A randomized placebo-controlled trial of prophylactic dexamethasone for transcatheter arterial chemoembolization.

This randomized, double-blind, placebo-controlled trial evaluated dexamethasone efficacy at preventing fever, anorexia, and nausea/vomiting, the most frequent adverse events of transcatheter arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC). Child-Pugh class A/B patients with HCC and no macrovascular invasion/extrahepatic metastases were randomly assigned to either a dexamethasone regimen (day 1, intravenous dexamethasone [20 mg] and granisetron [3 mg] before TACE; days 2 and 3, intravenous dexamethasone [8 mg]) or a control regimen (day 1, intravenous placebo [saline] and granisetron [3 mg]; days 2 and 3, intravenous placebo). The primary endpoint was complete response, defined as the absence of grade ≥1 fever, anorexia, or nausea/vomiting according to the Common Terminology Criteria for Adverse Events (version 4.0) and no use of rescue therapy for 120 hours after TACE. A total of 120 patients between October 2010 and June 2013 were randomly assigned to treatment groups. Overall the complete response rate was greater with the dexamethasone regimen than with the control regimen (47.5%, 95% confidence interval 34.3%-60.9%, versus 10.2%, 95% confidence interval 3.8%-20.8%; P < 0.001). Cumulative incidences of fever, anorexia, and nausea/vomiting were higher in the control regimen group compared with the dexamethasone group (P < 0.001, P < 0.001, and P = 0.095, respectively). The dexamethasone regimen was generally well tolerated by HCC patients including those with well-controlled diabetes mellitus and those with hepatitis B virus infection. Conclusion: The dexamethasone regimen was more effective than the control regimen at preventing TACE-induced fever, anorexia, and nausea/vomiting in patients with HCC. (Hepatology 2018;67:575-585).

Multicenter, randomized, controlled trial of S-1 monotherapy versus S-1 and interferon-α combination therapy for hepatocellular carcinoma with extrahepatic metastases.

AIM: No effective therapies for extrahepatic metastases from hepatocellular carcinoma (HCC) have yet been identified. Previous studies suggested a potentially promising antitumor effect of combination therapy of S-1, a novel oral dihydropyrimidine dehydrogenase inhibitor, and interferon (IFN)-α. The present study aimed to investigate the clinical efficacy of single agent S-1 and S-1/IFN-α for HCC patients with extrahepatic metastases in a randomized, open-label, multicenter trial. METHODS: A total of 103 patients with HCC with extrahepatic metastases were randomly assigned to the S-1/IFN-α group, receiving the combination of S-1 and IFN-α, or the S-1 group, receiving the single agent of S-1. Clinical efficacy and adverse events were compared between the two groups. RESULTS: A total of 49 patients in the S-1/IFN-α group and 51 patients in the S-1 group were included in the efficacy analysis. The response rate was 22.4% (11/49) in the S-1/IFN-α group and 13.7% (7/51) in the S-1 group; there was no significant difference. Overall and progression-free survival in the two groups were also not significantly different (1-year overall survival 50.8% vs. 72.4%, median progression-free survival 127 days vs. 157 days). The incidence of grade ≥3 adverse events in the S-1/IFN-α group was 62.7% (32/51), which tended to be higher than in the S-1 group (43.1% [22/51]). CONCLUSIONS: Oncological outcomes in both treatment groups were favorable compared with previous reports, though there was no significant beneficial effect of adding IFN-α to S-1 for the treatment of HCC patients with extrahepatic metastases.

Post-progression survival in patients with advanced hepatocellular carcinoma resistant to sorafenib.

BACKGROUND: Since the approval of sorafenib, no other agent has been proven to show survival benefits in clinical trials involving patients with advanced hepatocellular carcinoma (HCC) resistant to sorafenib. Prognostic factors for survival after tumor progression in sorafenib-treated patients are critical for designing second-line trials. METHODS: To determine the factors affecting the post-progression survival (PPS) after sorafenib treatment, additional analyses were conducted using fixed data obtained from our previous prospective study. Data on patients with advanced HCC treated with sorafenib were analyzed in view of patient characteristics at the time of tumor progression and the progression pattern (intra-/extrahepatic growth or emergence of new intra-/extrahepatic lesions). RESULTS: Of the 89 enrolled patients, 70 were diagnosed with disease progression according to the Response Evaluation Criteria in Solid Tumors version 1.1. Multivariate Cox's regression analysis revealed that Child-Pugh scores of ≥7, macrovascular invasion (MVI), and alpha-fetoprotein of >400 ng/mL were independent predictors of poor PPS. Although both extrahepatic metastasis (EHM) and MVI were characteristics of advanced HCC, EHM was not determined as a prognostic factor. Additionally, the emergence of new extrahepatic lesions also served as an independent indicator of a poor prognosis. The PPS of the patients was well stratified according to the index based on the sum of these prognostic factors, ranging from 0 to 4. CONCLUSIONS: Child-Pugh score of ≥7, AFP of >400 ng/mL, MVI, and new extrahepatic lesions at the time of progression may be utilized to assess the prognosis and taken into consideration when designing second-line trials.

Liver function assessment according to the Albumin-Bilirubin (ALBI) grade in sorafenib-treated patients with advanced hepatocellular carcinoma.

BACKGROUND: The Albumin-Bilirubin (ALBI) grade has been proposed as a new, simple, and objective method of assessing liver function. However, there is lack of data in sorafenib-treated patients with advanced hepatocellular carcinoma (HCC). METHODS: We evaluated the correlations between the ALBI grade and Child-Pugh score, adverse events, and survival in 89 patients with advanced HCC who were prospectively treated with sorafenib. RESULTS: Majority of patients with ALBI grade 1 (14/15 patients, 93%) had a Child-Pugh score of 5. Patients with ALBI grade 2 had a wide range of liver function according to the Child-Pugh scores, with scores of 5, 6, 7, and ≥ 8. We divided ALBI grade 2 patients into ALBI grade 2A and 2B groups according to the median ALBI score among patients with ALBI grade 2. Although no significant difference was observed, the incidence of liver dysfunction in sorafenib-treated patients with ALBI grades 1, 2A, and 2B was 7%, 19%, and 35%, respectively. Overall survival in the ALBI grade 2B group was significantly shorter than that in the ALBI grade 1 and 2A groups. Thus, ALBI grade 2B was an independent predictor of poor prognosis in addition to elevated serum aspartate aminotransferase levels, increased serum alpha-fetoprotein level, and macrovascular invasion. CONCLUSION: Sorafenib may be indicated for all patients with advanced HCC and ALBI grade 1 and for some with ALBI grade 2. The subdivision of patients with ALBI grade 2 increases the utility of ALBI in identifying patients indicated for sorafenib therapy.

Sorafenib treatment in Child-Pugh A and B patients with advanced hepatocellular carcinoma: safety, efficacy and prognostic factors.

BACKGROUND: We aimed to evaluate the safety, efficacy and prognostic impact of baseline and early clinical markers in both Child-Pugh A and B patients with advanced hepatocellular carcinoma (HCC). METHODS: We prospectively studied 89 Japanese patients with HCC (Child-Pugh A, n = 59; Child-Pugh B, n = 30) who were started with sorafenib between May 2010 and July 2013. RESULTS: Frequency of sorafenib-related adverse events was almost similar between Child-Pugh score 5, 6, and 7 patients. The rate of liver dysfunction, including any grade encephalopathy, ≥ grade 3 ascites, or ≥ grade 3 bilirubin increased, in Child-Pugh score ≥8 group was significantly higher than that in the other groups. The median overall survival of Child-Pugh score 5, 6, 7 and ≥8 patients were 14.5, 11.1, 8.7 and 4.6 months, respectively. Patients in Child-Pugh score 6 had significantly longer OS than those in Child-Pugh score 7 (P = 0.049). Multivariate analysis identified macrovascular invasion (MVI), alpha-fetoprotein (AFP), Child-Pugh score and aspartate aminotransferase (AST) as baseline predictors of survival. However, extrahepatic metastasis (EHM) was not a significant prognostic factor. In addition, decrease in AFP level and development of hand-foot skin reaction within 4 weeks after sorafenib initiation were closely associated with favorable survival. CONCLUSION: It is possible that not only Child-Pugh score 5 and 6 but also 7 patients are eligible for future clinical trials with sorafenib or similar drugs. Various survival predictors identified in this study might be considered as stratification factor. Although both MVI and EHM is a phenotype of advanced HCC, MVI should be discriminated from EHM because of the prognostic impact on survival in sorafenib-treated advanced HCC patients.

A phase I/II study of S-1 with sorafenib in patients with advanced hepatocellular carcinoma.

BACKGROUND: Sorafenib is the sole molecular-targeted agent showing a survival benefit in patients with advanced hepatocellular carcinoma (HCC). We evaluated the tolerability and effectiveness of a combination of S-1 with sorafenib in patients with advanced HCC. METHODS: S-1 was administered during days 1-14 and sorafenib was administered every day. This treatment was repeated every 21 days. In phase I, we determined the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs). The dose of each drug was planned as follows: cohort 1: S-1 48 mg/m(2)/day and sorafenib 400 mg/day, cohort 2a: S-1 48 mg/m(2)/day and sorafenib 800 mg/day, cohort 2b: S-1 64 mg/m(2)/day and sorafenib 400 mg/day, cohort 3: S-1 64 mg/m(2)/day and sorafenib 800 mg/day, and cohort 4: S-1 80 mg/m(2)/day and sorafenib 800 mg/day. In phase II, the patients were treated at the MTD to evaluate safety and efficacy. RESULTS: Nineteen patients were enrolled in phase I. One of the six patients in cohort 1 and one of the six patients in cohort 3 experienced DLT. None of the three patients in cohort 2a experienced DLT and three of the four patients in cohort 4 experienced DLT. Therefore, cohort 3 was considered the MTD. Subsequently, 26 patients were enrolled in phase II. The most common grade 3/4 toxicities were an increase of aspartate aminotransferase (38.5 %), thrombocytopenia (23.1 %), neutropenia (19.2 %), hyperbilirubinemia (15.4 %), an increase of alanine aminotransferase (15.4 %), hyponatremia (11.5 %), rash (11.5 %), and hypophosphatemia (11.5 %). Sudden death occurred in one patient (3.8 %). A patient (3.8 %) had a partial response, 15 (57.7 %) had stable disease, and 10 (38.5 %) had progressive disease. The median times to progression and overall survival were 2.4 and 10.5 months, respectively. CONCLUSION: The MTD of S-1 and sorafenib in patients with advanced HCC was 64 mg/m(2)/day and 800 mg/day, respectively. This dose/regimen demonstrated substantial clinical activity among patients with advanced HCC.

A phase I/II trial of capecitabine combined with peginterferon α-2a in Patients with sorafenib-refractory advanced hepatocellular carcinoma.

BACKGROUND: Several pilot studies have demonstrated the effectiveness of combination therapy with pyrimidine fluoride and interferon for advanced hepatocellular carcinoma.This study aimed to determine the recommended dose of capecitabine combined with peginterferon α-2a (Phase I) and evaluate its safety and efficacy for sorafenib-refractory advanced hepatocellular carcinoma (Phase II). METHODS: Capecitabine was administered daily on days 1-14, while peginterferon α-2a was administered on days 1, 8, and 15. The cycle was repeated every 21 days. The patients were scheduled to receive capecitabine [mg/(m(2)∙day)] and peginterferon α-2a (µg/week) at 3 dose levels in phase I: 1200 and 90 (level 1), 1600 and 90 (level 2), and 2000 and 90 (level 3), respectively. RESULTS: A total of 30 patients were enrolled. The recommended dose was level 3. Among the 24 patients receiving the drug at the recommended dosage, 2 (8 %) exhibited a partial response, 9 (38 %) exhibited stable disease, 10 (42 %) exhibited progressive disease, and 3 (13 %) were not evaluated. The median time to progression and overall survival were 3.0 months and 7.2 months, respectively. The most common toxicities were decreased white blood cell (88 %), neutrophil (88 %), and platelet counts (58 %); fatigue (50 %); and palmar-plantar erythrodysesthesia syndrome (42 %). Four patients (17 %) discontinued treatment because of severe adverse events. CONCLUSION: Capecitabine at 2000 mg/(m(2)∙day) combined with peginterferon α-2a (90 µg/week) exhibited moderate, albeit manageable, toxicity and was declared as the recommended phase II dose. Further research is required to refine the efficacy of this combination.

Efficacy of sorafenib in intermediate-stage hepatocellular carcinoma patients refractory to transarterial chemoembolization.

OBJECTIVE: We compared the benefits of sorafenib therapy with continued transarterial chemoembolization (TACE) in TACE-refractory patients with intermediate-stage hepatocellular carcinoma (HCC). METHODS: This retrospective study reviewed intermediate-stage HCC patients who underwent the first TACE. Patients were defined as TACE-refractory and divided into two cohorts: (1) patients who switched from TACE to sorafenib and (2) those who continued TACE. We evaluated the patient overall survival (OS) and time to disease progression (TTDP; the time patients reached Child-Pugh C or developed advanced-stage HCC). RESULTS: A total of 509 patients with HCC underwent TACE. Of 249 intermediate-stage HCC patients undergoing the first TACE, 122 were deemed refractory. At the time they were identified as refractory, 20 patients converted to sorafenib, whereas 36 patients continued TACE. We excluded patients with Child-Pugh scores of ≥ 8, those with advanced-stage HCC, those who had undergone hepatic arterial infusion chemotherapy or other systemic therapy, and those treated with best supportive care alone. The median TTDP and OS were 22.3 and 25.4 months, respectively, in the conversion group, and 7.7 and 11.5 months, respectively, in the continued group (p = 0.001 and p = 0.003, respectively). CONCLUSIONS: It is possible that sorafenib conversion might prolong OS and TTDP in TACE-refractory patients with intermediate-stage HCC.

Natural history of postvascular-phase iso-enhanced lesions on the sonogram in chronic liver diseases.

BACKGROUND AND AIM: This study examined the natural history of postvascular-phase iso-enhanced lesions (PIELs) on contrast-enhanced sonograms to determine the potential risk and predictive factors for developing hepatocellular carcinoma (HCC) in chronic liver diseases. METHODS: This prospective study included 87 PIELs on contrast-enhanced sonograms (postvascular-phase: 10 min post-injection of perflubutane microbubbles) in 72 patients with chronic liver diseases (45 males and 27 females; age 65.0 ± 10.8y; PIEL diameter 12.5 ± 4.2 mm). The PIELs were followed up by ultrasound/contrast-enhanced ultrasound, computed tomography, or magnetic resonance imaging at 3 to 6 months intervals. RESULTS: Twenty patients developed HCCs during the study period (median, 22.0 months). The cumulative risk of HCC occurrence was 7.9% at 1 year and 36.0% at 3 years. The presence of coexistent HCC (hazard ratio [HR], 4.975; 95% confidence interval [CI], 1.729-14.316; P = 0.003) and alpha-fetoprotein > 20 ng/mL (HR, 4.104; 95% CI, 1.621-10.392; P = 0.003) were significant factors for the risk of HCC occurrence. Fourteen of these lesions were diagnosed as HCCs that developed from iso-enhanced lesions. Cumulative HCC occurrence rates from PIEL > 14 mm was 23.5% at 1 year and 46.3% at 3 years. Cox regression analysis showed that PIEL > 14 mm (HR, 6.780; 95% CI, 2.060-22.32; P = 0.002) and alpha-fetoprotein > 20 ng/mL (HR, 4.892; 95% CI, 1.559-15.350; P = 0.007) were statistically significant factors for HCC occurrence. CONCLUSIONS: Patients with coexistent HCC, alpha-fetoprotein > 20 ng/mL, or PIEL > 14 mm should be carefully monitored because of the high potential for HCC occurrence.

Pretreatment microbubble-induced enhancement in hepatocellular carcinoma predicts intrahepatic distant recurrence after radiofrequency ablation.

OBJECTIVE: The purpose of this study is to examine whether pretreatment findings in hepatocellular carcinoma (HCC) using contrast-enhanced ultrasound can predict local or distant recurrence after radiofrequency ablation (RFA). SUBJECTS AND METHODS: Subjects of the prospective study were 54 patients with HCC lesions treated by RFA. Intensity differences between lesion and liver parenchyma at early arterial (4 seconds) and peak enhancement times and washout at late phase were provided on contrast-enhanced sonograms with perflubutane microbubble agent. The pretreatment findings were examined with respect to intrahepatic local and distant recurrence. RESULTS: Univariate analysis showed that intensity differences at the early arterial time (hazard ratio [HR], 2.2; 95% CI, 1.0-4.6; p = 0.042) and lesion frequency (HR, 2.3; 95% CI, 1.0-5.0; p = 0.044) were risk factors for distant recurrence. Multivariate analysis showed that intensity differences at the early arterial time (HR, 2.7; 95% CI, 1.2-5.8; p = 0.014) and lesion frequency (HR, 2.9; 95% CI, 1.3-6.5; p = 0.015) were risk factors for distant recurrence. The cumulative distant recurrence rate for patients with intensity differences at the early arterial time was greater at less than 10 dB than at 10 dB or higher (33.3% and 91.3% vs 23.9% and 65.1% at 1 and 2 years, respectively; p = 0.035). The cumulative distant recurrence rate was 16.5% and 61.1% at 1 and 2 years, respectively, in patients with solitary lesions and 54.7% and 77.4% at 1 and 2 years, respectively, in patients with multiple lesions (p = 0.0296). No pretreatment findings were predictive for local recurrence. CONCLUSION: HCC lesions with gradual enhancement in the early arterial time displayed potential distant recurrence risk after RFA, requiring careful posttreatment surveillance.

Adherence to medication is a more important contributor to viral breakthrough in chronic hepatitis B patients treated with entecavir than in those with Lamivudine.

Viral breakthrough is related to poor adherence to medication in some chronic hepatitis B patients treated with nucleos(t)ide analogues (NAs). Our study aimed to examine how adherence to medication is associated with viral breakthrough in patients treated with NAs. A total of 203 patients (135 ETV and 68 LAM) were analyzed in this retrospective analysis. Physical examination, serum liver enzyme tests, and hepatitis B virus marker tests were performed at least every 3 months. We reviewed medical records and performed medical interviews regarding to patients' adherence to medication. Adherence rates <90% were defined as poor adherence in the present study. Cumulative viral breakthrough rates were lower in the ETV-treated patients than in the LAM-treated patients (P<0.001). Seven ETV-treated (5.1%) and 6 LAM-treated patients (8.8%) revealed poor adherence to medication (P=0.48). Among ETV-treated patients, 4 (3.1%) of 128 patients without poor adherence experienced viral breakthrough and 3 (42.8%) of 7 patients with poor adherence experienced viral breakthrough (P<0.001). Only 3 of 38 (7.8%) LAM-treated patients with viral breakthrough had poor adherence, a lower rate than the ETV-treated patients (P=0.039). Nucleoside analogue resistance mutations were observed in 50.0% of ETV- and 94.1% of LAM-treated patients with viral breakthrough (P=0.047). Viral breakthrough associated with poor adherence could be a more important issue in the treatment with especially stronger NAs, such as ETV.

3-Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor-initiating hepatocellular carcinoma cells.

Recent advances in stem cell biology have identified tumor-initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self-renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self-renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor-initiating HCC cells remains unclear. In this study, we conducted loss-of-function assay of EZH2 using short-hairpin RNA and pharmacological inhibition of EZH2 by an S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep). Both EZH2-knockdown and DZNep treatment impaired cell growth and anchorage-independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)(+) tumor-initiating cells. Administration of 5-fluorouracil (5-FU) or DZNep suppressed the tumors by implanted HCC cells in non-obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5-FU predominantly reduced the number of EpCAM(+) cells and diminished the self-renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor-initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor-initiating HCC cells.

Percutaneous ethanol injection for hepatocellular carcinoma: 20-year outcome and prognostic factors.

BACKGROUND: Ethanol injection is the best-known image-guided percutaneous ablation for hepatocellular carcinoma (HCC) and a well-tolerated, inexpensive procedure with few adverse effects. However, there have been few reports on its long-term results. AIMS: We report a 20-year consecutive case series at a tertiary referral centre. METHODS: We performed 2147 ethanol injection treatments on 685 primary HCC patients and analysed a collected database. RESULTS: Final computed tomography demonstrated complete ablation of treated tumours in 2108 (98.2%) of the 2147 treatments. With a median follow-up of 51.6 months, 5-, 10- and 20-year survival rates were 49.0% [95% confidence interval (CI) = 45.3-53.0%], 17.9% (95% CI = 15.0-21.2%) and 7.2% (95% CI = 4..5-11.5%) respectively. Multivariate analysis demonstrated that age, Child-Pugh class, tumour size, tumour number and serum alpha-fetoprotein level were significant prognostic factors for survival. Five-, 10- and 20-year local tumour progression rates were 18.2% (95% CI = 15.0-21.4%), 18.4% (95% CI = 15.2-21.6%) and 18.4% (95% CI = 15.2-21.6%) respectively. Five-, 10- and 20-year distant recurrence rates were 53.5% (95% CI = 49.4-57.7%), 60.4 (95% CI = 56.3-64.5%) and 60.8% (95% CI = 56.7-64.9%) respectively. There were 45 complications (2.1%) and two deaths (0.09%). CONCLUSIONS: Ethanol injection was potentially curative for HCC, resulting in survival for more than 20 years. This study suggests that new ablation therapies will achieve similar or even better long-term results in HCC.

Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3.

Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. Using a tandem affinity purification (TAP) strategy and mass spectrometry, we have found that Gli1 interacts with 14-3-3epsilon, and that Gli2 and Gli3 also bind to 14-3-3epsilon through homologous sites. This interaction depends on their phosphorylation, and cAMP-dependent protein kinase (PKA), a known negative regulator of Hedgehog signaling serves as a responsible kinase. A Gli2 mutant engineered to eliminate this interaction exhibited increased transcriptional activity (2 approximately 3x). Transcriptional repression by 14-3-3 binding was also observed with Gli3, when its N-terminal repressor domain was deleted. The phosphorylation sites responsible for the binding to 14-3-3 are distinct from those required for proteolysis, the known mechanism for PKA-induced repression of Hh signaling. Our data propose a novel mechanism in which PKA down-regulates Hedgehog signaling by promoting the interaction between Gli and 14-3-3 as well as proteolysis. Given the certain neuronal or malignant disorders in human caused by the abnormality of 17p13 encompassing 14-3-3epsilon overlap with increased Hh signaling, the Gli-14-3-3 interaction may have pathological significance for those human diseases.

Altered composition of fatty acids exacerbates hepatotumorigenesis during activation of the phosphatidylinositol 3-kinase pathway.

BACKGROUND & AIMS: Some clinical findings have suggested that systemic metabolic disorders accelerate in vivo tumor progression. Deregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is implicated in both metabolic dysfunction and carcinogenesis in humans; however, it remains unknown whether the altered metabolic status caused by abnormal activation of the pathway is linked to the protumorigenic effect. METHODS: We established hepatocyte-specific Pik3ca transgenic (Tg) mice harboring N1068fs*4 mutation. RESULTS: The Tg mice exhibited hepatic steatosis and tumor development. PPARγ-dependent lipogenesis was accelerated in the Tg liver, and the abnormal profile of accumulated fatty acid (FA) composition was observed in the tumors of Tg livers. In addition, the Akt/mTOR pathway was highly activated in the tumors, and in turn, the expression of tumor suppressor genes including Pten, Xpo4, and Dlc1 decreased. Interestingly, we found that the suppression of those genes and the enhanced in vitro colony formation were induced in the immortalized hepatocytes by the treatment with oleic acid (OA), which is one of the FAs that accumulated in tumors. CONCLUSIONS: Our data suggest that the unusual FA accumulation has a possible role in promoting in vivo hepato-tumorigenesis under constitutive activation of the PI3K pathway. The Pik3ca Tg mice might help to elucidate molecular mechanisms by which metabolic dysfunction contributes to in vivo tumor progression.

Macroscopic morphologic subtypes of laterally spreading colorectal tumors showing distinct molecular alterations.

Recent advances in colonoscopic techniques have resulted in more frequent detection of superficial-type colorectal tumors, that is, laterally spreading tumors (LSTs), although little is known about the characteristic clinical features and genetic alterations of LSTs. To elucidate the molecular characteristics of LSTs, genetic alterations in the KRAS, BRAF and PIK3CA genes and abnormal expression of the p53, beta-catenin and MYC proteins were analyzed using direct DNA sequencing and immunohistochemistry for 50 protruded-type tumors (Protruded), 35 granular-type LSTs (LST-G) and 19 nongranular-type LSTs (LST-NG). In addition, loss of heterozygosity (LOH) close to the adenomatous polyposis coli (APC) gene (5q21) was examined in these tumors. In univariate analyses, significant differences were noted in the percentages with KRAS mutations (Protruded, LST-G, LST-NG = 30.0%, 54.3%, 21.1%, respectively, p = 0.0156), nuclear accumulation of beta-catenin (Protruded, LST-G, LST-NG = 50.0%, 37.1%, 68.4%, respectively, p = 0.0267), expression of MYC (Protruded, LST-G, LST-NG = 26.0%, 17.1%, 42.1%, respectively, p = 0.0456) and LOH at the APC gene locus (Protruded, LST-G, LST-NG = 22.0%, 20.0%, 47.4%, respectively, p = 0.0302). Multivariate analysis demonstrated that the macroscopic subtype of LST was significantly associated with KRAS mutation (for LST-NG: odds ratio [OR] 0.23, 95% CI 0.06-0.90) and nuclear accumulation of beta-catenin (for LST-NG: OR 4.05, 95% CI 1.11-14.8). Our data revealed that the 2 subtypes of LST have different molecular characteristics, suggesting that 2 or more different molecular mechanisms result in colorectal tumors with a similar growth pattern.

Prognostic significance of genetic alterations detected by high-density single nucleotide polymorphism array in gastric cancer.

We sought to identify genomic changes that could be useful for clinical application, focusing on chromosomal instability and using a high-density single nucleotide polymorphism (SNP) array. We analyzed 34 gastric cancer cell lines for areas of DNA that exhibited copy number changes using the Affymetrix GeneChip Human Mapping 50 K Arrays. The results obtained with the cell lines were verified in 42 gastric cancer tissues using genomic PCR, quantitative real-time PCR, and loss of heterozygosity (LOH) analyses. Twenty-six local homozygous deletion regions, including 13 novel loci, and 31 recurrent high-grade gain regions, encompassing 14 novel loci, were found in the gastric cancer cell lines. Among the genes detected for high-grade gain in the cell lines, MYC, PAK1, and ITGB4BP showed copy number gain in more than 40% of gastric cancer tissues. LOH of AB051467, PTPRD, A2BP1, and C20orf133 was detected in more than 35% of gastric cancer tissues. The number of LOH was significantly greater in tumors with lymph node metastasis. In the early stage, the prognosis of patients with LOH of less than two genes was significantly better than that of those with LOH of two genes or more. Using high-density SNP arrays, we identified several novel and minute genomic alterations. LOH of four genes could be useful for prediction of lymph node metastasis and prognosis in early stage gastric cancers.

Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.

BACKGROUND & AIMS: Although colorectal cancer (CRC) progression has been associated with alterations in KRAS and RAS signaling, not all CRC cells have KRAS gene mutations. RAS activity is modulated by RAS-GTPase-activating proteins (RASGAPs), so we investigated the role of RASGAPs in CRC progression. METHODS: The level of RASGAP expression in CRC cells was analyzed using quantitative real-time polymerase chain reaction. The expression of the RAS protein activator like-1 (RASAL1) was examined in clinical colorectal neoplasms using immunohistochemistry. The clinicopathologic (age, sex, and tumor site and grade) and molecular (KRAS gene mutation, as well as CTNNB1 and TP53 expression patterns) factors that could affect RASAL1 expression were examined. RESULTS: Of 12 RASGAPs examined, expression levels of only RASAL1 decreased in CRC cells; RASAL1 expression decreased in most CRC cells with wild-type KRAS gene but rarely in those with mutant KRAS gene. A transfection assay showed that RASAL1 repressed RAS/mitogen-activated protein kinase signaling in response to growth factor stimulation and reduced proliferation of CRC cells that contained wild-type KRAS gene. RASAL1 expression was detected in 46.9% (30/64) of adenocarcinoma, 17.4% (8/46) of large adenoma, and no (0/42) small adenoma samples. RASAL1 expression levels were correlated with the presence of wild-type KRAS gene in CRC tumor samples (P= .0010), distal location (P= .0066), and abnormal expression of TP53 (P= .0208). CONCLUSIONS: RASAL1 expression is reduced in CRC cells that contain wild-type KRAS gene. Reductions in RASAL1 expression were detected more frequently in advanced lesions than in small adenomas, suggesting that RASAL1 functions in the progression of benign colonic neoplasms.

Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion.

Receptor tyrosine kinases (RTKs) are involved in oncogenesis and disease progression for many cancers. Inhibitors targeting them are vigorously developed and some of them are tested in the clinical setting. Amplifications of certain RTKs (c-Met, FGFR2 and ErbB2) have been associated with human gastric cancer progression. According to our genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism genotyping microarrays, we confirmed that the c-met locus was amplified in four gastric cancer cell lines (Hs746T, MKN45, NUGC4 and SNU5). It was reported that somatic mutation is occasionally detected in tumor samples of a certain type of cancer with gene amplification. Previous reports showed gastric cancers harbored mutations of FGFR2 and ErbB2, but c-Met oncogenic mutation had not yet been reported. We performed mutational analysis of the cytoplasmic domains of c-Met using the genome DNA of the gastric cancer cell lines, and found that Hs746T cells had a splice site mutation of exon 14. By cDNA sequencing and Western blotting, we showed that the mutation caused juxtamembrane domain deletion. Previously, this mutation had been detected only in lung cancer specimens and this deletion resulted in the loss of Cbl E3-ligase binding causing decreased ubiquitination and delayed down-regulation. In conclusion, four gastric cancer cell lines harbored amplification of c-met locus, and among them, Hs746T had a putative oncogenic mutation with amplification. This information will be useful for screening of inhibitors targeting gastric cancer with c-Met aberration.

Prospective risk assessment for hepatocellular carcinoma development in patients with chronic hepatitis C by transient elastography.

UNLABELLED: Liver stiffness, noninvasively measured by transient elastography, correlates well with liver fibrosis stage. The aim of this prospective study was to evaluate the liver stiffness measurement (LSM) as a predictor of hepatocellular carcinoma (HCC) development among patients with chronic hepatitis C. Between December 2004 and June 2005, a total of 984 HCV-RNA positive patients, without HCC or a past history of it, visited the University of Tokyo Hospital. LSM was performed successfully in 866 patients, who gave informed consent. During the follow-up period (mean, 3.0 years), HCC developed in 77 patients (2.9% per 1 person-year). The cumulative incidence rates of HCC at 1, 2, and 3 years were 2.4%, 6.0%, and 8.9%, respectively. Adjusting for other significant factors for HCC development, patients with higher LSM were revealed to be at a significantly higher risk, with a hazard ratio, as compared to LSM < or =10 kPa, of 16.7 (95% confidence interval [CI], 3.71-75.2; P < 0.001) when LSM 10.1-15 kPa, 20.9 (95% CI, 4.43-98.8; P < 0.001) when LSM 15.1-20 kPa, 25.6 (95%CI, 5.21-126.1; P < 0.001) when LSM 20.1-25 kPa, and 45.5 (95% CI, 9.75-212.3; P < 0.001) when LSM >25 kPa. CONCLUSIONS: This prospective study has shown the association between LSM and the risk of HCC development in patients with hepatitis C. The utility of LSM is not limited to a surrogate for liver biopsy but can be applied as an indicator of the wide range of the risk of HCC development.