Non-classical Monocytes Enhance the Efficacy of Immune Checkpoint Inhibitors on Colon Cancer in a Syngeneic Mouse Model.

BACKGROUND/AIM: The response rate to immune checkpoint inhibitors (ICIs) is approximately 10%-30% and only in a few cancer types. In the present study, we determined whether non-classical monocytes (NCMs) could enhance ICI efficacy in colon cancer using a syngeneic mouse model. MATERIALS AND METHODS: The MC38 C57BL/6 mouse colon cancer model was used. Cells collected from the bone marrow of C57BL/6 mice were cultured, and NCMs were fractionated by cell sorting and administered via the tail veins to the mice implanted with MC38 cells. The anti-mouse PD-L1 antibody was administered three times, and tumor volume and overall survival were observed. RESULTS: More tumors were eradicated and more complete response occurred, after cotreatment with ICIs and NCMs than after treatment with ICIs alone. Moreover, no efficacy was observed when NCMs were administered alone. CONCLUSION: NCMs enhance ICI efficacy. The underlying mechanisms and clinical applications will be studied in the future.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors.

Introduction: Programmed cell death ligand 1 (PD-L1) expression in tumor tissues is measured as a predictor of the therapeutic efficacy of immune checkpoint inhibitors (ICIs) in many cancer types. PD-L1 expression is evaluated by immunohistochemical staining using 3,3´-diaminobenzidine (DAB) chronogenesis (IHC-DAB); however, quantitative and reproducibility issues remain. We focused on a highly sensitive quantitative immunohistochemical method using phosphor-integrated dots (PIDs), which are fluorescent nanoparticles, and evaluated PD-L1 expression between the PID method and conventional DAB method. Methods: In total, 155 patients with metastatic or recurrent cancer treated with ICIs were enrolled from four university hospitals. Tumor tissue specimens collected before treatment were subjected to immunohistochemical staining with both the PID and conventional DAB methods to evaluate PD-L1 protein expression. Results: PD-L1 expression assessed using the PID and DAB methods was positively correlated. We quantified PD-L1 expression using the PID method and calculated PD-L1 PID scores. The PID score was significantly higher in the responder group than in the non-responder group. Survival analysis demonstrated that PD-L1 expression evaluated using the IHC-DAB method was not associated with progression-free survival (PFS) or overall survival (OS). Yet, PFS and OS were strikingly prolonged in the high PD-L1 PID score group. Conclusion: Quantification of PD-L1 expression as a PID score was more effective in predicting the treatment efficacy and prognosis of patients with cancer treated with ICIs. The quantitative evaluation of PD-L1 expression using the PID method is a novel strategy for protein detection. It is highly significant that the PID method was able to identify a group of patients with a favorable prognosis who could not be identified by the conventional DAB method.

Low syntaxin 17 expression in donor liver is associated with poor graft prognosis in recipients of living donor liver transplantation.

AIM: Liver transplantation (LT) is the only curative therapy for decompensated liver cirrhosis. For recipients of living donor LT (LDLT), restoration of liver function after transplantation is highly dependent on liver regenerative capacity, which requires large amounts of intracellular energy. Mitochondrial metabolism provides a stable supply of adenosine 5'-triphosphate (ATP) for liver regeneration. Mitophagy is a selective process in which damaged, non-functional mitochondria are degraded and replaced with new functional mitochondria. We investigated the relationship between expression of Syntaxin17 (STX17), a key protein in mitophagy regulation, in donor livers and graft survival. METHODS: We examined STX17 expression in grafts from 143 LDLT donors who underwent right lobe resection and investigated the relationship between STX17 expression and graft function. We investigated the correlations among STX17 expression, mitochondrial membrane potential and cell proliferation, using a STX17-knockdown hepatocyte cell line. RESULTS: Recipients transplanted with low STX17-expression grafts had significantly lower graft survival rates than recipients transplanted with high STX17-expression grafts (88.9% vs. 100%, p < 0.01). Multivariate analysis showed that low STX17 expression (HR: 10.7, CI: 1.29-88.0, p < 0.05) and the absence of splenectomy (HR: 6.27, CI: 1.59-24.8, p < 0.01) were independent predictive factors for small-for-size graft syndrome, which is the severe complication in LDLT. In the vitro experiments, the percentage of depolarized damaged mitochondria was increased in the STX17-knockdown hepatocyte cell line, suggesting decreased mitophagy and ATP synthesis. Cell proliferation was significantly decreased in the STX17-knockdown hepatocyte cell line. CONCLUSION: STX17 contributes to mitophagy and maintenance of mitochondrial function in hepatocytes and may be a predictor of graft dysfunction in LDLT patients.

Ferroptosis is induced by lenvatinib through fibroblast growth factor receptor-4 inhibition in hepatocellular carcinoma.

The tyrosine kinase inhibitor lenvatinib is used to treat advanced hepatocellular carcinoma (HCC). Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Nuclear factor erythroid-derived 2-like 2 (Nrf2) protects HCC cells against ferroptosis. However, the mechanism of lenvatinib-induced cytotoxicity and the relationships between lenvatinib resistance and Nrf2 are unclear. Thus, we investigated the relationship between lenvatinib and ferroptosis and clarified the involvement of Nrf2 in lenvatinib-induced cytotoxicity. Cell viability, lipid ROS levels, and protein expression were measured using Hep3B and HuH7 cells treated with lenvatinib or erastin. We examined these variables after silencing fibroblast growth factor receptor-4 (FGFR4) or Nrf2 and overexpressing-Nrf2. We immunohistochemically evaluated FGFR4 expression in recurrent lesions after resection and clarified the relationship between FGFR4 expression and lenvatinib efficacy. Lenvatinib suppressed system Xc - (xCT) and glutathione peroxidase 4 (GPX4) expression. Inhibition of the cystine import activity of xCT and GPX4 resulted in the accumulation of lipid ROS. Silencing-FGFR4 suppressed xCT and GPX4 expression and increased lipid ROS levels. Nrf2-silenced HCC cells displayed sensitivity to lenvatinib and high lipid ROS levels. In contrast, Nrf2-overexpressing HCC cells displayed resistance to lenvatinib and low lipid ROS levels. The efficacy of lenvatinib was significantly lower in recurrent HCC lesions with low-FGFR4 expression than in those with high-FGFR4 expression. Patients with FGFR4-positive HCC displayed significantly longer progression-free survival than those with FGFR4-negative HCC. Lenvatinib induced ferroptosis by inhibiting FGFR4. Nrf2 is involved in the sensitivity of HCC to lenvatinib.

Impact of Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status.

We examined phosphorylated nuclear factor erythroid 2-related factor 2 (P-NRF2) expression in surgically resected primary hepatocellular carcinoma (HCC) and investigated the association of P-NRF2 expression with clinicopathological features and patient outcome. We also evaluated the relationship among NRF2, cancer metabolism, and programmed death ligand 1 (PD-L1) expression. In this retrospective study, immunohistochemical staining of P-NRF2 was performed on the samples of 335 patients who underwent hepatic resection for HCC. Tomography/computed tomography using fluorine-18 fluorodeoxyglucose was performed, and HCC cell lines after NRF2 knockdown were analyzed by array. We also analyzed the expression of PD-L1 after hypoxia inducible factor 1α (HIF1A) knockdown in NRF2-overexpressing HCC cell lines. Samples from 121 patients (36.1%) were positive for P-NRF2. Positive P-NRF2 expression was significantly associated with high alpha-fetoprotein (AFP) expression, a high rate of poor differentiation, and microscopic intrahepatic metastasis. In addition, positive P-NRF2 expression was an independent predictor for recurrence-free survival and overall survival. NRF2 regulated glucose transporter 1, hexokinase 2, pyruvate kinase isoenzymes L/R, and phosphoglycerate kinase 1 expression and was related to the maximum standardized uptake value. PD-L1 protein expression levels were increased through hypoxia-inducible factor 1α after NRF2 overexpression in HCC cells. Conclusions: Our large cohort study revealed that P-NRF2 expression in cancer cells was associated with clinical outcome in HCC. Additionally, we found that NRF2 was located upstream of cancer metabolism and tumor immunity.

Intrinsic activation of ß-catenin signaling by CRISPR/Cas9-mediated exon skipping contributes to immune evasion in hepatocellular carcinoma.

Comprehensive analysis of clinical samples has recently identified molecular and immunological classification of hepatocellular carcinoma (HCC), and the CTNNB1 (ß-catenin)-mutated subtype exhibits distinctive characteristics of immunosuppressive tumor microenvironment. For clarifying the molecular mechanisms, we first established human and mouse HCC cells with exon 3 skipping of ß-catenin, which promoted nuclear translocation and activated the Wnt/ß-catenin signaling pathway, by using newly developed multiplex CRISPR/Cas9-based genome engineering system. Gene set enrichment analysis indicated downregulation of immune-associated gene sets in the HCC cells with activated ß-catenin signaling. Comparative analysis of gene expression profiles between HCC cells harboring wild-type and exon 3 skipping ß-catenin elucidated that the expression levels of four cytokines were commonly decreased in human and mouse ß-catenin-mutated HCC cells. Public exome and transcriptome data of 373 human HCC samples showed significant downregulation of two candidate cytokine genes, CCL20 and CXCL2, in HCC tumors with ß-catenin hotspot mutations. T cell killing assays and immunohistochemical analysis of grafted tumor tissues demonstrated that the mouse Ctnnb1Δex3 HCC cells evaded immunosurveillance. Taken together, this study discovered that cytokine controlled by ß-catenin signaling activation could contribute to immune evasion, and provided novel insights into cancer immunotherapy for the ß-catenin-mutated HCC subtype.

Molecular and immunological paradigms of hepatocellular carcinoma: Special reference to therapeutic approaches.

The development of hepatocellular carcinoma (HCC) is a multistep process with a complex interaction of various genetic backgrounds and the tumor microenvironment. In addition to the development of rational approaches to epidemiologic research, early detection, and diagnosis, considerable progress has been made in systemic treatment with molecular-targeted agents for patients with advanced HCC. Moreover, encouraging reports of recent clinical trials of combination therapy with immune-checkpoint inhibitors (ICIs) has raised high hopes. Each HCC is the result of a unique combination of somatic alterations, including genetic, epigenetic, transcriptomic, and metabolic events, leading to conclusive tumoral heterogeneity. Recent advances in comprehensive genetic analysis have accelerated molecular classification and defined subtypes with specific characteristics, including immune-associated molecular profiles reflecting the immune reactivity in the tumor. In considering the development of therapeutic strategies in combination with immunotherapy, proper interpretation of molecular pathological characterization could lead to effective therapeutic deployment and enable individualization of the management of HCC. Here, we review distinctive molecular alterations in the subtype classification of HCC, current therapies, and representative clinical trials with alternative immune-combination approaches from a molecular pathological point.

Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma.

The processing of intracellular reactive oxygen species (ROS) by nuclear factor erythroid-derived 2-like 2 (Nrf2) and NADPH quinone oxidoreductase 1 (Nqo1) is important for tumor metastasis. However, the clinical and biological significance of Nrf2/Nqo1 expression in hepatocellular carcinoma (HCC) remains unclear. We aimed to clarify the clinical importance of Nrf2/Nqo1 expression in HCC and evaluate the association of Nrf2/Nqo1 expression with HCC metastasis. We also evaluated the impact of Nqo1 modulation on HCC metastatic potential. We used spheroids derived from HCC cell lines. In anchorage-independent culture, HCC cells showed increased ROS, leading to the upregulation of Nrf2/Nqo1. Futile stimulation of Nqo1 by ß-lapachone induces excessive oxidative stress and dramatically increased anoikis sensitivity, finally diminishing the spheroid formation ability, which was far stronger than depletion of Nqo1. We analyzed 117 cases of primary HCC who underwent curative resection. Overexpression of Nrf2/Nqo1 in primary HCC was associated with tumor size, high α-fetoprotein, and des-γ-carboxy-prothrombin levels. Overexpression of Nrf2/Nqo1 was also associated with multiple intrahepatic recurrences (P = .0073) and was an independent risk factor for poor prognosis (P = .0031). NADPH quinone oxidoreductase 1 plays an important role in anchorage-independent survival, which is essential for survival for circulation and distant metastasis of HCC cells. These results suggest that targeting Nqo1 activity could be a potential strategy for HCC adjuvant therapy.

Characterization of human pegivirus infection in liver transplantation recipients.

Approximately 2% of healthy persons are infected with human pegivirus (HPgV). HPgV is transmitted via vertical, sexual, and blood-borne routes. Recently, the association of HPgV infection with the risk of lymphoma was reported. Here, we examined the prevalence of chronic HPgV infection in liver transplantation (LT) recipients and patients with hepatectomy and the influence of HPgV infection after LT on clinical and perioperative factors. We enrolled 313 LT recipients and 187 patients with hepatectomy who received care at the Kyusyu University Hospital between May 1997 and September 2017. Of the 313 recipients and 187 patients enrolled in this study, 44 recipients (14.1%) and 2 patients (1.1%) had HPgV viremia, respectively. There was no significant association between HPgV infection and LT outcomes. Interestingly, one recipient was infected with HPgV during the peritransplant period, which was likely transmitted via blood transfusion because HPgV RNA was detected from the blood bag transfused to the recipient during LT. We reviewed the available literature on the prevalence HPgV infections in other organ-transplanted patients and whether they impacted clinical outcomes. They also had the higher prevalence of HPgV infection, while it appears to be of low or no consequences. In addition, HPgV infection induced the upregulation of interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells. LT recipients had higher HPgV viremia compared to patients with hepatectomy. Although HPgV infection was not associated with LT-related outcomes, it induced ISG expression in recipients.

The Role of DNA Repair Glycosylase OGG1 in Intrahepatic Cholangiocarcinoma.

BACKGROUND/AIM: The effects of oxidative stress on various carcinomas were reported in previous studies, but those in intrahepatic cholangiocarcinoma (ICC) have not been fully elucidated. The purpose of this study was, thus, to reveal the effects of oxidative DNA damage and repair enzymes on ICC. MATERIALS AND METHODS: The levels of 8-hydroxydeoxyguanosine (8-OHdG) and 8-OHdG DNA glycosylase (OGG1) were immunohistochemically evaluated in specimens resected from 63 patients with ICC. RESULTS: Low OGG1 expression was related to tumour depth T4 (p=0.04), venous invasion (p=0.0005), lymphatic vessel invasion (p=0.03), and perineural invasion (p=0.03). Compared to the high-OGG1-expression group, patients with low OGG1 expression had a significantly poorer prognosis (overall survival: p=0.04, recurrence-free survival: p=0.02). Unlike for OGG1, the expression levels of 8-OHdG showed no association with prognosis. CONCLUSION: Oxidative DNA damage and DNA repair enzymes may be closely related to ICC progression.

Liver-enriched transcription factor expression relates to chronic hepatic failure in humans.

The mechanisms by which the liver fails in end-stage liver disease remain elusive. Disruption of the transcription factor network in hepatocytes has been suggested to mediate terminal liver failure in animals. However, this hypothesis remains unexplored in human subjects. To study the relevance of transcription factor expression in terminal stages of chronic liver failure in humans, we analyzed the expression of liver-enriched transcription factors (LETFs) hepatocyte nuclear factor (HNF)4α, HNF1α, forkhead box protein A2 (FOXA2), CCAAT/enhancer-binding protein (CEBP)α, and CEBPß. We then selected downstream genes responsible for some hepatic functions (ornithine transcarbamylase [OTC], cytochrome P450 3A4 [CYP3A4], coagulation factor VII [F7], cadherin 1 [CDH1], phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) [p-ERM], phospho-myosin light chain [p-MLC], low-density lipoprotein receptor-related protein 1 [LRP1]) in liver tissue from patients at different stages of decompensated liver function based upon Child-Pugh classification, Model for End-Stage Liver Disease score, and degree of inflammatory activity/fibrosis. We first examined differential expression of LETF and determined whether a relationship exists between transcript and protein expression, and liver function. We found HNF4α expression was down-regulated and correlated well with the extent of liver dysfunction (P = 0.001), stage of fibrosis (P = 0.0005), and serum levels of total bilirubin (P = 0.009; r = 0.35), albumin (P < 0.001; r = 0.52), and prothrombin time activity (P = 0.002; r = 0.41). HNF4α expression also correlated with CYP3A4, OTC, and F7 as well as CDH1 RNA levels. The Rho/Rho-associated protein kinase pathways, which have been implicated in the regulation of HNF4α, were also differentially expressed, in concert with LRP1, a reported upstream regulator of RhoA function. Conclusion: HNF4α and other members of the LETFs appear to be important regulators of hepatocyte function in patients with chronic hepatic failure. (Hepatology Communications 2018;2:582-594).

Coexpression of SALL4 with HDAC1 and/or HDAC2 is associated with underexpression of PTEN and poor prognosis in patients with hepatocellular carcinoma.

Spalt-like transcriptional factor 4 (SALL4), a stem marker, is reactivated in several cancers. A previous study has demonstrated that SALL4 interacts with the nucleosome remodeling deacetylase complex, which contains histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2). In this study, we investigated the expression status of SALL4, HDAC1, and HDAC2 and their relationship with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by immunohistochemical analysis of the posthepatectomy specimens of 135 patients with hepatocellular carcinoma who were treated at our hospital. Ninety-two frozen samples were subjected to quantitative reverse-transcription polymerase chain reaction analysis to detect the messenger RNA levels of PTEN. Seventy-six (56%) of 135 patients were positive for SALL4, and this group had a higher prevalence of hepatitis B antigen, a higher value of α-fetoprotein (AFP) and protein induced by vitamin K absence (PIVKAII) and poor histologic differentiation. The 5-year survival rate was significantly lower in the SALL4-positive group. High HDAC1 expression (51%) was correlated with a poor histologic differentiation and a poor prognosis. High HDAC2 expression (46%) was associated with a higher prevalence of hepatitis B antigen positivity, a poor histologic differentiation and higher prevalence of vascular invasion, and a lower 5-year survival rate. Coexpression of SALL4 with HDAC1 and/or HDAC2 was correlated with underexpression of PTEN. Moreover, multivariable analysis revealed that coexpression of SALL4 with HDAC1 and/or HDAC2 was predictive of an unfavorable prognosis. Our data thus suggested that the combination of SALL4, HDAC1, and HDAC2 may provide a potential target for molecular therapy.

Liver transplantation for cryptogenic liver failure caused by diffuse hepatic angiosarcoma: case report.

BACKGROUND: Primary hepatic angiosarcoma is a non-epithelial malignancy derived from sinusoidal endothelial cells, accounting for approximately 1.8% of primary hepatic malignancies. Diagnosis of primary hepatic angiosarcoma is complicated by difficulties in the qualitative radiological assessment of these tumors. Prognosis is very poor due to local recurrence and distant metastasis after liver resection or liver transplantation (LT). CASE PRESENTATION: This case report describes two patients with primary hepatic angiosarcoma who were diagnosed by histopathological examination of the explanted liver after LT. One patient had undergone living donor LT, and the other had undergone deceased donor LT. Neither showed evidence of malignancy on the pre-operative imaging tests. CONCLUSIONS: Hepatic angiosarcoma has a very high relapse rate after LT. Pre-transplant liver biopsy may be necessary to distinguish diffuse hepatic angiosarcoma from tumors of other origin in patients with cryptogenic liver failure.

The learning curves in living donor hemiliver graft procurement using small upper midline incision.

The learning curve for performing living donor hemiliver procurement (LDHP) via small upper midline incision (UMI) has not been determined. Living donors (n=101) who underwent LDHP via UMI were included to investigate the learning curve using cumulative sum analysis. The cumulative sum analysis showed that nine cases for right lobe (case #23) and 19 cases for left lobe (case #32 in the whole series) are needed for stable and acceptable surgical outcomes in LDHP via UMI. The established phase (n=69, since case #33) had a significantly shorter operative time, a smaller incision size, and less blood loss than the previous learning phase (n=32, serial case number up to the last 19th left lobe case). Multivariate analysis showed that the learning phase, high body mass index ≥25 kg/m2 , and left lobe graft procurement are the factors associated with surgical events including operative blood loss ≥400 mL, operative time ≥300 minutes, or surgical complications ≥Clavien-Dindo grade II. There is an obvious learning curve in performing LDHP via UMI, and 32 cases including both 19 cases for left lobe and nine cases for right lobe are needed for having stable and acceptable surgical outcomes.

Sarcopenia is a poor prognostic factor following hepatic resection in patients aged 70 years and older with hepatocellular carcinoma.

AIM: The present study investigated the effect of sarcopenia on short- and long-term surgical outcomes and identified potential prognostic factors for hepatocellular carcinoma (HCC) following hepatectomy among patients 70 years of age and older. METHODS: Patient data were retrospectively collected for 296 consecutive patients who underwent hepatectomy for HCC with curative intent. Patients were assigned to two groups according to age (younger than 70 years, and 70 years and older), and the presence of sarcopenia. The clinicopathological, surgical outcome, and long-term survival data were analyzed. RESULTS: Sarcopenia was present in 112 of 296 (37.8%) patients with HCC, and 35% of patients aged 70 years and older. Elderly patients had significantly lower serum albumin levels, prognostic nutrition index, percentage of liver cirrhosis, and histological intrahepatic metastasis compared with patients younger than 70 years. Overall survival and disease-free survival rates in patients with sarcopenia correlated with significantly poor prognosis in the group aged 70 years and older. Multivariate analysis revealed that sarcopenia was predictive of an unfavorable prognosis. CONCLUSION: This retrospective analysis revealed that sarcopenia was predictive of worse overall survival and recurrence-free survival after hepatectomy in patients 70 years of age and older with HCC.

Telaprevir versus simeprevir for the treatment of recurrent hepatitis C after living donor liver transplantation.

AIM: Our aim was to evaluate the clinical outcomes of telaprevir (TVR)- or simeprevir (SMV)-based triple therapy for recurrent hepatitis C after living donor liver transplantation. METHODS: Twenty-six patients received antiviral therapy, consisting of either TVR (n = 12) or SMV (n = 14) in combination with pegylated interferon and ribavirin, plus cyclosporin. RESULTS: More patients had a dose reduction of the direct-acting agent (36.3% vs 0.0%, P = 0.02) or required blood transfusion for anemia (58.3% vs 7.1%, P < 0.01) in the TVR group. The cyclosporin trough/dose ratio increased significantly from week 0 to week 4 in the TVR group (1.6 ± 0.4 to 5.1 ± 2.0, P < 0.01), but not in the SMV group (1.2 ± 0.3 to 1.3 ± 0.2, P = 0.68). The 24-week cumulative viral clearance rate was 91.7% and 85.7% in the TVR and in SMV groups, respectively. The early viral response and sustained viral response rates were 91.7% and 83.3%, respectively, in the TVR group, compared with 85.7% and 64.3%, respectively, in the SMV group. Interferon-mediated graft dysfunction occurred in four and five patients in the TVR and SMV groups, respectively; two patients were treated by oral steroids, five by steroid pulse and two by thymoglobulin, resulting in viral breakthrough in one case. CONCLUSION: SMV-based triple therapy was associated with fewer adverse events and drug interactions with cyclosporin, and possibly less antiviral properties to TVR. Interferon-mediated graft dysfunction is a significant clinical problem that warrants particular caution following living donor liver transplantation.

Local therapy improvement of outcome in a patient with recurrent hepatocellular carcinoma after living donor liver transplantation: a case report.

We describe a very rare case of recurrent hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT). A 47-year-old female underwent LDLT for HCC within Milan criteria, receiving a left liver lobe from her 38-year-old husband with an incompatible blood type. Thirty months after LDLT, however, enhanced computed tomography (CT) showed a tumor in her left adrenal gland; after another six months, enhanced CT and positron-emission tomography detected a frontal head bone tumor; enhanced CT performed 54 months after LDLT revealed a recurrent HCC in the liver graft. Each of these three tumors was surgically resected. Two months after hepatic partial resection, an enhanced CT showed another HCC in her liver graft, which was treated with local ablation therapy. One year after the last ablation treatment, the recipient is doing well without evidence of recurrence. At the time of detection of each of these tumors, the patient's serum concentration of hepatitis B virus surface antigen was elevated. Pathological examination showed that the resected tumors were moderately differentiated HCCs. Genotyping by microsatellite analysis confirmed their origin to be metastases of the primary HCC.