Michel Gillet

Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer.

The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14ARF and p16INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14ARF and p16INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1β of p14ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14ARF, documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14ARF and p16INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14ARF was inversely correlated with p53 overexpression (p = 0.02). p14ARF and p16INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p = 0.02). Methylation of the p14ARF gene occurred more frequently in well-differentiated adenocarcinomas (p = 0.005), whereas the p16INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p = 0.002). The present results underline the role of p14ARF and p16INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14ARF and p16INK4a, might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways.

Experience of liver transplantation for incurable alveolar echinococcosis: a 45-case European collaborative report.

Background. Alveolar echinococcosis (AE) of the liver is a rare and severe parasitic disease. It behaves like a slow-growing liver cancer, and liver transplantation (LT) has been proposed in advanced cases since 1985. The aim of this retrospective study was to collect all AE transplant cases in Europe, analyze the results, and specify the usefulness of LT for this unusual indication. Methods. A questionnaire was sent to 83 LT centers from July 1996 to December 1999. Results. Sixty-five centers responded: 45 AE patients (mean age, 45.8 years) underwent an LT procedure at 16 LT centers. The mean interval between diagnosis and LT was 5 years. One patient died during the hepatectomy phase. Five-year survival was 71%. Five-year survival without recurrence was 58%. The nine early deaths were mostly related to bacterial or fungal infections, or both, in patients in bad condition when LT was performed. Six patients had a graft AE reinfection. Five late deaths were related directly to ongoing AE. In the other cases, benzimidazole (BZM) therapy seemed to stabilize AE residues. Conclusions. This unique experience indicates that LT is feasible for life-threatening AE. Specific management is needed to optimize the results: earlier decision for LT in incurable symptomatic biliary AE, pre- and post-LT BZM therapy, meticulous pre-LT evaluation to identify extrahepatic extension, and an immunosuppressive regimen kept to a minimum.

Naturally Acquired MAGE-A10- and SSX-2-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2+ melanoma patients. In two of six HLA-A2+ HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8+ T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Combined laparoscopic-endoscopic method using an omental plug for therapy of gastroduodenal ulcer perforation

BACKGROUND Laparoscopic techniques have been proposed as an alternative to open surgery for therapy of peptic ulcer perforation. They provide better postoperative comfort and absence of parietal complications, but leakage occurs in 5% of cases. We describe a new method combining laparoscopy and endoluminal endoscopy, designed to ensure complete closure of the perforation. METHODS Six patients with anterior ulcer perforations (4 duodenal, 2 gastric) underwent a concomitant laparoscopy and endoluminal endoscopy with closure of the orifice by an omental plug attracted into the digestive tract. RESULTS All perforations were sealed. The mean operating time was 72 minutes. The mean hospital stay was 5.5 days. There was no morbidity and no mortality. At the 30-day evaluation all ulcers but one (due to Helicobacter pylori persistence) were healed. CONCLUSIONS This method is safe and effective. Its advantages compared with open surgery or laparoscopic patching as well as its cost-effectiveness should be studied in prospective randomized trials.

Determinants of protracted cytomegalovirus infection in solid-organ transplant patients.

Background. Recurrent infection frequently follows the response to the initial treatment of cytomegalovirus (CMV) infection in solid-organ transplant (SOT) recipients. The objective of this study was to describe the course of CMV infection in SOT patients and to identify factors that would predict protracted CMV infection with recurrences. Methods. Quantitative polymerase chain reaction (PCR) assay for CMV DNA in leukocytes and in plasma were used to assess viral load changes retrospectively in consecutive SOT patients, whose CMV infection episodes had been attended therapeutically or preemptively using quantitative blood culture. Results. Among 101 SOT patients, CMV infection occurred in 63, of whom 32 developed recurrent infection after the initial episode. In patients with recurrent infection, PCR indicated that a majority (27) of recipients had high level of CMV DNA in peripheral blood leukocytes and plasma throughout a protracted (≥1 month) period including after preemptive or therapeutic ganciclovir courses. Predictors of protracted high-level infection were increasing age, CMV donor seropositivity, and all measures of viral load during the initial episode. CMV recipient seropositivity protected strongly against protracted infection. End of treatment plasma CMV DNA best discriminated between patients who did or did not develop protracted infection. Conclusions. In SOT patients, protracted CMV infection is associated with increasing age, donor seropositivity, recipient seronegativity, and high viral load during the first episode. End of therapy plasma CMV DNA level best predicts the occurrence of protracted infection. In patients with a high risk of protracted infection, prophylaxis is likely to be particularly cost effective.

Angiogenesis of liver metastases

PURPOSE: Tumor-induced angiogenesis requires migration and remodeling of endothelial cells derived from pre-existing blood vessels. Vascular endothelial growth factor is the growth factor most closely implicated in the development of neovessels in colon cancer. However, vascular endothelial growth factor-specific receptors flt-1 and KDR mRNA expression are absent in normal sinusoid vessels surrounding vascular endothelial growth factor-producing secondary hepatic tumors. Thus, the potential role of sinusoidal endothelial cells in the mechanism of neovessel formation within liver metastatic carcinomas remains unclear. The purpose of this study was to determine whether sinusoidal endothelial cells are involved in tumor angiogenesis in a syngeneic model of liver metastases from colorectal cancer. METHODS: Sinusoidal endothelial cells were identified by fluorescence microscopy after uptake of acetylated low density lipoprotein labeled with a fluorescent probe (dioctadecylindocarbocyanine). One hundred microliters of dioctadecylindocarbocyanine acetylated low density lipoprotein were injected intraportally at the start of experiment in BD IX rats. Two days later, intraportal injection of 107 DHD K12, a chemically induced colon carcinoma cell line, was performed in syngeneic BD IX rats. Animals were killed one week later and the livers were processed for routine histologic examination and immunohistochemistry using the rat endothelial cell antigen-1 monoclonal antibody. RESULTS: In normal parenchyma fluorescence was associated with sinusoidal cells but not with endothelium of large blood vessels. Thus, specific acetylated low density lipoprotein uptake allowed histological differentiation of sinusoidal endothelial cells from other large-vessel endothelial cells present in the hepatic parenchyma. In tumor-bearing liver a spatial gradient of fluorescence was generated. Labeled cells accumulated at the periphery of the metastases. When tumors grow beyond 200 µm, neovessel formation was observed; there was an invasion of fluorescent-labeled cells from the periphery, which were arranged in a tubular formation within neoplasia. CONCLUSION: In liver metastases tumor vessels are lined with sinusoidal endothelial cells. Identification of a specific cell type involved in the formation of the stromal compartment of tumors has important implications. Sinusoidal endothelial cells express well-characterized surface receptors and differ morphologically and metabolically from large-vessel endothelia. They should be considered as attractive targets for future and existing antiangiogenic strategies directed against the stromal compartment of liver metastases.

Hepatic nonoxidative disposal of an oral glucose meal in patients with liver cirrhosis.

Seven patients with liver cirrhosis and five healthy subjects were studied over 4 hours after ingestion of a glucose meal to determine whether alterations of hepatic nonoxidative glucose disposal participate in the pathogenesis of impaired glucose tolerance. Hepatic uridyl-diphosphoglucose (UDPG) turnover was calculated from the isotopic enrichment of urinary acetaminophen glucuronide during continuous infusion of 13C-galactose and used as an index of hepatic glycogen synthesis. Patients with cirrhosis had postprandial hyperglycemia and decreased glucose clearance, but hepatic UDPG turnover was not altered (1.84 +/- 0.29 mg/kg fat-free mass min v 1.76 +/- 0.15 in controls, nonsignificant). It is concluded that hepatic postprandial glycogen synthesis is unaltered in patients with advanced cirrhosis, demonstrating important hepatic functional reserve.

Esophageal cancer as second primary tumor after breast cancer radiotherapy

BACKGROUND An increased risk of esophageal cancer has been reported in survivors of breast cancer treated with radiotherapy. This study further characterizes this association. METHODS Through hospital databases, 118 patients (109 men, 9 women) treated for esophageal cancer between 1985 and 1993 were identified, of whom 37 had 60 synchronous or metachronous cancers. 5 women had primary esophageal cancer after having breast cancer, and are the subjects of this case-control study. RESULTS All 5 women had been treated with radical mastectomy and adjuvant radiotherapy; none received chemotherapy. Their ages at the time of breast cancer ranged from 36 to 82 years; at esophageal cancer, 61 to 95 years. Time between radiotherapy and esophageal cancer varied from 13 to 31 years. All esophageal cancers were squamous cell carcinomas. Mean survival after esophageal cancer was 14.2 months. CONCLUSIONS Radio-induced esophageal cancer can occur as a second primary cancer in women who survive at least 1 decade after mastectomy and adjuvant radiotherapy.

Preoperative hyperfractionated accelerated radiotherapy (HART) and concomitant CPT-11 in locally advanced rectal carcinoma: A Phase I study

PURPOSE Patients with locally advanced rectal carcinoma are at risk for both local recurrence and distant metastases. We demonstrated the efficacy of preoperative hyperfractionated accelerated radiotherapy (HART). In this Phase I trial, we aimed at introducing chemotherapy early in the treatment course with both intrinsic antitumor activity and a radiosensitizer effect. METHODS AND MATERIALS Twenty-eight patients (19 males; median age 63, range 28-75) with advanced rectal carcinoma (cT3: 24; cT4: 4; cN+: 12; M1: 5) were enrolled, including 8 patients treated at the maximally tolerated dose. Escalating doses of CPT-11 (30-105 mg/m(2)/week) were given on Days 1, 8, and 15, and concomitant HART (41.6 Gy, 1.6 Gy bid x 13 days) started on Day 8. Surgery was to be performed within 1 week after the end of radiochemotherapy. RESULTS Twenty-six patients completed all preoperative radiochemotherapy as scheduled; all patients underwent surgery. Dose-limiting toxicity was diarrhea Grade 3 occurring at dose level 6 (105 mg/m(2)). Hematotoxicity was mild, with only 1 patient experiencing Grade 3 neutropenia. Postoperative complications (30 days) occurred in 7 patients, with an anastomotic leak rate of 22%. CONCLUSIONS The recommended Phase II dose of CPT-11 in this setting is 90 mg/m(2)/week. Further Phase II exploration at this dose is warranted.

Postprandial Hepatic Glycogen Synthesis In Liver Transplant Recipients1

BACKGROUND The liver plays a central role in glucose homeostasis by releasing glucose in the fasting state and by taking up and converting into glycogen part of the glucose absorbed from the gastrointestinal tract after meal ingestion. METHODS To determine whether the hepatic denervation that accompanies liver transplantation interferes with these functions, we assessed glucose tolerance to an oral glucose load in seven patients at 2-6 weeks after orthotopic liver transplantation, in six patients after kidney transplantation, and in six healthy controls. Hepatic glycogen synthesis was non-invasively assessed over the 4 hours after ingestion of a glucose load by monitoring hepatic uridine diphosphoglucose turnover with 13C galactose and acetaminophen. RESULTS Liver and kidney transplant recipients had increased postprandial glucose concentrations but normal hepatic uridine diphosphoglucose turnover, indicating an unaltered hepatic glycogen synthesis. CONCLUSIONS These results indicate that denervated liver transplants have an adequate glucoregulatory function. Postprandial hyperglycemia in liver transplant recipients is therefore not due to alterations of liver glucose metabolism.