirrhosis include radiological evidence of nodular liver surface contour, formation of collateral vessels, splenomegaly and ascites. AFP, α-fetoprotein; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; LAT, local ablation therapy; TAC-ADR, transarterial therapy using adriamycin; TAC-EC, transarterial therapy using combined epirubicin and cisplatin; TAC-EC+RT, transarterial chemotherapy using combined epirubicin and cisplatin with radiotherapy. Table 2. Risk factors for HBV reactivation and hepatitis due to HBV reactivation during anti-cancer therapy for hepatocellular carcinoma HBV reactivation Univariate Multivariate Factor P-value HR (95% CI) P-value Male sex.227 Age years ( ).54 HBeAg seropositivity ( ).384 HBV DNA> 4 copies/ml ( ).41 AFP level > ng/ml.565 ALT level > IU/l.9 Child Pugh class B.427 Tumour size >5 cm ( ).429 Tumor multiplicity ( ).433 Portal vein thrombosis ( ). Extrahepatic metastasis ( ).798 Treatment option used < ( ).1 AFP, α-fetoprotein; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HR, hazard ratio. frequent episodes of HBV reactivation during the follow-up than those with a low viraemia (HBV DNA 4 copies/ml; P=.67, log-rank test). Kaplan Meier plots for treatment option over time showed a significant trend toward the increasing incidence of HBV reactivation with increasing intensity of locoregional therapy (Figure 1B). The risk of developing viral reactivation was the highest in patients undergoing TAC-EC+RT, followed by TAC-EC, TAC-ADR and LAT (P<.1, log-rank test). The estimated probabilities of HBV reactivation at 6 months were 3.9%,.1%, 34.% and 66.7% for the LAT, TAC-ADR, TAC-EC and TAC-EC+RT groups, respectively. There was a significant dose risk relationship between the degree of treatment intensity and HBV reactivation, with an adjusted HR of 2.45 (95% CI ) for TAC-ADR, 4.19 (95% CI ) for TAC-EC and.17 (95% CI ) for TAC-EC+RT, as International Medical Press

5 HBV reactivation in HCC patients Figure 1. The cumulative probabilities of HBV reactivation according to HBV DNA level and treatment option HBV reactivation, % A B Treatment intensity HBV DNA levels > 4 copies/ml 4 copies/ml Time, months HBV reactivation, % TAC-EC+RT TAC-EC TAC-ADR LAT Time, months DNA> copies/ml DNA copies/ml TAC-EC RT TAC-EC TAC-ADR LAT (A) Patients with a high viraemia tended to experience more frequent incidences of HBV reactivation during the follow-up than those with a low viraemia, with estimated 6- and 12-month rates of 33.1% versus 24.3% and 46.6% versus.7%, respectively (P=.67, log-rank test). (B) There is a dose risk relationship between treatment intensity and HBV reactivation. The estimated HBV reactivation rates at 6 and 12 months were 3.9% and 8.3% for local ablation therapy (LAT),.1% and 34.7% for transarterial chemotherapy using adriamycin (TAC-ADR), 34.% and 52.9% for transarterial chemotherapy using combined epirubicin and cisplatin (TAC-EC), and 66.7% and.9% for TAC-EC with radiotherapy (TAC-EC+RT) groups, respectively (P<.1, log-rank test). Table 3. Risk for HBV reactivation according to treatment options for hepatocellular carcinoma Treatment options Crude HR (95% CI) P-value Adjusted HR (95% CI) a P-value LAT 1. (reference) <.1 b 1. (reference).1 b TAC-ADR 2.79 ( ) ( ).71 TAC-EC 4.32 ( ) ( ).13 TAC-EC+RT 9.47 ( ) <.1.17 ( ) <.1 a Adjustment for age, HBV DNA level and tumour stage. b P-value for trend. HR, hazard ratio; LAT, local ablation therapy; TAC-ADR, transarterial therapy using adriamycin; TAC-EC, transarterial therapy using combined epirubicin and cisplatin; TAC-EC+RT, transarterial chemotherapy using combined epirubicin and cisplatin with radiotherapy. compared with patients treated with LAT (P-value for trend =.1; Table 3). Patterns of HBV reactivation risk stratified by treatment intensity between patients with a low and high viraemia Additionally, a stratified analysis by treatment option was done separately in patients with a low and high viraemia, using a cutoff HBV DNA level of 4 copies/ ml. The estimated HBV reactivation rates at 6 months in patients with a low viraemia were %, 12.%,.% and 52.9% for the LAT, TAC-ADR, TAC-EC and TAC- EC+RT groups, respectively, while the corresponding rates in patients with a high viraemia were 9.1%, 23.%, 36.6% and 81.6%, respectively (Figure 2A and 2B). For the LAT group, only one of the patients with a low viraemia developed HBV reactivation, whereas those with a high viraemia often eventually developed HBV reactivation during extended follow-up, with 2-year estimated probabilities of 9.1% versus 35.4% (Figure 2A and 2B). Incidence of hepatitis and hepatic decompensation in patients with HBV reactivation For the reactivated patients, the median times to viral reactivation and hepatitis due to viral reactivation were 2.83 months (range ) and 4.37 months (range ) from the initiation of Antiviral Therapy

6 JW Jang et al. Figure 2. Comparison of the probabilities of HBV reactivation by treatment option in patients with a low and high HBV DNA level A HBV reactivation, % HBV DNA 4 copies/ml B HBV reactivation, % HBV DNA> 4 copies/ml Time, months Time, months TAC-EC+RT TAC-EC TAC-ADR LAT A low HBV DNA level was defined as 4 copies/ml and a high HBV DNA level as > 4 copies/ml. (A) Trends for increasing risk of HBV reactivation with treatment intensity are shown in patients with a low viraemia (P=.2, log-rank test for trend). There was a relatively low risk of reactivation in low-level viraemic patients receiving local ablation therapy (LAT), with a 2-year estimated probability of 9.1%. (B) The overall risks of HBV reactivation in patients with high-level viraemia were very high, irrespective of treatment option, with a dose risk relationship between treatment intensity and HBV reactivation (P<.1, log-rank test for trend). TAC-ADR, transarterial chemotherapy using adriamycin; TAC-EC, transarterial chemotherapy using combined epirubicin-cisplatin; TAC-EC+RT, transarterial chemotherapy using combined epirubicin and cisplatin with radiotherapy. therapy, respectively. The incidence rates of reactivation hepatitis among the 62 patients with HBV reactivation were.% (1/5) for LAT, 41.7% (/24) for TAC-ADR,.% (4/8) for TAC-EC and 68.% (17/25) for TAC-EC+RT. The corresponding rates for hepatic decompensation were % (/5) for LAT, 4.2% (1/24) for TAC-ADR, 12.5% (1/8) for TAC-EC and 32.% (8/25) for TAC-EC+RT. Trends were significant for a higher risk of reactivation hepatitis and hepatic decompensation with the increasing intensity of treatment option (P-value for trend =. and.6, respectively; Figure 3). Antiviral therapy was immediately administered to all the 32 patients with hepatitis due to HBV reactivation. Ten patients developed hepatic decompensation resulting from viral reactivation, and one of them in the TAC-EC+RT group died of hepatic failure days after HBV reactivation, despite antiviral therapy. For ethical reasons and whenever clinically warranted, 22 of the patients with viral reactivation only eventually had antiviral therapy during extended follow-up, even though there was no evidence of definitive reactivation hepatitis. Among the remaining eight virally reactivated patients without antiviral therapy, two died of tumour progression, two were lost to further follow-up, and four had fluctuating HBV DNA levels ranging from 3 to 5 copies/ml without decompensation events during the follow-up. Discussion Studies regarding HBV reactivation have identified several risk factors for reactivation, including the types of diseases, other host factors, viral factors and chemotherapeutic drugs used [5,14,15]. Among these, a high viral load has been consistently found to be the most strongly associated with HBV reactivation in multiple publications [12,16,17]. The present study also showed a strong positive relationship between high viraemia and an increased risk for reactivation in patients with HCC. The definitive pretreatment cutoff level of HBV DNA predicting HBV reactivation may vary with the types of malignancies or degree of immune suppression by treatment used. In studies of patients with lymphoma and breast cancer who were treated with steroid- and anthracycline-containing regimens, a higher level of serum HBV DNA, ranging from copies/ml, was suggested as an optimal cutoff predicting HBV reactivation [16,17]. By contrast, the current data indicated that the relatively low HBV DNA level of 4 copies/ml is a better cutoff predictive of viral reactivation during loco-regional therapy for HCC, and further confirmed the results of our previous study [12]. In addition, this appears to be in line with current management guidelines for hepatitis B, indicating that a lower, or any detectable level of HBV DNA, be the initiating point for International Medical Press

7 HBV reactivation in HCC patients antiviral therapy in patients with advanced fibrosis or cirrhosis [6,7,18]. One of the most important findings emerging from our analyses is that the treatment option is one of the strongest risk factors for reactivation, with a linear dose-risk relationship between treatment intensity and HBV reactivation. Indeed, we found increasing HRs for HBV reactivation by more intense regional treatment, as follows: 1. for LAT, 2.45 for TAC-ADR, 4.19 for TAC-EC and.17 for TAC-EC+RT, which are thought to be in increasing order of aggressiveness of treatment. Particularly, the estimated probability of HBV reactivation in the TAC-EC+RT group was as high as 66.7% at 6 months, which is comparable to the reported rates of >% in transplant settings that are believed to be at the highest risk for HBV reactivation [5,14,15]. Another Korean study of intra-arterial chemoembolization with radiotherapy for HCC has formerly reported a 21.8% HBV reactivation rate [19], which is modest in comparison to our data. However, the study used a less intense chemotherapy regimen and a strict definition of viral reactivation (viral increase of >2 log of baseline level), as compared with the current study. Thus, these results are in agreement as to the high risk of HBV reactivation during chemo-radiation therapy for HCC. The mechanism of radiation-induced reactivation of HBV is not well-understood. Recently, radiation was reported to induce viral reactivation via a bystander mechanism involving cytokines, such as interleukin-6, released during radiotherapy []. Additionally, intensified treatment itself is an important factor for the degree of immunosuppression and decreases the number of immune cells [21,22]. Indeed, a grade 3 or higher leukopaenia in our analysis was the most common in the TAC-EC+RT group than the other treatment groups (39.5% versus %, P<.5; JWJ et al., data not shown). This may reflect, in part, a considerable compromise in the host immune system by such intensive chemo-radiation, although a relationship between the level of immunosuppression and the number of circulating WBCs, which are a major part of the immune system, should be further investigated. Thus, it is speculated that the downgraded immunity and cytokinebased mechanism played additive roles in causing such an extremely high rate of reactivation in the chemoradiotherapy group. In light of this, patients with HBVrelated HCC undergoing chemo- radiotherapy deserve special attention as high-risk situations. Pre-emptive antiviral therapy should be initiated in all patients who are to receive chemo-radiotherapy. Not all patients with HBV reactivation have acute ALT flares [5]. In theory, cytotoxic therapy suppresses the immune function, and the more potent the immunosuppression, the greater the capability of viral replication potentially leading to extensive liver damage [23,24]. Figure 3. Incidence of hepatitis and hepatic decompensation among patients with HBV reactivation Incidence rate, %. P= Reactivation hepatitis LAT TAC-ADR P= Decompensation TAC-EC TAC-EC+RT 32. There were significant trends for increasing incidences of both reactivation hepatitis and decompensation episodes with the increasing intensity of treatment (P-value for trend =. and.6, respectively). The mean ±sd and median (range) alanine aminotransferase levels in the reactivated cases were 75.6 ±34.5 U/l and 63 U/l (45 115) for the local ablation therapy (LAT), 116. ±152. U/l and 74 U/l (27 7) for the transarterial chemotherapy using adriamycin (TAC-ADR), ±258.5 U/l and 93 U/l (39 855) for the transarterial chemotherapy using combined epirubicin and cisplatin (TAC-EC), and ±247.3 U/l and 136 U/l (41 1,) for the TAC-EC with radiotherapy (TAC-EC+RT) groups. The median (range) HBV DNA levels at the time of reactivation were copies/ml ( ) for the LAT,.1 6 copies/ml (.6 6 1, ) for the TAC-ADR, copies/ml (.3 6 1,2.5 6 ) for the TAC-EC and copies/ml (.3 6 1, ) for the TAC-EC+RT groups. Indeed, we found in our analysis that a substantial number of reactivated patients receiving TAC-EC or TAC- EC+RT with more intense immunosuppressive effect eventually developed overt hepatitis and decompensation episodes, whereas only a small number of reactivated patients receiving less-intense TAC-ADR or LAT developed the hepatic events. Taken together, the overall findings further represent the major role of the degree of treatment intensity compromising host immunity in inducing reactivation hepatitis and its severity. The implications for treatment intensity on viral reactivation and the usefulness of pre-emptive therapy may be debatable by a viral status in various locoregional settings. From additional analyses, it seems reasonable to opt for a pre-emptive therapy approach based on treatment intensity in patients with a low viraemia because a dose risk relationship between treatment intensity and reactivation is apparent. Indeed, the risk of HBV reactivation in patients with a low viraemia receiving LAT alone was low in our results, and thus pre-emptive antiviral therapy may not be highly indicated in this population. However, antiviral therapy would still be beneficial in those patients with low-level but detectable viral load, given Antiviral Therapy

8 JW Jang et al. that the therapy can reduce necro-inflammation in the microenvironment of tumour or the adjacent liver tissues. Thus, the decision-making as to antiviral therapy in patients with a low viraemia undergoing RFA or PEIT could be individualized, taking into account the potential additional benefits besides pre-emptive intent. By contrast, such an individualized pre- emptive approach may be no longer relevant in patients with a high viraemia because the overall risk of HBV reactivation in those patients was indeed high during extended follow-up, irrespective of treatment options (Figure 2B). Thus, all patients with a high viraemia should be a high-risk group and considered for preemptive antiviral therapy. There are some limitations in our study. The types of loco-regional therapy for HCC widely vary, depending on the centre expertise. Accordingly, the clinical features of HBV reactivation can also vary among institutions. In some instances of our patients, the possibility of spontaneous reactivation of HBV cannot be completely excluded [25,26], particularly in those with delayed HBV reactivation. In conclusion, this study highlights that high-level viraemia and high-level immune suppression caused by intense treatment are the major risk factors for HBV reactivation during loco-regional therapy for HCC. Trends are evident for the increased risk and severity of reactivation with the aggressiveness of treatment. Based on our data, pre-emptive antiviral therapy should be considered for all patients with a high viraemia irrespective of treatment option, as well as for all patients undergoing any intense anti-cancer therapy. Acknowledgements JWJ and JYC were involved in the study concept and design. JWJ, JHK, JDK and HYW were involved in the acquisition of data. JWJ, SHB, JYC and SKY were involved in the analysis and interpretation of data. JWJ was involved in the drafting of the manuscript. CRY and JWJ were involved in the statistical analysis. JYC was involved in the study supervision. This study was supported by a grant from the Research Foundation of Physicians, The Catholic University of Korea. Disclosure statement The authors declare no competing interests. References 1. Bosch FX, Ribes J, Borras J. Epidemiology of primary liver cancer. Semin Liver Dis 1999; 19: Nagamatsu H, Kumashiro R, Itano S, et al. Investigation of associating factors in exacerbation of liver damage after chemotherapy in patients with HBV-related HCC. Hepatol Res 3; 26: Jang JW, Choi JY, Bae SH, et al. Transarterial chemorlipiodolization can reactivate hepatitis B virus replication in patients with hepatocellular carcinoma. J Hepatol 4; 41: Yeo W, Lam KC, Zee B, et al. Hepatitis B reactivation in patients with hepatocellular carcinoma undergoing systemic chemotherapy. Ann Oncol 4; 15: Yeo W, Johnson PJ. 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