Chronic Hepatitis B (CHB) related cirrhosis and hepatocellular carcinoma (HCC) account for more than 750,000 deaths per year. Current therapies for CHB are limited in achieving HBsAg decline/loss and thus there remains a pressing need for curative treatment strategies. Although, Pegylated Interferon-α (Peg-IFNα) may be used, the majority of patients progress to nucleos(t)ide analogue (NUC) therapy due to treatment failure. Peg-IFNα and NUCs used in isolation act differentially on the immune response; Peg-IFNα induces NK cell activation and NUC therapy may partially restore T cell function. NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined the NK cell pool in HBeAg-positive CHB patients treated with Peg-IFNα and whether changes in the NK cell repertoire are induced when patients are 'primed' with Peg-IFNα and importantly, whether these changes are sustained or further modulated long-term after switching to sequential NUC therapy. The cumulative expansion of CD56bright NK cells driven by 48-weeks of Peg-IFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Peg- IFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors during sequential NUCs. The expansion in proliferating, functional NK cells and HBsAg reduction was greater and more pronounced following sequential NUCs than in patients treated with de novo NUCs. This highlights the potential benefit of Peg-IFNα- priming, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses. Sustained boosting of NK cells on sequential NUCs following Peg-IFNα-priming has not previously been described raising the potential of 'long-lived' NK cell populations in keeping with their emerging adaptive features. These findings provide a mechanistic and immunological rationale to explore combination/sequential treatment strategies for CHB, including on-treatment immune responses in the liver, whilst awaiting the emergence of new therapies in the field.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

Cancer is the second leading cause of death in the USA, following cardiovascular disease. Treating cancer using conventional therapies is associated with low response rates and high toxicity, because these therapies usually lack specific tumor accumulation. Loading anticancer drugs into intelligently designed polymeric nanoparticles (NPs) can serve in delivering these drugs specifically to the tumor site, thus boosting their efficacy and reducing any associated off target toxicity. Targeting NPs to the tumor site can occur through either passive or active means. In passive targeting, NPs of specific size and surface characteristics can exploit the tumor’s erratic vasculature and occluded lymphatic drainage to extravasate the systemic circulation and accumulate preferentially at the tumor site. Active targeting mandates grafting the surface of NPs with a ligand that specifically interacts with a protein expressed at higher levels at the tumor site, in comparison to elsewhere in the body. In the current research, we independently investigated the utilization of passive and active targeting strategies to treat aggressive forms of cancer. Initially, passively targeted poly(lactic-co-glycolic acid) (PLGA) NPs to treat aggressive forms of endometrial cancer (EC) were investigated. A novel combination of soluble paclitaxel (PTX), a first line chemotherapy for EC, and soluble BIBF1120 (BIBF, nintedanib), an antiangiogenic molecular inhibitor, was first tested against three EC cell lines bearing different p53 mutations. The results showed that only EC cells with loss of function (LOF) p53 were sensitive to the combination therapy, indicating the potential of this combination to engender synthetic lethality to PTX. Next, NPs loaded with PTX were investigated with respect to the impact of varying the polymer lactic acid to glycolic acid ratio and the surfactant type on the major physicochemical properties of the prepared nanoparticles, drug loading, cellular uptake, cytotoxicity, and drug release. The optimum formulation was then loaded with BIBF and the combination of independently loaded passively targeted NPs were further evaluated for in vivo activity against a xenograft model of LOF p53 EC. The combination of independently loaded NPs exhibited the highest reduction in tumor volume and prolonged survival when compared to soluble PTX, PTX NPs or untreated control. These data highlight this specific combination of NPs as a novel promising therapy for LOF p53 EC. In a second study, the use of actively targeted NPs to treat liver cancer was explored. In this study, a combination of small interfering RNA (siRNA) against astrocyte elevated gene-1 (AEG-1), and all-trans retinoic acid (ATRA) was investigated as a new therapy for hepatocellular carcinoma (HCC). AEG-1 is a highly expressed oncogene that is directly involved in HCC progression and aggressiveness, in addition to reducing the ability of retinoic acid to induce apoptosis in HCC cells. First, a new conjugate was synthesized that was capable of delivering siRNA selectively to HCC cells, using galactose as a targeting moiety. The conjugate was prepared by linking poly(amidoamine) (PAMAM) dendrimers, polyethylene glycol (PEG) and lactobionic acid (Gal, disaccharide containing galactose) (PAMAM-PEG-Gal). We confirmed the synthesis of the conjugate using 1H-NMR, Mass spectrometry and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Next, nanoplexes of the synthesized conjugate, PAMAM-PEG-Gal, and AEG-1 siRNA were prepared. Nanoplexes were further characterized for their size, surface charge, morphology, and electrophoretic mobility to identify the optimum complexation ratio between PAMAM-PEG-Gal and the siRNA. Then, mice bearing orthotopic luciferase expressing HCC cells were treated with the optimum nanoplex formulation. Results showed that a combination of AEG-1 nanoplexes and ATRA results in a significant reduction in luciferase expression, reduced liver weight, lower AEG-1 mRNA levels and increased apoptosis, when compared to utilizing nanoplexes with silencing control (siCon), siCon+ATRA, or AEG-1 nanoplexes alone. The results indicate that the combination of liver-targeted AEG-1 nanoplexes and ATRA may be a potential treatment for aggressive HCC. These data place targeted NPs as a promising efficient delivery system for cancer treatment.

The goniothalamin (GTN) and related styryl-lactones were found to be cytotoxic and apoptotic to a variety of tumor cell lines including breast cancer MCF-7, cervical cancer HeLa and leukemia HL60. In HL60 cells, GTN triggers mitochondria dysfunction, caspase activation and eventually, apoptosis. In this study, we demonstrated that GTN was able to induce apoptosis in hepatocellular carcinoma derived cells, SK-Hep1 and Hep-3B cells via upregulation of the phorbol-12-myristate-13-induced 1 (PMAIP1) protein , alternation of mitochondrial membrane potentials (P < 0.05) via TP53-dependent and -independent transactivations. Treatment with GTN induced DNA damage, formation of reactive oxygen species (ROS, P < 0.05) and activated cleaved CASP8, CASP9, CASP3 and poly (ADP-ribose) polymerase 1 proteins. However, cleaved BH3 interacting domain (BID) death agonist protein was not identified, suggesting that an intrinsic cellular stress was produced after GTN treatments in both cell lines. A pan caspase inhibitor, z-VAD-FMK, suppressed GTN-induced apoptotic cell percentages (P < 0.05), demonstrating that GTN-induced apoptosis was mediated by the activation of the caspase cascade protein. The GTN induced ROS formation prior to DNA damage in SK-Hep1, yet in reverse order in Hep-3B cells. Moreover, GTN induced DNA damages through activation of the gamma H2A histone family member X (£^H2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2 checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15), and £^H2AFX/pCHEK2 (Thr68), in SK-Hep1 and Hep-3B cells,respectively. Among five integral outer mitochondrial membrane proteins that blocks or induces apoptotic death, PMAIP1 protein and PMAIP1 mRNA levels were upregulated after GTN treatments for 4 to 6 h in both cell lines (P < 0.05). Transfection of shRNA interference plasmids targeting PMAIP1 gene downregulated PMAIP1 mRNA (P < 0.05) and PMAIP1 protein (P < 0.05) levels, as well as GTN-induced apoptotic cell percentages (P < 0.05) in both cell lines. In parallel, transfection of the shRNA interference plasmid targeting TP53 gene in SK-Hep1 cells, downregulated TP53 and PMAIP1 mRNA (P < 0.05) and TP53, pTP53, PMAIP1, cleaved PARP1 protein levels and apoptotic cell percentages (P < 0.05). Treatment with the TP53 inhibitor, PFT-£\ or transfection of a dominant negative TP53 plasmid, pTP53mt135, repressed TP53, pTP53 and PMAIP1 protein and/or TP53, PMAIP1 mRNA levels (P < 0.05), however, significantly augmented GTN-induced apoptotic cell percentages (P < 0.05). Cytosol/mitochondria fractionation identified that TP53, along with PMAIP1 proteins, were translocated to mitochondria after GTN treatment in time-dependent manners. Taken together, our studies demonstrated that GTN induced apoptosis via PMAIP1 via both TP53-dependent and -independent transactivation pathways. In TP53-positive SK-Hep1 cells, PMAIP1 and TP53 proteins conducted synergic effects.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world and Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC had been explored in recent years. An extensive array of growth factors and their receptors had been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. Current therapeutic approaches for HCC include surgical resection (include liver transplantation), trans-arterial embolization (TAE), alcohol injection, etc. However, the effect is limited due to most of the HCC patients present with advanced stages of the disease. Therefore, this underscores the need for the development of novel therapeutic strategies. It is pivotal to set up an orthotopic hepatoma model for the development of novel intervention strategies for HCC. Under the guidance of ultrasound, we are able to create hepatoma in the liver lobe of Sprague-Dawley (SD) rats by injection of Novikoff (N1-S1) hepatoma cells. In addition, sonographic technique was employed for the monitoring of tumor growth in this animal model in the following subprojects. The continuous, non-invasive measurement of orthotopic hepatoma development will be a valuable tool for the evaluation of effects of drugs for treatment of HCC. In Chapter 1, the study employed a relatively non-invasive approach to establish an orthotopic HCC model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic HCC in 60.4% of the SD rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. Moreover, there was a significant correlation in tumor size measurement by ultrasound and computerized tomography. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in microvessel density in tumors. In chapter 2, we investigated the chemopreventive effects of celecoxib in the growth of orthotopic rat HCC and the possible signal pathways involved. The status of COX-2 expression in rat Novikoff HCC was consistent with that in human HCC. Both Western blot and PCR tests had proved that N1-S1 was a HCC model presenting with low COX-2 enzymes in tumor cells. Then, low doses of celecoxib was shown to effectively inhibited the proliferation and increased the apoptosis of N1-S1 cells in vitro, which were also safe to the normal hepatocytes. Moreover, chemoprevention by celecoxib inhibiting the HCC tumor growth was shown in rat orthotropic HCC model. Tumor incidence was not affected by the celecoxib prevention, but, tumor weight was found significantly suppressed by the drug. Possible mechanisms of chemoprevention by celecoxib seen in the animal model were thought to be related to the anti-angiogenic, anti-proliferative and anti-hCSC characters of the drug. In chapter 3, we tried to test the combined inhibitory effects of low doses of celecoxib and epirubicin on the growth of HCC. Combined low doses of epirubicin and celecoxib was effective in inhibiting the hepatic cancer stem cells, tumor angiogenesis, tumor cell proliferation, as well as promoting cancer apoptosis. These are compatible with the effects of the individual drugs on HCC growth shown in the previous two chapters. In general, combination therapy expressed more effectiveness in tumor suppression and less bone marrow suppression than the individual drugs used alone. Taken together, ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic HCC in SD rats, which is suitable and convenient for therapeutic trial of anti-HCC treatment. In the current study, we had proved the efficacies of low doses of two drugs, epirubicin and celecoxib, acting individually, as well as the combined effects of them in treating HCC in this model.

Leukocyte cell-derived chemotaxin 2 (LECT2) is first isolated as a 16-kDa secreted protein from cultured fluid of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells. Recently LECT2 has shown to be synthesized by human hepatocytes and stimulates the growth of chondrocytes. LECT2 is involved in chemotactic factor to neutrophils and may be associated with rheumatoid arthritis. Besides, LECT2 is evolutionarily conserved and acts as a repressor in the Wnt/£]-catenin signaling pathway. Wnt/£]-catenin signaling is implicated in liver carcinogenesis. However, the exact roles of LECT2 in liver carcinogenesis are not yet well characterized. This study is to investigate the extra roles of LECT2 in Wnt signaling. Our results showed that adenoviral administration of LECT2 over-expression suppress oncogenic processes such as migration, invasion, proliferation and colony formation, as well as alteration in cell cycle distributions. In animal model significantly suppress liver malignancies in orthotopic Novikoff hepatoma. In conclusion, we show that ad-LECT2 gene delivery attenuated cell carcinogenesis process via downregulated Wnt/£]-catenin signaling in vitro and suppressed tumor growth in vivo. Besides LECT2 over-expression represents a novel therapeutically factor for hepatocelluar carcinoma.

By Western blotting and immunohistochemical analyses, we have demonstrated that Pin1 was overexpressed in 71.4% of hepatocellular carcinoma (HCC) and its levels correlated with the clinical survival rate. This conclusion was supported by the results from examining Pin1 protein in HCC cancer cell lines. RT-PCR was performed to examine the Pin1 transcription level in tumor part and was compared with that in non-tumor part. Our results indicated that pin1 overexpression was due to the upregulation of Pin1 transcription. Interestingly, most of the cases with upregulation of Pin1 have been shown to correlate with £]-catenin and Cyclin D1 accumulation in HCC specimens. These results were consistent with the previous studies that Pin1 caused £]-catenin and Cyclin D1 elevation in breast cancer. The concordance between hepatitis virus chronic infection and Pin1 overexpression of HCC patients was also analysis. Taken together, these data indicated that Pin1 overexpression leading to £]-catenin and Cyclin D1 accumulation might play a critical role in hepatocellular carcinogenesis and tumor progression. Pin1 levels therefore can be used as a prognostic marker for HCC, and our results suggested that Pin1 is a potential target for therapeutic intervention in hepatocellular carcinoma.

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does ¡§differentiation status¡¨ of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.

Introduction: Most literatures researched radiofrequency ablation (RFA) for early hepatocellular carcinoma (HCC) defined the early tumor size as 3cm or less. However, detection rate of HCC smaller than 2 cm became increasing since high risk patients had received regular screening and the imaging techniques has been much improved. Whether RFA or percutaneous ethanol injection (PEI) is better for a patient with such a small HCC is still controversial. Methods: We retrospectively obtained patients with single HCC 2 cm in diameter or smaller from the computerized medical records database in a local teaching hospital located at southern Taiwan, diagnosed during January 1, 2002 to April 30, 2008. Those patients received RFA (RFA group) or PEI (PEI group) as the first-line nonsurgical treatments were enrolled for further analysis. We compared baseline characteristics of RFA and PEI groups, including gender, age, possible risk factors of recurrence, and prognostic factors. Then, we analyzed recurrent rate, time to recurrence, survival rate, complication rate, mean cost of each treatment, and hospital stay of RFA and PEI groups. Results: There were 32 patients qualified for the study design, including 22 in PEI group¡G13 males and 9 females with mean age was 63.73 years; and 10 in RFA group¡G7 males and 3 females with mea age was 58.30 years¡CNo statistically significant differences between RFA and PEI groups were observed with respect to baseline characteristics. Nevertheless, there was significant difference between these two groups with respect to mean hospital stay (p=0.007) and mean cost (p¡Õ0.001): mean cost of PEI was NTD $16934.7; mean cost of RFA was NTD $51677.6, the difference was NTD $34732.9. There was no difference respect to complication rate, recurrent rate, time to recurrence and overall survival rate between RFA and PEI groups. Conclusion: For patients with single HCC 2 cm in diameter or smaller (i.e. very early HCC), we concluded that: if under similar basic background, the cost of RFA was much higher than that of PEI, but no difference in the complication rate, recurrent rate, time to recurrence and overall survival rate between these two treatment.