Immunohistochemical variability of epidermal growth factor receptor (EGFR) in liver metastases from colonic carcinomas.

AIMS: To follow and compare immunohistochemical expression of epidermal growth factor receptor (EGFR) in tumour cells during the entire natural history of colonic carcinoma, from primary tumour to paired lymph node and sequentially resected liver metastases; and to test interobserver reproducibility of EGFR analysis. METHODS AND RESULTS: Forty patients had resection of colonic adenocarcinoma (27 with metastatic lymph nodes) and at least one partial hepatectomy (PH) for liver metastases; a second and a third PH were performed, respectively, in 14 and three patients; seven patients had tumour liver biopsy. EGFR immunohistochemistry (n = 130) was analysed independently by two pathologists. EGFR expression (membranous staining detected in > or = 1% of tumour cells) was detected in 38/40 colonic carcinomas, 23/26 lymph nodes and 51/64 liver metastases. Both primary tumours and related metastases were EGFR+ in 28 patients (73%). Discrepancies were found in EGFR status between liver and lymph node (23%) and among the different liver samples (31%). Interobserver agreement was very good (intraclass correlation coefficients of 0.81, 0.91 and 0.85, respectively, for interpretation of staining in colon, lymph node and liver metastases). CONCLUSIONS: Since immunohistochemical detection of EGFR remains a prerequisite for EGFR-targeted therapy eligibility, different tumour samples should be tested to allow every patient a chance to take advantage of this treatment.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma.

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Primary liver carcinoma in genetic hemochromatosis reveals a broad histologic spectrum.

Hepatocellular carcinoma (HCC) is a well-known complication of genetic hemochromatosis (GH). However, the frequency of primary liver carcinoma (PLC) with biliary differentiation, such as cholangiocarcinoma (CC) and combined hepatocholangiocarcinoma (CHCC), in GH remains unclear We analyzed the histologic type of 20 PLCs occurring in the background of GH; all patients were homozygotic for the C282Y mutation. Ten were depleted of iron by successive phlebotomies, while the remaining 10 were untreated. Histologically, 13 cases were classified as HCC, 3 as CC, and 4 as CHCC. Immunohistochemical detection of Hep Par 1, cytokeratin 19 (CK19), and MUC1 supported this classification; PLC with biliary differentiation was immunoreactive for MUC1 in 86% (6/7) of cases and for CK19 in 100% (7/7) of cases. The nontumoral liver exhibited no cirrhosis or extensive fibrosis in 6 cases. Von Meyenburg complexes were present in 11 cases and intraparenchymal bile duct adenomas in 3. These data suggest that PLCs in patients with GH present a wide histologic spectrum, with tumors showing frequent biliary differentiation; may arise on a nonfibrotic or a cirrhotic liver; and often are associated with Von Meyenburg complexes and to a lesser extent with bile duct adenomas.

Frequent FGFR3 mutations in papillary non-invasive bladder (pTa) tumors.

We recently identified activating mutations of fibroblast growth factor receptor 3 (FGFR3) in bladder carcinoma. In this study we assessed the incidence of FGFR3 mutations in a series of 132 bladder carcinomas: 20 carcinoma in situ (CIS), 50 pTa, 19 pT1, and 43 pT2-4. All 48 mutations identified were identical to the germinal activating mutations that cause thanatophoric dysplasia, a lethal form of dwarfism. The S249C mutation, found in 33 of the 48 mutated tumors, was the most common. The frequency of mutations was higher in pTa tumors (37 of 50, 74%) than in CIS (0 of 20, 0%; P < 0.0001), pT1 (4 of 19, 21%; P < 0.0001) and pT2-4 tumors (7 of 43, 16%; P < 0.0001). FGFR3 mutations were detected in 27 of 32 (84%) G1, 16 of 29 (55%) G2, and 5 of 71 (7%) G3 tumors. This association between FGFR3 mutations and low grade was highly significant (P < 0.0001). FGFR3 is the first gene found to be mutated at a high frequency in pTa tumors. The absence of FGFR3 mutations in CIS and the low frequency of FGFR3 mutations in pT1 and pT2-4 tumors are consistent with the model of bladder tumor progression in which the most common precursor of pT1 and pT2-4 tumors is CIS.

Carcinological risks and retroperitoneal laparoscopy.

PURPOSE: To determine the incidence of trocar site spillage, local recurrence, and metastatic disease associated with retroperitoneal laparoscopic tumor resection. METHODS: From 1994 to 1999, 228 retroperitoneal laparoscopic procedures were performed at our institution. Fifty-six procedures (24.6%) were for malignancies and comprised 41 radical nephrectomies and 8 partial nephrectomies for renal tumors, and 7 nephro-ureterectomies for renal pelvis tumors. The pathological stage and the status of surgical margins were noted according to the 1997 TNM classification. Postoperative follow-up data were obtained by means of physical and radiological examinations after 1 and 3 months, and then half-yearly. Trocar site seeding, local recurrence and metastatic disease were recorded. Kaplan-Meier actuarial analysis was used to determine the disease-free survival likelihood. RESULTS: The mean follow-up was 24.9+/-13.85 months. All the patients had tumor-free surgical margins. No laparoscopic trocar site recurrences were identified. For laparoscopic radical nephrectomy: one patient had a local recurrence with hepatic metastasis of a pT(3)G(2) tumor after 9 months and died 19.7 months after the procedure. One patient with a pT(3a)G(3)M+ tumor died 23.1 months after radical nephrectomy without any sign of local recurrence. For laparoscopic nephro-ureterectomy: one patient with a pT(3)G(3) tumor had a local recurrence 12.1 months after the procedure and died 26.6 months after surgery. One patient with a pT(1)G(2) renal pelvis tumor had bone metastasis at 9 months and died 29 months after the procedure. The Kaplan-Meier actuarial disease-free survival rate was 91% at 54 months for radical nephrectomy, 61% at 30 months for nephro-ureterectomy and 100% at 49 months for partial nephrectomy. CONCLUSION: Malignancies of the upper urinary tract can be managed by means of retroperitoneal laparoscopy. Short-term results suggest that this procedure is not associated with an increased risk of portsite or local recurrence, and that disease-free survival is equivalent to that obtained with open surgery.

In vivo retrovirus-mediated gene transfer into lamb liver.

TOPIC: Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo has been previously reported in the rat. Before considering human applications of these techniques in the treatment of inherited liver diseases, it was necessary to document its efficiency in a large animal model. Lamb was choosen because the liver was similar to human liver regarding size and anatomy. MATERIALS AND METHODS: To induce hepatocyte division which is necessary for infection with retroviral particles, animals were subjected to a left hepatectomy. Kinetics of liver regeneration were assessed on sequential liver biopsies after partial hepatectomy in order to provide an evaluation of the peak of maximal liver regeneration in a first animal group. Recombinant retroviruses encoding a reporter gene (E. coli beta galactosidase) were then perfused through the portal vein of the regenerating liver in a second animal group. RESULTS: The more intense liver regeneration occurred from one to 6 days after partial hepatectomy, with the highest thymidine kinase rate and MIB-1 antibody staining on the second day. The proportion of genetically modified lamb hepatocytes expressing the reporter gene was less than 1%, despite the use of higher titers of retroviral particles than those described in previous reports. CONCLUSION: The results obtained in rodent livers with this in vivo gene transfer methodology cannot currently be scaled up in a large ruminant model. The efficacy of vectors has to be tested in other large mammals before planning gene therapy trials for the treatment of inherited liver diseases.

Hepatocellular carcinoma occurring in nonfibrotic liver: epidemiologic and histopathologic analysis of 80 French cases.

Hepatocellular carcinoma (HCC) occurring in nonfibrotic liver represents a rare, ill-defined subgroup of HCC without cirrhosis in which mechanisms of hepatocarcinogenesis remain unclear. The aim of our study was to assess epidemiological factors and detailed histopathologic changes in the nontumoral liver of patients developing such tumors. Of 330 HCCs resected in our institution between 1985 and 1998, we retrospectively analyzed 80 cases (53 men, 27 women; mean age, 51 +/- 16 years) in which the nontumoral liver showed no (n = 28) or minimal (n = 52) portal fibrosis without any septal fibrosis. In the group with no portal fibrosis there was no male predominance, and patients were significantly younger (44 +/- 19 years vs. 54 +/- 14 years) than those with minimal portal fibrosis. Sixty-seven tumors were typical HCCs, 8 were of fibrolamellar type, and 5 were hepatocholangiocarcinomas. Mean tumor size was 10 +/- 5 cm. Risk factors for HCC development were found in 30 patients: hepatitis B (n = 17) or C (n = 2) virus infections, alcohol consumption (n = 11), and hemochromatosis (n = 1). In the nontumoral liver, periportal and lobular necrosis, mild portal inflammation, steatosis, and iron overload were present in 15%, 57%, 52%, and 54% of cases, respectively. Liver cell changes were noted in 6%. This study emphasizes the need for strict criteria to classify HCC without cirrhosis. HCC in nonfibrotic liver is a distinct subgroup in which nontumoral liver shows nonspecific minimal changes without regeneration or premalignant lesion. Etiologic factors are often unidentified, although presence of HBV infection in 21% suggests a direct oncogenic role of this virus.

[Diagnosis and histologic surveillance of hepatitis C]. / Diagnostic et surveillance histologiques de l'hépatite C.

The role of liver biopsy in hepatitis C viral infection is diagnostic and prognostic. It states diagnoses of chronic hepatitis, cirrhosis and hepatocellular carcinoma. The association of portal lymphoid nodules, inflammatory bile duct lesions and steatosis suggests an hepatitis C viral etiology. Liver biopsy allows grading (extent of necro-inflammatory lesions) and staging (amount of fibrosis) of the disease using scoring systems proposed by Knodell et al. and (or) by METAVIR group. It can be helpful in confirming (or refuting) the presence of secondary diagnoses such as alcohol-induce liver disease or haemochromatosis and in assessing the efficacy of antiviral treatments.

Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model.

Gene therapy for hepatocellular carcinoma (HCC) has shown some promise, but its evaluation requires relevant experimental models. With this aim, we present an evaluation of the interest of using the woodchuck model of HCC to assess in vivo gene transfer efficiency. We tested the transduction efficacy of the adenoviral vectors directing lacZ gene product expression under the control of the cytomegalovirus and alpha-fetoprotein (AFP) regulatory sequences. We have also investigated whether an adenoviral cytomegalovirus-thymidine kinase (Tk) vector might induce an antitumoral effect in this model. Our results demonstrate that with direct intratumoral and intrahepatic arterial injections, transduction of a significant proportion of tumor cells occurred even in large HCC nodules. Furthermore, due to intra-arterial anastomoses, direct intratumoral injection led to transduction of some noninjected HCC nodules. Moreover, direct intratumoral injection of a herpes simplex virus-1 Tk-encoding vector induced, on ganciclovir administration, a significant antitumoral effect in the two animals evaluated. However, in one animal, massive hepatic failure occurred due to Tk expression in nontumor cells. These results emphasize the need to target the expression of the Tk gene to tumor cells using a hepatoma-specific promoter such as AFP promoter. However, we showed that, in vivo, lacZ expression as driven by the AFP promoter was extremely low, thus emphasizing some potential pitfalls when using this approach. Altogether, our data stress the need to test gene therapy-based strategies in such in vivo animal models of HCC and evaluate gene transduction, expression, and biological activity, as well as its potential toxicity.

Hepatocellular carcinoma in WHV/N-myc2 transgenic mice: oncogenic mutations of beta-catenin and synergistic effect of p53 null alleles.

The intronless N-myc2 gene was originally identified as the major target of hepatitis virus insertion in woodchuck liver tumors. Here we report that transgenic mice carrying the N-myc2 gene controlled by woodchuck hepatitis virus (WHV) regulatory sequences are highly predisposed to liver cancer. In a WHV/N-myc2 transgenic line, hepatocellular carcinomas or adenomas arose in over 70% of mice, despite barely detectable expression of the methylated transgene in liver cells. Furthermore, a transgenic founder carrying unmethylated transgene sequences succumbed to a large liver tumor by the age of two months, demonstrating the high oncogenicity of the woodchuck N-myc2 retroposon. Stabilizing mutations or deletions of beta-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of beta-catenin activation in Myc-induced tumorigenesis. The ability of the tumor suppressor gene p53 to cooperate with N-myc2 in liver cell transformation was tested by introducing a p53-null allele into WHV/N-myc2 transgenic mice. The loss of one p53 allele in transgenic animals markedly accelerated the onset of liver cancer (P=0.0001), and most tumors of WHV/N-myc2 p53+/Delta mice harbored either a deletion of the wt p53 allele or a beta-catenin mutation. These findings provide direct evidence that activation of N-myc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that different genetic pathways may underlie liver carcinogenesis initiated by a myc transgene. Oncogene (2000).

Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes.

Transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene followed by the administration of ganciclovir (GCV) into hepatocellular carcinoma (HCC)-derived cell lines either in vitro or transplanted into nude mice has been shown to provide a potential strategy for HSV-tk-based gene therapy of HCC. We report herein an analysis of the antitumoral efficacy of two recombinant adenoviruses (Ads), Ad.CMVtk and Ad.AFPtk, in a relevant model of multifocal hepatic lesions induced in rats by a potent alkylating chemical carcinogen, diethylnitrosamine. Two routes of administration of the Ad were studied: intratumoral and intrahepatic artery injections. Both recombinant Ads, Ad.CMVtk and Ad.AFPtk, express the HSV-tk gene under the control of the early enhancer/promoter cytomegalovirus and alpha-fetoprotein regulatory gene sequences, respectively. The antitumor response was assessed by magnetic resonance imaging and by autopsy and histological analysis following postmortem. Tumor growth cessation was demonstrated by magnetic resonance imaging in large tumor nodules of size 5-8 mm treated by intratumoral administration of 2x10(9) pfu Ad.CMVtk plus i.p. treatment with GCV. We also show an antitumor efficacy in small tumor nodules of size <3 mm treated with 2x10(9) pfu Ad.CMVtk plus GCV by the intrahepatic artery route, albeit associated with an adverse toxicity. In vivo targeting of the HSV-tk gene to diethylnitrosamine-induced HCC cells with the recombinant Ad.AFPtk suppresses the hepatic toxicity in the nontumoral liver. The lower antitumor response would argue for the use of multiple injections of such adenoviral constructs. These observations may lead to potential approaches for designing gene therapy destined for early treatment of dysplastic nodules or advanced HCC in cirrhosis.

Epithelioid hemangioendothelioma, multiple focal nodular hyperplasias, and cavernous hemangiomas of the liver.

Malignant vascular neoplasms of the liver are uncommon. We report the case of a young woman who developed an epithelioid hemangioendothelioma of the liver associated with multiple focal nodular hyperplasias and hepatic cavernous hemangiomas. Such an unusual association is probably not fortuitous and could support the theory that focal nodular hyperplasia is a reaction to an abnormal vascular supply rather than a true neoplasm.

Squamous cell carcinoma and lipomatous pseudohypertrophy of the pancreas.

A 68-year-old woman who had been treated for non-insulin-dependent diabetes mellitus for the past 20 years was admitted to hospital because of abdominal pain and weight loss. Radiological investigation revealed a tumour in the body of the pancreas and numerous intraductal calcifications in both the tail and the head of the pancreas. Left-sided pancreatectomy was performed to remove the tumour. The resection specimen showed fatty enlargement of the parenchyma and numerous intraductal calcifications in the tissue adjacent to the tumour, which was 7 cm in diameter and was found to be a primary squamous cell carcinoma with a spindle cell component. There was also lipomatous pseudohypertrophy.

Enhanced in vivo adenovirus-mediated gene transfer to rat hepatocarcinomas by selective administration into the hepatic artery.

Adenovirus-mediated gene therapy of experimental hepatocarcinoma is hindered by low transduction efficacy in vivo. We evaluated the extent of gene expression following various routes of administration of recombinant adenovirus AdCMVlacZ in diethylnitrosamine-induced rat hepatocarcinoma. We first characterized the vascularization of diethylnitrosamine-induced hepatocarcinomas using a computerized tomography scanner approach. The efficacy of gene transfer was then evaluated by three routes of administration: intraportal, selective injection through the hepatic artery and direct injection into the tumor. Diethylnitrosamine-induced hepatocarcinomas had predominantly an arterial blood supply, 67% of the total liver blood supply. Compared with intraportal administration, arterial injection improved gene transfer into tumors whereas that to the non-tumor areas was diminished. In addition, this route of injection allowed the efficient transduction of dysplastic nodules. Diethylnitrosamine-induced hepatocarcinoma in rats is a relevant model for the study of human hepatocarcinoma due to its vascularization. Arterial infusion improved the ratio of transduced tumorous to nontumorous cells and allowed targeting of gene transfer to dysplastic nodules. This will be useful in the design of gene therapy for hepatocarcinoma.

Histopathologic impact of GB virus C infection on chronic hepatitis C.

BACKGROUND & AIMS: Dual infection by hepatitis GB virus type C (GBV-C) and hepatitis C virus (HCV) is common. To assess the histopathologic impact of GBV-C infection on liver lesions, liver biopsy specimens of 105 patients chronically infected with HCV, 17 of whom (15%) were also infected with GBV-C, were reviewed. METHODS: Semiquantitative histopathologic assessment of liver lesions was performed using the Knodell's score and the METAVIR grading system. RESULTS: Hepatitis activity was mild, moderate, or severe in 3 (18%), 11 (64%), and 3 (18%) patients, respectively, infected with GBV-C and HCV vs. 26 (29%), 56 (64%), and 6 (7%) patients, respectively, infected with HCV alone (no significant difference). Cirrhosis was present in 4 (24%) coinfected patients vs. 19 (22%) HCV-positive patients (no significant difference). No significant difference in fibrosis, presence of portal lymphoid aggregates, steatosis, and hemosiderosis was observed between the two groups. There was no significant difference in the evaluation of each item of the Knodell's score. CONCLUSIONS: This detailed histopathologic evaluation of GBV-C infection in chronic hepatitis C shows that GBV-C infection does not affect histopathologic severity and characteristics of chronic hepatitis C, thus suggesting a minor role of GBV-C infection in liver disease.

In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.