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The diagnostic accuracy of 18F-FDG-PET (fluoro-2-deoxyglucose-positron emission tomography) remains questionable for primary hepatocellular carcinoma (HCC) but seems to be more promising for restaging and therapy control. Yet, there are no data on FDG-PET in 131I-lipiodol treatment for primary liver cancer. The aim of this study was to relate baseline FDG-PET findings to histologic data and to assess, for the first time, the role of repetitive FDG-PET imaging for follow-up of 131I-lipiodol treatment. Eighteen (18) patients (16 HCC, 2 cholangiocellular carcinoma; CCC) with 36 treatment courses (up to four per patient) had 35 PET exams, including 18 post-treatment follow-up scans in 10 patients (up to three per patient, one without baseline PET; n = 17). Histopathologic results were available in 15 patients. PET results were retrospectively related to histopathologic type, grading, presence of cirrhosis, and tumor size at baseline and compared with computed tomography (CT) during follow-up. Prior to 131I-lipiodol treatment, 8 patients were PET positive and 9 PET negative. Most of the large HCCs were PET positive and most small tumors PET negative (p < 0.05), despite an overlap below 11 cm. There was no identifiable correlation between PET results and degree of tumor differentiation. Overall, 9 of 10 patients with 17 of 18 follow-up scans showed concordant results with CT. The one discrepant case became PET negative after the first treatment course, despite CT-proven tumor growth (false negative). Patient management was not changed due to PET results. In conclusion, large HCCs were significantly more often PET positive, but there was no correlation with the degree of differentiation. Follow-up PET may be useful if the tumor is first demonstrated to be FDG positive.

Background: The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, and new alternative treatments are needed., Aims: To comparatively test the angiostatic and antitumour effects of adenoviral gene transfer of angiostatin (PlgK1-4, amino acids 1-440) and full kringles 1-5 (PlgK1-5, amino acids 1-546) in a model of subcutaneously transferred HCC in mice., Methods: PlgK1-4 and PlgK1-5 were generated from human WtPlg cDNA and used for adenovirus construction. Vector function and angiostatic effects were confirmed in vitro and in vivo. Antitumoral efficacies of intratumoral vector injections were studied in a model of subcutaneously transferred HCC model., Results: Cell supernatants containing PlgK1-4 and PlgK1-5 reduced endothelial tube formation in vitro by about 30%, whereas WtPlg exerted no inhibitory effect. Endothelial cell infiltration in vivo was decreased by about 60%, but not in AdWtPlg-treated animals. Intratumoral treatment of subcutaneous HCC tumours inhibited growth by 40% for AdPlgK1-4 and 63% for AdPlgK1-5 in surviving mice 12 days after initiation of treatment, whereas treatment with AdWtPlg even led to accelerated growth. Although PlgK1-4 and PlgK1-5 have similar inhibitory effects on intratumoral microvessels, PlgK1-5 markedly improved the survival time compared with PlgK1-4., Conclusion: PlgK1-5 and PlgK1-4 effectively inhibited HCC growth. As PlgK1-5 could also prolong the survival time, inducing complete tumour elimination in half of the AdPlgK1-5-treated mice, PlgK1-5 might be the most potential plasminogen fragment for treatment of experimental HCC.

Aim: To evaluate the efficacy and tolerance of iodine-131-lipiodol ((131)I-lipiodol) for hepatocellular carcinoma (HCC) in German long term patients and comparison with medically treated controls., Patients, Methods: 38 courses of intra-arterial (131)I-lipiodol therapy with a total activity up to 6.7 GBq were performed in 18 patients with HCC (6 with portal vein thrombosis). Liver and tumour volume and lipiodol deposition were measured by computed tomography and (131)I activity by scintigraphy. Therapeutic efficacy was determined by tumour volume change and matched-pairs analysis in comparison to medically (i.e. tamoxifen or medical support) treated patients., Results: Tumour volume decreased in 20/32 index nodules (63%) after the first course. Repeated therapy frequently resulted in further tumour reduction. Overall response to treatment was partial in 11 nodules, minor response in 4 nodules, and disease was stable in 12 and progressive in 5. Significant response was associated with pretherapeutic nodule volume up to 150 ml (diameter of 6.6 cm). Survival rate after 3, 6, 9, 12, 24 and 36 months was 78, 61, 50, 39, 17, and 6%. Matched-pairs analysis of survival revealed (131)I-lipiodol to be superior to medical treatment. The most important side effect was a pancreatitis-like syndrome whereas overall tolerance was good., Conclusion: The long term results confirm that HCC therapy with (131)I-lipiodol is effective and probably superior to medical treatment. Tumour nodules of up to 6 cm diameter are well suited for this therapy even in the presence of portal vein thrombosis.

Methods: We applied an experimental approach employing two recombinant adenoviral vectors (Ad) that express interleukin-12 (IL-12) and angiostatin-like molecule (K1-3) respectively to a subcutaneous hepatoma model in mice., Results: Injection of AdK1-3 into tumour nodules established by subcutaneous (s.c.) implantation of Hepa129 hepatoma cells in C3H mice resulted in a significant dose-dependent reduction in tumour growth by 57% in the high dosage group (5x10(9) plaque-forming units [pfu], n=8) 10 days after treatment initiation. Similar antitumoural effects were found for the intratumoural mono-therapy with IL-12 (2.5x10(9) pfu, n=8) resulting in 60% tumour inhibition at the same time point. The survival rate was significantly (p=0.009) improved in the IL-12 but not in the K1-3 treatment group. A combination therapy of AdK1-3 and AdIL-12 was also effective, but did not further improve antitumour efficacy compared with the monotherapy., Conclusion: In conclusion, both mono- and combination therapy of K1-3 and IL-12 significantly inhibited tumour progression in this experimental tumour model. The co-administration of both compounds did not result in additive antitumour effects. We hypothesise that the lack of additive antitumour effects of the combination treatment might be attributed to partially counteracting antitumour effects and further studies are needed to illustrate the interference of tumour angiogenesis and tumour inflammation in this tumour model.

Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well-recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk-1), AdsFlk-1, to control pre-established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse-transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk-1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk-1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial-like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor-bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk-1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral-mediated gene transfer of an Flk-1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment.

Background/aims: Hepatocellular carcinomas (HCC) often show resistance to the effects of transforming growth factor-beta (TGF-beta). This study focuses on molecular mechanisms of this resistance to explore ways to overcome it., Methods: Transcription and protein expression of TGF-beta type I and type II receptors (TGF-betaRI/RII) were analyzed in clinical HCCs and the human hepatoma cell lines HuH-7 and HepG2. HuH-7 cells were transiently and stably transfected with a constitutively active TGF-betaRI mutant (CA TGF-betaRI). Resulting growth kinetics, integrin expression, invasiveness, TGF-beta-mediated activation of human plasminogen activator inhibitor type-1 (PAI-1) promoter and Smad expression were determined., Results: In clinical HCCs, there was less TGF-betaRII (6/10 cases) and more TGF-betaRI (8/10 cases) protein expression detectable in tumor compared to adjacent liver tissue. In HuH-7 cells, TGF-betaRII expression was likewise decreased. Cells transiently transfected with CA TGF-betaRI exhibited strong TGF-beta-related PAI-1 promoter activation. Stably transfected cells showed an attenuated response of the PAI-1 promoter, but increased Smad7 expression. Proliferation of stable clones was decreased. There was no change in integrin expression or invasiveness., Conclusions: Decreased TGF-betaRII protein expression might cause TGF-beta resistance in a subset of clinical HCCs. Stable transfection with CA TGF-betaRI reverses this in HuH-7 cells without increasing invasiveness., (Copyright 2005 S. Karger AG, Basel.)

Background and Objectives: Hepatocellular carcinoma (HCC) is a common cancer worldwide. As prognosis of HCC patients depends not only on tumour extension but also on liver function, TNM staging of HCC is of limited value. The Okuda score incorporating the variables of liver function and tumour extension is used widely. However, among patients with an intermediate Okuda score, survival varies considerably. Several newer scores promise to perform better than the Okuda score in stratifying HCC patients. We therefore tested the ability of several newer scores to predict survival in comparison to the Okuda score in a European cohort of HCC patients., Patients and Methods: A total of 120 patients with sufficient follow-up data were identified retrospectively among the 130 patients with HCC first seen between 1997 and 2000 in our department. Child-Pugh score, Okuda score, Vienna survival model for HCC (VISUM-HCC) score, Chevret score, Barcelona clinic liver cancer (BCLC) classification and cancer of the liver Italian programme (CLIP) score were calculated. Survival analysis was performed for all eligible patients stratified according to each scoring system. Receiver operating characteristics analysis was performed using six months survival as the outcome measure. Univariate and stepwise logistic regression analyses were performed to identify prognostic factors., Results: Survival times of HCC patients grouped according to all scores were significantly different. All scores performed similarly to the Okuda score in the receiver operating characteristic analysis. Prognostic factors for survival were albumin concentration and the presence of portal obstruction., Conclusion: In our central European cohort, there was no advantage of using the newer scores instead of the Okuda score.

Background and Aims: Adeno-associated virus (AAV) is the only known virus capable of site-specific genomic integration in human cells. Thus, AAV-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. We therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells., Methods: Plasmids were constructed that contained a GFPneo fusion transgene with or without the AAV-inverted terminal repeats (ITRs). In addition, Rep protein was either encoded in CIS or supplied in TRANS by co-transfections. Stable clones were analyzed by Southern blotting for site-specific integration., Results: The ITRs alone conferred neither stable nor site-specific transgene integration. Expression of Rep protein in CIS or TRANS resulted in an increased frequency of integration regardless of the presence of ITRs. It was shown that in the absence of the ITRs, other Rep-binding site (RBS) like sequences such as the ColE1 sequence present in plasmid backbones can function as RBS. Site-specific integration was achieved in up to 26% of clones derived from hepatoma cells., Conclusion: Both expression of Rep proteins and inclusion of a RBS are necessary for enhanced and stable integration of AAV-based non-viral vectors. A novel two-plasmid system capable of achieving stable and site-specific gene transfer in hepatoma cells is introduced., (Copyright 2003 S. Karger AG, Basel)

Background: Alpha-fetoprotein (AFP) is re-expressed in 60%-70% of hepatocellular carcinomas (HCC) and may therefore be a potential target for a prophylactic or therapeutic tumour-specific vaccination. A prerequisite for this approach is the possibility to induce AFP-specific T-lymphocytes in patients with HCC and/or cirrhosis., Methods: Peripheral blood was examined for the presence of AFP-specific T-lymphocytes using a FACS-based interferon-gamma secretion assay., Results: In a group of healthy volunteers, the presence of AFP-specific CD4- and CD8-lymphocytes was demonstrated. Screening of blood of 14 cirrhotic patients without HCC and 23 cirrhotic patients with HCC showed that patients with liver diseases that represent targets for vaccination also harbour CD4-positive as well as CD8-positive AFP-specific Tlymphocytes. AFP reactivity in patients' lymphocytes was not significantly influenced by soluble serum AFP. The median stimulation factors for CD4-positive T-lymphocytes were significantly higher (P = 0.0365) in cirrhotic patients without HCC (median 2.08, range 0.50-4.40) compared to cirrhotic patients with HCC (median 1.15, range 0.24-8.50)., Conclusion: AFP-specific T-lymphocytes that may be instrumental in HCC vaccination strategies are present in humans. This study suggests that immunopreventive vaccination of cirrhotic patients rather than immunotherapeutic vaccination of HCC patients may be preferable.

Unresectable hepatocellular carcinoma (HCC) are associated with a poor prognosis. Recently, one controlled study reported a strikingly prolonged survival of patients with HCC who were treated with octreotide. Until other randomised controlled trials become available, this multicentric retrospective study therefore assesses the outcome of HCC-patients who received octreotide treatment. 63 patients (53 males, 10 females) who had been treated with octreotide at 13 participating German centres were included in the analysis. In 54 cases liver cirrhosis was present (25 Child-Pugh A, 20 Child-Pugh B, 7 Child-Pugh C, 2 unknown). The liver disease was associated with alcohol abuse in 19 patients, alcohol and viral hepatitis in four patients, while 12 patients had only markers of past or present hepatitis B infection, 11 patients suffered of chronic hepatitis C infection, and four patients were seropositive for both hepatitis B and hepatitis C markers. The Okuda stage was stage I in 23, stage II in 33, and stage III in 7 patients. The patients initially received octreotide as a long-acting release formula (20-30 mg/month) in 43 cases or through subcutaneous injection (dose 3 x 50-3 x 300 ug/day) in the remaining cases. 11 of the patients receiving subcutaneous treatment were later converted to the long-acting release form of the drug. At three months, a partial remission was achieved in two patients, while 22 tumours showed no change and 26 tumours progressed. At six months, 11 tumours showed no change, while 15 tumours progressed. The patients' median survival was 9 months (Okuda stage I 16 months, stage II 9 months, stage III 4 months). In conclusion, octreotide treatment did not result in markedly prolonged survival in this retrospective series of 63 patients.

Aim: To conduct a cohort study of 101 patients with hepatocellular carcinoma (HCC) presenting to a tertiary care medical referral center in Germany between 1997 and 1999., Methods and Results: Data were retrospectively analyzed by chart review. In 95 cases (72 males and 23 females) sufficient data were available for analysis. Twenty five (29%) of 85 patients were HBsAg or anti HBc positive, 21/85 (25%) were anti HCV positive, and 6/85 (7%) were positive for both HBV and HCV-markers. Age was significantly lower in HBV positive patients than in the other two groups. Thirty one (34%) of 90 patients had histories of alcohol abuse. In 79/94 (84%) patients, cirrhosis was diagnosed. Of these cirrhotic patients, 29/79 (37%) belonged to Child Pugh's group (CHILD) A, 32/79 (40%) to CHILD B, and 18/79 (23%) to CHILD C. AFP was elevated in 61/91 (67%) patients. A single tumor nodule was found in 38/94 (40%), more than one nodule in 31/94 (34%), and 25/94 (26%) had a diffusely infiltrating tumor, i.e. the tumor margins could not be seen on imaging procedures. Portal vein thrombosis was present in 19/94 (20%). Imaging data consistent with lymph node metastases were found in 10/92 (11%), while distant metastases were found in 8/93 (9%). According to Okuda 28/94 (30%) were grouped to stage I, 53/94 (56%) were grouped to stage II, and 13/94 (14%) were grouped to stage II. Survival data were available for 83 patients. The Kaplan-Meier estimate for median survival was 8 4 months. Factors influencing survival were the Okuda score, the presence of portal vein thrombosis, and the presence of ascites. The presence of non complicated liver cirrhosis by itself, distant metastases, or infection with hepatitis viruses did not influence survival. AFP positivity by itself did not influence survival, though patients with an AFP value greater than 100 microg/L did experience shortened survival. Treatment besides tamoxifen or supportive care was associated with prolonged survival. The influence of therapy on survival was most pronounced in Okuda stage II patients. There was longer survival in those Okuda stage II patients who were treated with percutaneous ethanol injection., Conclusion: Even in a low incidence area such as Germany, the majority of HCC is caused by viral hepatitis and therefore potentially preventable. Reflecting the high proportion of advanced stage tumors in our patients, the median survival was poor. Patients who received active therapy had a longer survival.

While the natural intact protein does not possess any transactivator function, C-terminal truncation of the middle hepatitis B surface (MHBs) protein yields a novel transactivator function. We have previously found that the truncated transactivator protein, MHBs(t167), is not secreted but retained within the secretory pathway. Here, we provide evidence that when full-length MHBs is coexpressed with the truncated MHBs(t167) protein, the secretion of the full-length protein is inhibited and both proteins accumulate within the cell. We further show that MHBs, forcibly retained in the cell by C-terminal fusion to the endoplasmic reticulum retention signal KDEL (MHBsKDEL), mimics the effects of MHBs(t167) in enhancing the nuclear-binding activity of transcription factors NFkappaB and AP-1, and activation of NFkappaB- and AP-1-dependent transcription of reporter genes. As is the case for MHBs(t167), MHBsKDEL-dependent activation of NFkappaB is inhibited by the antioxidant N-acetyl-L-cysteine indicating the involvement of reactive oxygen intermediates and suggesting a similar mechanism of activation. This study suggests that the intracellular retention and accumulation of the normally secreted MHBs leads to oxidative stress and activation of transcription. This may be an important but not exclusive mechanism in hepatocarcinogenesis., (Copyright 2002 S. Karger AG, Basel)

Hepatitis B infection is associated with an increased risk of hepatocellular carcinoma development. Hepatitis B proteins, such as the hepatitis B x protein, the large hepatitis B surface protein, or truncated middle hepatitis B surface proteins, regulate transcription of many candidate genes for liver carcinogenesis by trans-mechanisms. They also alter mechanisms of apoptosis and interfere with nucleotide excision repair of damaged DNA. Together with an influence on cellular signaling, these mechanisms may favor the cell's clonal expansion., (Copyright 2002 S. Karger AG, Basel)