Safety margin of radiofrequency ablation for hepatocellular carcinoma: a prospective study using magnetic resonance imaging with superparamagnetic iron oxide.

PURPOSE: In radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC), it is difficult to assess the ablative margin (AM) precisely by comparing pre- and post-RFA CT images. We prospectively studied the AMs using magnetic resonance imaging (MRI) with pre-administered superparamagnetic iron oxide (SPIO). SPIO is safe for kidney disease patients. MATERIALS AND METHODS: Hepatocellular carcinoma patients were treated with RFA within 8 h of SPIO administration. On T2*-weighted MRI performed 4-7 days later, AM was visualized as a hypointense rim. The ablation status was classified as AM(+) if the rim completely surrounded the tumor, AM(0) if the rim was partly discontinuous without tumor protrusion, and AM(-) if the rim was partly discontinuous with tumor protrusion. The minimal thickness of AM was measured. AM(-) tumors were re-treated consecutively. RESULTS: In total, 85 HCCs ablated in 76 patients were evaluated. The local recurrence rate at 3 years was 2% for AM(+) tumors and 34% for AM(0) tumors (p < 0.01). In addition, no local recurrence was seen in the tumors with an AM of ≥ 2 mm. CONCLUSION: MRI with pre-administered SPIO is useful for determining the AM precisely, and an AM of ≥ 2 mm is recommended for curative RFA. TRIAL REGISTRATION NUMBER: This study was registered with UMIN Clinical Trials Registry (UMIN 000025406).

Oncolytic Virotherapy and Gene Therapy Strategies for Hepatobiliary Cancers.

Advanced liver cancers and biliary cancers represent diseases with dismal prognosis because of frequent local invasion and metastasis. Effective therapeutic agents for these cancers have not been established. Oncolytic viruses (OVs) constitute a novel class of promising, selective anticancer agents and recent studies have elucidated their unique features. Moreover, clinical trials are demonstrating promising results. Numerous OVs are being tested in preclinical models of hepatocellular carcinoma (HCC). The lead agent Pexa-Vec (pexastimogene devacirepvec, JX-594), a recombinant Wyeth strain vaccinia virus, has demonstrated preliminary evidence of safety and efficacy for HCC in clinical trials. Few other OVs have entered clinical testing. Relatively few preclinical studies and clinical trials exist for biliary cancers. In this review, we introduce various approaches using OVs to treat the intractable hepatobiliary cancers.

Oncolytic Viruses: The Best is Yet to Come.

Oncolytic viruses are a promising anti-cancer platform, achieving significant pre-clinical and clinical milestones in recent years. A full arsenal of selective, safe, and effective viruses has been developed with some emerging pre-clinical research focusing on optimizing these therapies in the face of remaining challenges, both in the bloodstream and in the tumour microenvironment. Herein we discuss the recent progress in pre-clinical virotherapy research to address these challenges, with special focus on innovative strategies that seek to complement the current strengths of virotherapy, ensuring an optimal multi-faceted attack on cancer. This review highlights the research areas that we believe provide the most potential to increase the efficacy of this exciting biotherapy platform: cell carriers, tumour vascular destruction, microenvironment modulation, combination therapies, and virus-mediated anti-tumour immune responses.

Analysis of repeated proton beam therapy for patients with hepatocellular carcinoma.

PURPOSE: To evaluate the feasibility and efficacy of repeated proton beam therapy (PBT) and to analyze the dose-volume relationship. PATIENTS AND METHODS: A retrospective analysis was performed in 83 patients who received definitive repeated PBT. Proton beams were delivered with expiratory gating. The numbers of treatment courses were 2, 3, and 4 in 68, 12, and 3 patients, respectively. MIM software was used for dose analysis. RESULTS: The planned median total dose was 70.5 GyE for all tumors and the median doses for the 1st, 2nd, 3rd and 4th treatments were 71.0, 70.0, 70.0, and 69.3 GyE, respectively. There was no severe acute toxicity, and no radiation-induced liver dysfunction (RILD) was observed. The median overall survival (OS) period from the first PBT was 61months (95% CI: 50-71months), and the 2- and 5-year OS rates were 87.5% (95%CI: 80.2-94.8%) and 49.4% (95%CI: 37.6-61.2%), respectively. The maximal delivered dose to the liver ranged from 66.7 to 248.1 GyE (mean: 124.93 GyE) and the mean liver dose ranged from 5.4 to 66.5 GyE (mean: 24.23 GyE). CONCLUSION: Repeated PBT was well tolerated and safe, even though the liver doses in many patients deviated substantially from well-known critical levels for RILD.

Long-term outcomes of proton beam therapy in patients with previously untreated hepatocellular carcinoma.

Long-term efficacy of proton beam therapy (PBT) remains unclear for patients with previously untreated hepatocellular carcinoma (HCC). We aimed to study the long-term outcomes of PBT according to Barcelona Clinic Liver Cancer (BCLC) staging classifications in patients with previously untreated HCC. The major eligibility criteria of this observational study were an Eastern Cooperative Oncology Group performance status (PS) 0-2, Child-Pugh grade A or B, previously untreated HCC covered within an irradiation field, and no massive ascites. A total of 66.0-77.0 GyE was administered in 10-35 fractions. Local tumor control (LTC), defined as no progression in the irradiated field, progression-free survival (PFS), and overall survival (OS) were assessed according to BCLC staging. From 2002 to 2009 at our institution, 129 patients were eligible. The 5-year LTC, PFS, and OS rates were 94%, 28%, and 69% for patients with 0/A stage disease (n = 9/21), 87%, 23%, and 66% for patients with B stage disease (n = 34), and 75%, 9%, and 25% for patients with C stage disease (n = 65), respectively. The 5-year LTC and OS rates of 15 patients with tumor thrombi in major vessels were 90% and 34%, respectively. Multivariate analyses revealed that PS (0 versus 1-2) was a significant prognostic factor for OS. No grade 3 or higher adverse effects were observed. PBT showed favorable long-term efficacies with mild adverse effects in BCLC stage 0 to C, and can be an alternative treatment for localized HCC especially when accompanied with tumor thrombi. This study was registered with UMIN Clinical Trials Registry (UMIN000025342).

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

BACKGROUNDS: Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. METHODS: Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. RESULTS: Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone. CONCLUSIONS: We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.

Clinical features of hepatitis E virus infection in Ibaraki, Japan: autochthonous hepatitis E and acute-on-chronic liver failure.

Hepatitis E caused by hepatitis E virus (HEV) is a serious public health concern in developing countries where HEV is mainly transmitted through contaminated water. Recently, in industrialized countries, autochthonous hepatitis E, a porcine zoonosis, has been increasingly recognized. In Japan, the number of national notifications of acute hepatitis E has increased since the introduction of anti-HEV IgA antibody measurement, covered by the national health insurance program, in 2011. In the past three years, we examined five patients of acute hepatitis or acute-on-chronic liver failure caused by HEV infection who presented various clinical courses in the southern area of Ibaraki prefecture in Japan. Of these patients, 78-year-old and 63-year-old male patients presented acute hepatitis E and recovered by only bed rest. The latter patient had a history of consuming grilled or undercooked pork and shellfish prior to the onset of hepatitis E. Among the five patients examined, the infection route was detected only in this patient. Of note, a 65-year-old female patient presented severe hepatitis associated with painless thyroiditis. The patient was diagnosed with probable autoimmune hepatitis and was successfully treated with prednisolone (40 mg/day). Lastly, 58-year-old and 62-year-old male patients, both of whom had a history of diabetes mellitus and alcoholic liver disease, developed acute-on-chronic liver failure, and the latter patient with pre-existing liver cirrhosis died due to liver failure. Thus, patients with clinical HEV infection who display multiple underlying diseases can develop acute-on-chronic liver failure. In conclusion, HEV infection manifests the diverse clinical courses.

Proton beam therapy for unresectable intrahepatic cholangiocarcinoma.

BACKGROUND AND AIM: Treatment for unresectable intrahepatic cholangiocarcinoma (ICC) has not been established. The aim of the study was to evaluate the outcome of proton beam therapy (PBT) for patients with unresectable ICC. METHODS: Up to 2010, 20 patients (11 males, 9 females, median age 63 years old) with unresectable ICC (two, seven, seven, and four in stages II, IIIA, IIIC, and IV, respectively) were treated with PBT. The largest dimensions of the tumors ranged from 15 to 140 mm (median: 50 mm). The intrahepatic region and lymph nodes received median total proton doses of 72.6 GyE in 22 fractions and 56.1 GyE in 17 fractions, respectively. Four patients received concurrent chemotherapy (tegafur, gimeracil, and oteracil; TS-1) during PBT. Twelve patients were treated curatively, and eight were treated palliatively because tumors were present outside the irradiation field. RESULTS: In the curative group, nine tumors within the irradiated field were controlled in follow-up of 8.6-62.6 months (median: 20.8 months). Median survival rates in the curative and palliative groups were 27.5 and 9.6 months, respectively, and overall 1- and 3-year survival rates were 82% and 38%, and 50% and 0%, respectively. Eight patients survived for > 2 years, and there was no distant metastasis in five of these patients after 2 years. No severe side-effects occurred. CONCLUSIONS: The results suggest that long-term survival can be achieved using PBT for patients with unresectable ICC without distant metastasis. Further studies are required to determine the optimal treatment schedule and best combination of PBT and chemotherapy.

Glasgow Prognostic Score predicts poor prognosis among advanced biliary tract cancer patients with good performance status.

Advanced cancer patients with good performance status (PS) sometimes show poor prognosis despite receiving some chemotherapies. We evaluated prognosis of chemo-naïve advanced biliary tract cancer (ABTC) patients with good PS by Glasgow Prognostic Score (GPS). Sixty-two patients with Eastern Cooperative Oncology Group PS 0 or 1 were retrospectively analyzed, using multivariate Cox regression. GPS was defined with serum levels of two parameters, albumin >3.5 g/dl and C-reactive protein <1.0 mg/dl (both as 0, either as 1, and neither as 2). PS 0 (n = 32) and 1 (n = 30) patients had similar survival (P = 0.98). The median overall survival (OS) was 17.0 months for GPS 0 (n = 19), 14.2 months for GPS 1 (n = 17), and 6.4 months for GPS 2 (n = 26). GPS 2 had significantly shorter OS than GPS 0 (P = 0.002) or 1 (P = 0.033). Multivariate analysis identified two independent prognostic factors: GPS (hazard ratio 0.60, 95 % confidence interval 0.40-0.90, P = 0.012) and liver metastasis (hazard ratio 0.43, 95 % CI 0.20-0.90, P = 0.026). GPS was useful for chemo-naïve ABTC patients with good PS.

Association between pretreatment retention rate of indocyanine green 15 min after administration and life prognosis in patients with HCC treated by proton beam therapy.

PURPOSE: The Child-Pugh score is often used to judge the outcome of radiotherapy for hepatocellular carcinoma (HCC). The retention rate of indocyanine green 15 min after administration (ICG R15) can also be used to predict prognosis after liver resection. We evaluated the utility of ICG R15 for prediction of outcomes after proton beam therapy (PBT) for HCC. METHODS AND MATERIALS: A retrospective evaluation was performed in 250 patients who received PBT between 2002 and 2007. The patients (178 males and 72 females) had a median age of 71 years (range: 43-88). Child-Pugh categories were A (score 5-6), B (7-9), and C (10-15) in 197, 51, and 2 patients, respectively. ICG scores were 0-<10, 10-<20, 20-<30, 30-<40 and ⩾40 in 27, 99, 59, 28 and 37 patients, respectively; including 26, 92, 45, 16 and 18 Child-Pugh A patients and 1, 8, 14, 11, and 17 Child-Pugh B patients, respectively. Survival times from the start of PBT were compared between Child-Pugh A and B patients, and among each ICG group. RESULTS: The median survival times were 61 months (95% CI: 50-72 months) in all patients, and 64 and 20 months in Child-Pugh A and B patients, respectively (p=0.001), The 3-year survival rates were 72%, 72%, 75%, 63%, and 26% in patients with ICG scores of 0-<10, 10-<20, 20-<30, 30-<40, and ⩾40 (p=0.001); 70%, 75%, 77%, 65%, and 38% in these respective groups in Child-Pugh A patients (p=0.02); and 100%, 57%, 67%, 36%, and 14% in Child-Pugh B patients (p=0.173, not significant). Multivariate analysis showed that low ICG R15 and the absence of portal vein tumor thrombus were associated with good survival. CONCLUSIONS: Pretreatment ICG R15 is a useful prognostic factor for prediction of outcome of PBT in HCC patients, especially in those with Child-Pugh A liver function.

Antitumor effects of a sirtuin inhibitor, tenovin-6, against gastric cancer cells via death receptor 5 up-regulation.

Up-regulated sirtuin 1 (SIRT1), an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53). Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5). In the KatoIII cell line (TP53-null), DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

MDM4 expression as an indicator of TP53 reactivation by combined targeting of MDM2 and MDM4 in cancer cells without TP53 mutation.

MDM2 and MDM4, a structurally related MDM2 homolog, negatively regulates expression and functions of TP53 tumor suppressor gene. To explore the precise expression patterns and function of MDM2 and MDM4 in wild-type (wt) TP53 cancer cells, we analyzed 11 various cancer cell lines with wt TP53. All cell lines exhibited deregulated expression of MDM2 and MDM4, and were divided into two distinct types; the one expressing high levels of MDM4 and another expressing low levels of MDM4. The low MDM4 type expressed higher MDM2 levels than the high MDM4 type. In cells with high MDM4 expression, knockdown of MDM4 or MDM2 reactivated TP53, and simultaneous knockdown of MDM2 and MDM4 synergistically reactivated TP53. In contrast, in cells with low MDM4 expression, knockdown of only MDM2 reactivated TP53. These results suggest that both MDM2 and MDM4 are closely involved in TP53 inactivation in cancer cells with high MDM4 expression, whereas only MDM2, and not MDM4, is a regulator of TP53 in cells with low MDM4 expression. MDM4 expression in wt TP53-tumors is a potential indicator for TP53 reactivation cancer therapy by simultaneous targeting of MDM4 and MDM2. Specific knockdown of MDM2 and MDM4 might be applicable for TP53 restoration therapy.

A phase I study on combined therapy with proton-beam radiotherapy and in situ tumor vaccination for locally advanced recurrent hepatocellular carcinoma.

BACKGROUND: Proton-beam radiotherapy (PBT) has been shown to be effective to hepatocellular carcinoma (HCC) as a nonsurgical local treatment option. However, HCC still remains as one of the most difficult cancers to be cured because of frequent recurrences. Thus, methods to inhibit the recurrence need to be explored. To prevent the HCC recurrence, we here report on a prospective phase I study of 'in situ' tumor vaccination using CalTUMP, a newly developed immunoadjuvant consisting of BCG extract bound to hydroxyapatite and microparticulated tuberculin, following local PBT for HCC. METHODS: Patients with locally advanced recurrent HCC, which had been heavily pretreated with various treatments, were enrolled. PBT was performed with the conventional method to the target HCC. Subsequently, CalTUMP was injected into the same irradiated-tumor three times at one-week intervals. Three dose-levels of CalTUMP (1/10, 1/3, and 1/1) were administered to 3 patients each. Vital signs, blood samples, ultrasound, and computed tomographic scans were monitored to evaluate the safety. RESULTS: Three intratumoral injections of CalTUMP following PBT (median dose: 72.6 GyE) were accomplished in 9 patients. Transient low-grade fever and minor laboratory changes were observed in 7 patients after CalTUMP injections. No other treatment-related adverse events were observed. Median progression-free survival was 6.0 months (range: 2.1-14.2) and 4 patients were progression-free for more than 1 year. CONCLUSIONS: Intratumoral injection of CalTUMP following PBT was feasible and safe in patients with heavily pre-treated HCC. Further clinical studies to evaluate the efficacy of this in situ tumor vaccination are warranted.

Effects of dipyridamole coadministration on the pharmacokinetics of ribavirin in healthy volunteers.

  Ribavirin (RBV), a guanosine analog for treatment of hepatitis C, is a substrate of a nucleoside transporter, solute carrier family 29 member 1 (SLC29A1). To clarify the impact of SLC29A1 on the pharmacokinetics of RBV, an open-label, crossover study of single-dose RBV (200 mg, p.o.) with and without coadministration of dipyridamole (DP), an inhibitor of SLC29A1, was performed. Plasma and erythrocyte concentrations of RBV in the control phase and DP phase (25 mg, 3 times daily for 4 days) were compared in 10 healthy volunteers. SLC29A1 mRNA expression in peripheral blood mononuclear cells was also determined. In the DP phase, area under the concentration-time curves (AUC) of RBV in plasma and erythrocytes showed reductions of 23% and 17%, respectively (p < 0.05), with increases in apparent oral clearance of 18% and 25%, respectively (p < 0.05). The reduction rate of the AUC of erythrocyte RBV in the DP phase was associated with SLC29A1 mRNA expression: higher mRNA expression showed greater AUC reduction. The elimination half-life of both plasma and erythrocyte RBV did not differ between the 2 phases. These results suggest that RBV/DP coadministration reduces the concentration of RBV in blood by inhibiting an important role of SLC29A1 in gastrointestinal absorption of RBV.

Cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing uracil phosphoribosyltransferase (UPRT) in immunocompetent Syrian hamsters.

Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing uracil phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5-fluorouracil (5-FU), in immunodeficient, Ad-nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5-FU therapy and of its combination with CP for syngenic HaP-T1 pancreatic cancers in immunocompetent, Ad-permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5-FU in HaP-T1 cells in vitro. I.t. AxE1CAUP/5-FU treatment inhibited the growth of subcutaneous HaP-T1 allografts. The combination with high-dose CP inhibited serum Ad-neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5-FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically-approved, intermediate-dose CP also enhanced the efficacy of i.t. AxE1CAUP/5-FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad-permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV-mediated gene therapy.

Impact of solute carrier family 29 member 1 (SLC29A1) single nucleotide polymorphisms on mRNA expression in peripheral blood mononuclear cells.

The effects of solute carrier family 29 member 1 (SLC29A1) single nucleotide polymorphism (SNP), rs6932345 and rs747199, on SLC29A1 mRNA expression were examined. The expression levels of SLC29A1 mRNA in peripheral blood mononuclear cells (PBMCs) isolated from 46 healthy subjects (28 males and 18 females) was compared between wild-type and mutant carriers. The mRNA levels in the rs6932345 wild-type (AA genotype) was 1.71 times that in the mutation carriers (AC/CC genotype) (p<0.05). Similar results were observed for rs747199, because rs747199 was linked with rs6932345 at a frequency of 84.8%. It was confirmed that wild-type for rs6932345 and rs747199 showed higher SLC29A1 mRNA expression in PBMCs.

Evaluation of liver function after proton beam therapy for hepatocellular carcinoma.

PURPOSE: Our previous results for treatment of hepatocellular carcinoma with proton beam therapy (PBT) revealed excellent local control. In this study, we focused on the impact of PBT on normal liver function. METHODS AND MATERIALS: The subjects were 259 patients treated with PBT at the University of Tsukuba between January 2001 and December 2007. We evaluated the Child-Pugh score pretreatment, on the final day of PBT, and 6, 12, and 24 months after treatment with PBT. Patients who had disease progression or who died with tumor progression at each evaluation point were excluded from the analysis to rule out an effect of tumor progression. An increase in the Child-Pugh score of 1 or more was defined as an adverse event. RESULTS: Of the 259 patients, 241 had no disease progression on the final day of PBT, and 91 had no progression within 12 months after PBT. In univariate analysis, the percentage volumes of normal liver receiving at least 0, 10, 20, and 30 GyE in PBT (V0, 10, 20, and 30) were significantly associated with an increase of Child-Pugh score at 12 months after PBT. Of the 91 patients evaluated at 12 months, 66 had no increase of Child-Pugh score, 15 had a 1-point increase, and 10 had an increase of ≥2 points. For the Youden index, the optimal cut-offs for V0, V10, V20, and V30 were 30%, 20%, 26%, and 18%, respectively. CONCLUSION: Our findings indicate that liver function after PBT is significantly related to the percentage volume of normal liver that is not irradiated. This suggests that further study of the relationship between liver function and PBT is required.

Investigation of the geometric accuracy of proton beam irradiation in the liver.

PURPOSE: To investigate the geometric accuracy of proton beam irradiation to the liver by measuring the change in Hounsfield units (HUs) after irradiation. METHODS AND MATERIALS: We examined 21 patients with liver tumors who were treated with respiratory-gated proton beam therapy (PBT). The radiation dose was 66 GyE in 12 patients and 72.6 GyE in 9 patients. Image registration and reslicing of the computed tomography (CT) results obtained within 1 month before and 3 months after PBT was performed, referring to the planning CT image. The resliced CT images obtained after PBT were subtracted from the images obtained before PBT. We investigated whether the area of the large HU change was consistent with the high-dose distribution area using the location of the largest change in HU around the tumor (peak) on the subtracted CT image and the 90% dose distribution area of the planning CT image. RESULTS: The number of patients (n = 20) whose left-right peaks were within the 90% dose distribution area was significantly larger than the number of patients whose anterior-posterior peaks and superior-inferior peaks were within the 90% dose distribution area (n = 14, n = 13, p = 0.034, and p = 0.02, respectively). Twelve patients exhibited a peak within the 90% dose distribution area in all directions. Nine of the 11 patients with smaller 90% confidence intervals of the percent normalization of the beam cycle (BC; 90% BC) showed a peak within the 90% dose distribution area in six directions, and this percentage was higher than that among the patients with larger 90% BC (3/10, p = 0.03). CONCLUSION: The geometric accuracy of proton beam irradiation to the liver was higher in the left-right direction than in the other directions. Patients with an irregular respiratory rhythm have a greater risk of a reduced geometric accuracy of PBT in the liver.

EpCAM- and EGFR-targeted selective gene therapy for biliary cancers using Z33-fiber-modified adenovirus.

A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by ß-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.