Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.

PURPOSE: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage. EXPERIMENTAL DESIGN: Tumor pieces (2 x 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21(Cip1), p27(Kip1), p16(INK4a), pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression. RESULTS: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27(kip1) overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete alpha-fetoprotein, and has TP53 missense mutations in codons 192 and 242. CONCLUSIONS: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.     

Combined targeting of epidermal growth factor receptor and MDM2 by gefitinib and antisense MDM2 cooperatively inhibit hormone-independent prostate cancer.

PURPOSE: The epidermal growth factor receptor (EGFR) may play a relevant role in the progression, hormone therapy resistance, and prognosis of prostate cancer patients. Also MDM2, a negative p53 regulator that interacts with retinoblastoma (Rb), E2F, p19(arf) and the ras-mitogen-activated protein kinase(MAPK) cascade plays an important role in prostate cancer progression and prognosis. On the basis of the EGFR and MDM2 role in integrating signaling pathways critical for prostate cancer progression, we investigated whether their selective combined blockade may have a cooperative antitumor effect in prostate cancer. For this purpose, we have used the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) and a second generation hybrid oligonucleotide antisense MDM2 (AS-MDM2), respectively. EXPERIMENTAL DESIGN: Gefitinib and AS-MDM2 were administered to hormone-refractory and hormone-dependent human prostate cancer cells in vitro and to mice bearing tumor xenografts, evaluating the effects on growth, apoptosis, and protein expression, in vitro and in vivo. RESULTS: We demonstrated that the combination of gefitinib and AS-MDM2 synergistically inhibits the growth of hormone-independent prostate cancer cells in vitro. This effect is accompanied by the inhibition of MDM2, phosphorylated Akt (pAkt), phosphorylated MAPK (pMAPK), and vascular endothelial growth factor (VEGF) expression and by Rb hypophosphorylation. The combination of the two agents in nude mice bearing the same hormone-independent tumors caused a potent cooperative antitumor effect. Tumor samples analysis confirmed the inhibition of MDM2, pAkt, pMAPK, VEGF, and basic fibroblast growth factor expression. CONCLUSIONS: This study shows that EGFR and MDM2 play a critical role in the growth of prostate cancer, especially hormone-dependent, and that their combined blockade by gefitinib and AS-MDM2 causes a cooperative antitumor effect, supporting the clinical development of this therapeutic strategy.     

HMGA1 protein overexpression in human breast carcinomas: correlation with ErbB2 expression.

We measured, by immunohistochemistry, HMGA1 protein expression in 212 breast tissue specimens: 6 normal samples, 28 hyperplastic lesions (13 with cellular atypia), 11 fibroadenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, and 13 lobular carcinomas. HMGA1 was not expressed in normal breast tissue; HMGA1 staining was intense in 40% of hyperplastic lesions with cellular atypia and in 60% of ductal carcinomas and weak in fibroadenomas and in hyperplastic lesions without cellular atypia. Because HMGA1 expression was similar among ductal breast carcinomas with different histologic grading, we evaluated the association between HMGA1 expression and that of other markers of breast carcinoma invasion (estrogen and progesterone receptors, Ki-67 antigen, and ErbB2) in 21 cases of grade 3 breast ductal carcinomas and 7 cases of breast lobular carcinomas. We found that HMGA1 expression tended to be associated only with c-erbB-2 expression (Spearman rho: 0.36; P=0.065). Taken together, these results suggest that HMGA1 expression might be a novel indicator for the diagnosis and prognosis of human breast cancer.     

Prognostic value of CD40 in adult soft tissue sarcomas.

PURPOSE: The purpose is to evaluate the expression of CD40, a membrane protein predominantly expressed on B cells, dendritic cells, and macrophages, in a series of adult soft tissue sarcomas and to test its possible prognostic value. EXPERIMENTAL DESIGN: CD40 expression was studied by immunohistochemistry. Correlations with other baseline characteristics of patients and tumors were analyzed with chi(2) test. The prognostic value was studied with univariable and multivariable analysis adjusted by age, sex, tumor size, grade, location, and distant metastases. RESULTS: Eighty-two patients, between January 1994 and May 2001, were analyzed. Membrane or cytoplasmic staining for CD40 protein was absent in 30% of the tumors but present in <10% of cells in 22 (27%), in 10% to 50% in 23 (28%), and in >50% of cells in 12 (15%) tumors. There was no correlation between CD40 expression and age, sex, size, grade, and location of the primary tumor and distant metastases. With 61 patients (74.4%) progressed and 31 (37.8%) dead, CD40 expression was a significant prognostic factor for disease-free and overall survival at univariable and multivariable analysis. Patients with tumors expressing CD40 in >50% of cells had a dramatically unfavorable prognosis with median disease-free and overall survival of 7 and 17 months, respectively, and hazard ratios of relapse and death as compared with patients with CD40-negative tumors of 2.89 (95% confidence interval: 1.26-6.60) and 6.92 (95% confidence interval: 2.18-22.0), respectively. CONCLUSIONS: These data suggest that expression of CD40 protein in >50% of cells might indicate an unfavorable prognosis in adult soft tissue sarcomas.     

Use of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients.

PURPOSE: To evaluate safety and preliminary efficacy of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy (RT) in unresectable head and neck cancer patients. Secondary end points were the measurement of h-R3 serum levels and the assessment of the potential mechanisms of antitumor effect on patient biopsies. Anti-idiotypic response to h-R3 was assessed. To predict pharmacologic effect, a mathematical model for antibodies recognizing antigens expressed in tumors and normal tissues was built. PATIENTS AND METHODS: Twenty-four patients with advanced carcinomas of the head and neck received six once-weekly infusions of h-R3 at four dose levels in combination with RT. Pretreatment tumor biopsies were obtained to evaluate epidermal growth factor receptor expression as an enrollment criterion. Second biopsies were taken to evaluate the proliferative activity and angiogenesis in comparison with the pretreatment samples. Patient serum samples were collected to measure h-R3 levels and anti-idiotypic response. RESULTS: The combination of h-R3 and RT was well tolerated. Antibody-related adverse events consisted in infusion reactions. No skin or allergic toxicity appeared. Overall survival significantly increased after the use of the higher antibody doses. Immunohistochemistry studies of tumor specimens before and after treatment revealed that antitumor response correlated with antiproliferative and antiangiogenic effect. One patient developed antibodies to h-R3. The mathematical model predicted that the maximum difference between the area under the curve in tumors and normal tissues is reached when the antibody has intermediate affinity. CONCLUSION: h-R3 is a well-tolerated drug that may enhance radiocurability of unresectable head and neck neoplasms.     

Helicobacter pylori VacA toxin up-regulates vascular endothelial growth factor expression in MKN 28 gastric cells through an epidermal growth factor receptor-, cyclooxygenase-2-dependent mechanism.

PURPOSE: Helicobacter pylori causes gastric damage and is involved in gastric carcinogenesis. Vascular endothelial growth factor (VEGF) plays a major role in gastric mucosa repair and is overexpressed in gastric cancer. We investigated: (a) whether H. pylori, and in particular H. pylori VacA toxin, affected VEGF expression in gastric epithelial cells in culture; and (b) the signal transduction pathway involved in any effect exerted by H. pylori. EXPERIMENTAL DESIGN: MKN-28 cells were incubated with uninoculated BCF (control) or with BCF obtained from VacA-producing wild-type H. pylori 60190 strain or from its isogenic mutant 60190:v1, specifically lacking vacA gene in the presence or absence of ZD 1839, a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, PD098059, a selective inhibitor of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase, the kinase responsible for ERK phosphorylation, or SC-236, a selective inhibitor of cyclooxygenase (COX)-2 for 24-48 h. RESULTS: (a) Toxigenic H. pylori up-regulated VEGF mRNA and protein expression and caused a 2.5-fold increase in VEGF release compared with control, whereas nontoxigenic H. pylori did not; (b) H. pylori VacA toxin-induced up-regulation of VEGF was counteracted by selective inhibition of EGFR tyrosine kinase; (c) toxigenic H. pylori activated the ERK/MAP kinase cascade, and inhibition of MAP kinase activation counteracted H. pylori-induced VEGF up-regulation; (d) toxigenic H. pylori up-regulated COX-2 expression, and this effect was counteracted by blockade of EGFR tyrosine kinase; and (e) COX-2 selective inhibition counteracted H. pylori-induced up-regulation of VEGF. CONCLUSION: (a) H. pylori up-regulates VEGF expression in gastric epithelial cells; and (b) this effect is specifically related to VacA toxin and seems to depend on the activation of an EGFR-, MAP kinase-, and COX-2-mediated pathway.     

Phase I trial and pharmacokinetics of fenretinide in children with neuroblastoma.

PURPOSE: Fenretinide (4HPR), a synthetic retinoid, induces apoptosis in neuroblastoma cells. A Phase I study in children with neuroblastoma was designed to determine maximum tolerated dose, toxicity, and pharmacokinetics. EXPERIMENTAL DESIGN: Fifty-four patients received oral 4HPR, once daily, for 28 days, followed by a 7-day interruption, for up to 6 courses. The starting dose was 100 mg/m(2)/day. At least 3 patients were entered at each escalating 4HPR dose level. Pharmacokinetic sampling was performed on days 1 and 28 of the first course. RESULTS: Fifty-four patients, of whom 53 were evaluable, received doses between 100 and 4000 mg/m(2)/day for a total of 168 courses. Additional dose escalation was precluded by capsule number intake. A total of 34 of 53 evaluable patients showed manageable, reversible toxicities, which were not dose related. One dose-limiting toxicity (nyctalopia grade 3) occurred after the 1000 mg/m(2)/day dose. Twelve patients showed grade 2 toxicity: skin xerosis (6 cases); nyctalopia (3 cases); hepatic toxicity (1 case); diarrhea (1 case); and headache (1 case). Stable disease was observed in 41 patients for a median period of 23 months (range 2-35+). After first administration, average 4HPR peak plasma levels ranged from 0.6 to 6 micro M (after 100 and 4000 mg/m(2)/day, respectively) and increased 2-fold (to 1.3 and 12.9 micro M, respectively) after the 28-day treatment. 4HPR half-life increased from 17 h after the first administration to 25 h after the 28(th) administration. Incidence of grade 2-3 toxicity was 0 of 12 (0%), 7 of 22 (31%), and 4 of 8 (50%) with peak 4HPR concentrations <3 micro M, 3-10 micro M, and >10 micro M, respectively. After repeated treatment, retinol levels decreased from 20 to 10% of pretreatment levels after all of the doses. CONCLUSIONS: In children, 4HPR administration up to 4000 mg/m(2)/day over 28 days, followed by a 7-day interruption, results in manageable toxicity and in drug plasma concentrations comparable with those that induce apoptosis in neuroblastoma cell lines.     

Foramen Thyroideum: A Comparative Study in Embryos,Fetuses, and Adults

The incidence and characteristics of foramen thyroideum (FT) in embryonic and/or fetal larynges have not been established. In the present study, 90 adult larynges and 53 embryonic-fetal larynges were studied. The incidence of FT during the embryonic-fetal period (57%) was statistically different from the adult period (31%) (P = 0.005). All the FT found in the adult period contained vessels and/or nerves, while in the embryonic and fetal period only 63% presented neurovascular elements (P < 0.001). The origin of FT in the embryonic period and its persistence during adult life is discussed.     

Evaluation of neurodevelopmental outcome in highrisk infants in Australia

An understanding of the neurodevelopmental outcome of long-term survivors of neonatal intensive care is essential for the informed management of preterm or high risk infants. This annotation looks at the current status of neonatal follow-up services in Australasia and highlights problems in the collection and interpretation of data. It suggests that we should work towards achieving a consensus on standard definitions and test regimes and on national data collection.