Gideon Steinbach

THE EFFECT OF CELECOXIB, A CYCLOOXYGENASE-2 INHIBITOR, IN FAMILIAL ADENOMATOUS POLYPOSIS

BACKGROUND Patients with familial adenomatous polyposis have a nearly 100 percent risk of colorectal cancer. In this disease, the chemopreventive effects of nonsteroidal antiinflammatory drugs may be related to their inhibition of cyclooxygenase-2. METHODS We studied the effect of celecoxib, a selective cyclooxygenase-2 inhibitor, on colorectal polyps in patients with familial adenomatous polyposis. In a double-blind, placebo-controlled study, we randomly assigned 77 patients to treatment with celecoxib (100 or 400 mg twice daily) or placebo for six months. Patients underwent endoscopy at the beginning and end of the study. We determined the number and size of polyps from photographs and videotapes; the response to treatment was expressed as the mean percent change from base line. RESULTS At base line, the mean (+/-SD) number of polyps in focal areas where polyps were counted was 15.5+/-13.4 in the 15 patients assigned to placebo, 11.5+/-8.5 in the 32 patients assigned to 100 mg of celecoxib twice a day, and 12.3+/-8.2 in the 30 patients assigned to 400 mg of celecoxib twice a day (P=0.66 for the comparison among groups). After six months, the patients receiving 400 mg of celecoxib twice a day had a 28.0 percent reduction in the mean number of colorectal polyps (P=0.003 for the comparison with placebo) and a 30.7 percent reduction in the polyp burden (the sum of polyp diameters) (P=0.001), as compared with reductions of 4.5 and 4.9 percent, respectively, in the placebo group. The improvement in the extent of colorectal polyposis in the group receiving 400 mg twice a day was confirmed by a panel of endoscopists who reviewed the videotapes. The reductions in the group receiving 100 mg of celecoxib twice a day were 11.9 percent (P=0.33 for the comparison with placebo) and 14.6 percent (P=0.09), respectively. The incidence of adverse events was similar among the groups. CONCLUSIONS In patients with familial adenomatous polyposis, six months of twice-daily treatment with 400 mg of celecoxib, a cyclooxygenase-2 inhibitor, leads to a significant reduction in the number of colorectal polyps.

Regulation of cyclooxygenase-2 pathway by HER2 receptor.

Emerging lines of evidence suggest that in addition to growth factors, the process of colorectal tumorigenesis may also be driven by the upregulation of the inducible form of cyclooxygenase-2 (COX-2), an enzyme responsible for the conversion of arachidonic acid to PGEs. The present study was undertaken to investigate the expression and activation of the HER family members, and to explore the regulation of COX-2 expression by the HER2 pathway in human colorectal cancer cells. Here, we report that human colorectal cancer cell lines express abundant levels of HER2 and HER3 receptors, and are growth-stimulated by recombinant neu-differentiation factor-beta 1 (NDF). NDF-treatment of colorectal cancer cells was accompanied by increased tyrosine phosphorylation and heterodimerization of HER3 with HER2. In addition, we demonstrated that HER2 and HER3 receptors in colorectal cancer cells are constitutively phosphorylated on tyrosine residues and form heterodimeric complexes in the absence of exogenous NDF. Inhibition of HER2/HER3 signaling by an anti-HER3 mAb against the ligand binding site resulted in a decrease in the levels of constitutively activated HER2/HER3 heterodimers, and the unexpected reduction of COX-2 expression. Activation of the HER2/HER3 pathway by NDF induced the activation of COX-2 promoter, expression of COX-2 mRNA, COX-2 protein and accumulation of prostaglandin E2 in the culture medium. Finally, we demonstrated that NDF promotes the ability of colorectal cancer cells to survive in an extracellular matrix milieu, such as Matrigel, and also to invade through a 8 μm porous membrane. These biological activities of NDF and its stimulation of cell proliferation are blocked by a specific inhibitor of COX-2. Taken together, our findings provide the first biochemical evidence of a possible role of the COX-2 pathway in the mitogenic action of NDF in colorectal cancer cells where it may be constitutively upregulated due to the autocrine/paracrine activation of HER2/HER3 heterodimers.

Antibiotic Treatment of Gastric Lymphoma of Mucosa-Associated Lymphoid Tissue: An Uncontrolled Trial

Gastric low-grade B-cell lymphoma is related to Helicobacter pylori infection according to histopathologic, epidemiologic, and clinical characteristics. Although normal gastric mucosa does not contain organized lymphoid tissue, lymphoid follicles develop with H. pylori infection (1) and with autoimmune diseases, such as the Sjgren syndrome (2). Low-grade B-cell lymphoma has been postulated to arise within this mucosa-associated lymphoid tissue (MALT) and is often called low-grade MALT lymphoma (3, 4). The incidence of gastric low- and high-grade MALT lymphoma is increased in populations with a high prevalence of H. pylori infection, and H. pylori infection has been reported in up to 90% of patients with low-grade MALT lymphoma (4-6). In addition, investigators have shown that growth of this tumor may depend on antigenic stimulation by H. pylori: They demonstrated that the proliferation of lymphoma B cells in cell culture can be stimulated by H. pylori-specific T cells and related cytokines in the presence of H. pylori (7). On the basis of these findings, trials of antibiotic treatment of gastric low-grade MALT lymphoma have been initiated, and the regression of lymphoma after cure of H. pylori infection has been reported in a high proportion of patients with low tumor burden (8-13). Data on patients with significant tumor infiltration are still forthcoming (13, 14). Because MALT lymphoma has only recently been approached as a distinct clinicopathologic entity (15, 16), its natural history and clinical course are not fully defined. Current data suggest that it is often an indolent tumor with long periods of mild activity and confinement to the stomach. Patients often present with nonspecific upper intestinal discomfort, ulcer-associated symptoms, or gastric bleeding. The endoscopic appearance may suggest benign gastritis, and extensive biopsies may be required for diagnosis. Progression to significant tumor mass, dissemination, or transformation to high-grade, aggressive lymphoma occur in an undefined subset of patients, who may present with early satiety or weight loss. Spontaneous remissions are rare (17-20). Because low-grade MALT lymphoma is an uncommon, often indolent form of cancer with few clinical findings and because the risk for progression to high-grade MALT lymphoma is still unknown (15, 16), definitive data on cure require long-term follow-up of large cohorts from standardized clinical trials. This report presents an interim analysis of an ongoing trial designed to determine the long-term response of low-grade gastric MALT lymphoma to antibiotic treatment and to define criteria for treatment and follow-up. Methods Patients Patients with gastric MALT lymphoma restricted to the stomach and perigastric lymph nodes (modified Ann Arbor stage I and II N1) were eligible for the study. The University of Texas, M.D. Anderson Cancer Center (MDACC), Internal Review Board approved the study, and all patients provided written informed consent. The National Cancer Institute and the institutional review boards of participating institutions approved the multi-institutional protocol. To enable a follow-up period of at least 18 months, only patients treated before May 1997 were included in this analysis. Study Design and Treatment The interim data are derived from an ongoing, prospective, uncontrolled treatment trial with third-party patient registry. Patients were studied at four participating centers. Pathologic diagnosis and resolution of MALT lymphoma were confirmed at MDACC, and all but one patient were examined endoscopically at MDACC. Study design included 1) tumor staging with bilateral bone marrow biopsies and aspirates and computed tomography of the abdomen and pelvis done at baseline and yearly; 2) endoscopy at baseline, at weeks 6 to 8, at 3- to 4-month intervals thereafter until resolution of MALT lymphoma was seen on two consecutive endoscopies, at 6-month intervals thereafter for 2 years, and then yearly thereafter; and 3) endoscopic ultrasonography at baseline and at each endoscopy until resolution of masses or infiltration of the muscularis propria, if present (defined as thickness>2 mm or obliteration of wall architecture), and then at 6- to 12-month intervals. The treatment protocol consisted of two of the following three oral antibiotic regimens1] amoxicillin, 750 mg three times daily, and clarithromycin, 500 mg three times daily; 2) tetracycline, 500 mg four times daily, and clarithromycin, 500 mg three times daily; or 3) tetracycline, 500 mg four times daily, and metronidazole, 500 mg three times dailyadministered sequentially for 21 days at baseline and at 8 weeks along with a proton-pump inhibitor (lansoprazole, 30 mg twice daily [n=29], or omeprazole, 20 mg twice daily [n=5]), and bismuth subsalicylate, two tablets four times daily. Patients who were allergic to penicillin received the tetracycline-based regimens. Tumor Staging Tumors were staged endoscopically to separate superficial gastritis from significant ulcers and infiltration and from mass lesions. A modified Ann Arbor staging system that incorporated changes proposed by Blackledge, Musshoff, and Rohatiner and their coworkers was used initially (21-23). The TNM (tumor, node, metastasis) classification of the American Joint Committee on Cancer and Union Internationale Contre le Cancer (24, 25), which corresponds to the modified Ann Arbor staging, was subsequently adapted and applied (Table 1). The extent of tumor (T) infiltration through the stomach wall and to adjacent organs corresponds to T staging of gastric cancer. Modified Ann Arbor stage I corresponds to stage I T1-4 N0 M0. Modified Ann Arbor stage II1 (22) (involvement of perigastric lymph nodes) corresponds to stage II T1-4 N1 M0. Modified Ann Arbor stage II2 (22) (involvement of distant lymph nodes caudal to the diaphragm, including para-aortic and retroperitoneal lymph nodes) corresponds to stage II T1-4 N2 M0. Ann Arbor stage III (lymph node involvement on both sides of the diaphragm) is designated by stage III T1-4 N3 M0. Ann Arbor stage IV (organ metastasis or involvement of a second extranodal site) is designated by stage IV T1-4 N0-3 M1. Table 1. Staging of Gastric Lymphoma of Mucosa-Associated Lymphoid Tissue Criteria for Response Response to treatment was evaluated at 3- to 4-month intervals beginning with the fifth month after treatment. Treatment was considered to have failed if patients did not meet criteria for improvement; these patients were removed from the study. In stage I T2, I T3, and II N1 tumors, improvement was defined as regression to a lower stage, 30% reduction in abnormal wall thickness, or 30% reduction in the size of the tumor mass (product of the greatest diameters). These patients remained in the study if sequential improvement was evident at each 3-month interval. Patients with persistent mucosal disease (stage I T1) documented by histopathology were formally reviewed at 12-month intervals; a consensus on withdrawal from or continuation in the study was based on clinical considerations, current knowledge, and patient preference. Initially, patients with persistent stage I T1 disease were withdrawn from the study at 12 months (n=2). Subsequently, patients with persistent stage I T1 disease were observed for more than 36 months if improvement was documented at 12-month intervals. Criteria for improvement in stage I T1 disease included reduction in the number of affected gastric sites or affected biopsy specimens or improved histologic score (8), endoscopic appearance of progressive atrophy and scarring, or resolution of abnormal wall thickness on endoscopic ultrasonography. Complete remission was defined as the absence of histopathologic evidence of lymphoma and an endoscopic appearance of gastritis or better. Partial remission was defined as a reduction in endoscopic stage in stage I T2 disease or at least 50% reduction in the size of the mass lesions in stage I T3 or II T3 N1 disease. In stage I T1 disease, partial remission was defined as at least 75% reduction in the number of gastric sites or biopsy specimens showing lymphoma. Treatment was considered to have failed in patients who met criteria for failure or who were withdrawn from the study before complete remission occurred. Endoscopy and Biopsies The gastric mapping protocol included 2 or more maximum-capacity biopsies from each of 7 to 12 areas of the gastric map (26) and at least 6 biopsies from 2 or more of the most abnormal areas. The more extensive mapping was done at baseline and at clinically relevant time points. Studies, done at defined intervals, included routine histopathology, H. pylori testing by Genta stain, rapid urease test (CLO-test, Delta West, Bentley, Australia) or serology, Southern blot or polymerase chain reaction (PCR) for immunoglobulin gene rearrangement analysis, and immunoglobulin light-chain immunocytochemistry. Diagnostic Criteria Low-grade B-cell MALT lymphoma was diagnosed by established histologic criteria [15, 27], including 1) a dense diffuse infiltrate of marginal-zone centrocyte-like B cells with round to slightly irregular nuclear contours, often with abundant pale cytoplasm; 2) presence of lymphoepithelial lesions, characterized by infiltration and disruption of gastric glands or crypts by groups of neoplastic lymphoid cells; and 3) absence of any areas where large cells predominate. Minor criteria supporting but not essential for diagnosis included presence of immunoglobulin light-chain restriction; presence of residual secondary follicle centers with or without intact mantles; and presence of follicular colonization, defined as replacement of follicle centers by neoplastic lymphoid cells. Immunophenotypic expression of pan-B-cell antigens, such as CD20, and lack of expression of CD5 or CD10 supported the diagnosis. Patients with foci of large-cell transformation were excluded from the study. Southern Blot and Polymerase Chain Reaction High-molec

Defibrotide for the treatment of severe hepatic veno-occlusive disease and multiorgan failure after stem cell transplantation: a multicenter, randomized, dose-finding trial.

Therapeutic options for severe hepatic veno-occlusive disease (VOD) are limited and outcomes are dismal, but early phase I/II studies have suggested promising activity and acceptable toxicity using the novel polydisperse oligonucleotide defibrotide. This randomized phase II dose-finding trial determined the efficacy of defibrotide in patients with severe VOD following hematopoietic stem cell transplantation (HSCT) and identified an appropriate dose for future trials. Adult and pediatric patients received either lower-dose (arm A: 25 mg/kg/day; n = 75) or higher-dose (arm B: 40 mg/kg/day; n = 74) i.v. defibrotide administered in divided doses every 6 hours for > or =14 days or until complete response, VOD progression, or any unacceptable toxicity occurred. Overall complete response and day +100 post-HSCT survival rates were 46% and 42%, respectively, with no significant difference between treatment arms. The incidence of treatment-related adverse events was low (8% overall; 7% in arm A, 10% in arm B); there was no significant difference in the overall rate of adverse events between treatment arms. Early stabilization or decreased bilirubin was associated with better response and day +100 survival, and decreased plasminogen activator inhibitor type 1 (PAI-1) during treatment was associated with better outcome; changes were similar in both treatment arms. Defibrotide 25 or 40 mg/kg/day also appears effective in treating severe VOD following HSCT. In the absence of any differences in activity, toxicity or changes in PAI-1 level, defibrotide 25 mg/kg/day was selected for ongoing phase III trials in VOD.

Regression of gastric lymphoma of mucosa-associated lymphoid tissue with antibiotic therapy for Helicobacter pylori

Regression of low-grade B cell gastric lymphoma of mucosa-associated lymphoid tissue after eradication of Helicobacter pylori with antibiotic therapy was recently shown in a small number of patients with low-volume tumors. A patient with a > 10 cm nodular gastric mucosa-associated lymphoid tissue lymphoma that caused hematemesis and weight loss is described. Antibiotic therapy of H. pylori resulted in full clinical recovery and resolution of the mass lesion and morphological features of lymphoma on routine histological examination. However, monotypic immunostaining of plasma cells persisted in a separate and grossly normal-appearing region of the stomach. Antibiotic therapy may be of benefit in patients with mucosa-associated lymphoid tissue lymphoma with mass lesions and significant signs and symptoms, but periodic search for residual lymphoma is needed.

Colorectal Cancer Screening

During the past decade we have seen dramatic advances in colon cancer screening. Reduction in mortality in average risk screening for colorectal cancer has now been shown in multiple trials. Efforts to increase public awareness and compliance with evidence-based screening guidelines are underway. Recent guidelines have incorporated family history, as it has been identified as a common risk factor. The genes responsible for the inherited syndromes of colon cancer have been identified and genetic testing is available. Currently, screening the average risk population over the age of 50 would reduce mortality from colon cancer by 50%. Future advances will likely include improved screening tests, and the development of familial genetic testing.

Phase 3 trial of defibrotide for the treatment of severe veno-occlusive disease and multi-organ failure.

Hepatic veno-occlusive disease (VOD), also called sinusoidal obstruction syndrome (SOS), is a potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). Untreated hepatic VOD/SOS with multi-organ failure (MOF) is associated with >80% mortality. Defibrotide has shown promising efficacy treating hepatic VOD/SOS with MOF in phase 2 studies. This phase 3 study investigated safety and efficacy of defibrotide in patients with established hepatic VOD/SOS and advanced MOF. Patients (n = 102) given defibrotide 25 mg/kg per day were compared with 32 historical controls identified out of 6867 medical charts of HSCT patients by blinded independent reviewers. Baseline characteristics between groups were well balanced. The primary endpoint was survival at day +100 post-HSCT; observed rates equaled 38.2% in the defibrotide group and 25% in the controls (23% estimated difference; 95.1% confidence interval [CI], 5.2-40.8;P= .0109, using a propensity-adjusted analysis). Observed day +100 complete response (CR) rates equaled 25.5% for defibrotide and 12.5% for controls (19% difference using similar methodology; 95.1% CI, 3.5-34.6;P= .0160). Defibrotide was generally well tolerated with manageable toxicity. Related adverse events (AEs) included hemorrhage or hypotension; incidence of common hemorrhagic AEs (including pulmonary alveolar [11.8% and 15.6%] and gastrointestinal bleeding [7.8% and 9.4%]) was similar between the defibrotide and control groups, respectively. Defibrotide was associated with significant improvement in day +100 survival and CR rate. The historical-control methodology offers a novel, meaningful approach for phase 3 evaluation of orphan diseases associated with high mortality. This trial was registered at www.clinicaltrials.gov as #.

Inactivation of Germline Mutant APC Alleles by Attenuated Somatic Mutations: A Molecular Genetic Mechanism for Attenuated Familial Adenomatous Polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in familial adenomatous polyposis (FAP). Patients with FAP typically develop hundreds to thousands of benign colorectal tumors and early-onset colorectal cancer. A subset of germline APC mutations results in an attenuated FAP (AFAP) phenotype, in which patients develop fewer tumors and develop them at an older age. Although a genotype-phenotype correlation between the locations of APC germline mutations and the development of AFAP has been well documented, the mechanism for AFAP has not been well defined. We investigated the mechanism for AFAP in patients carrying a mutant APC allele (APC(AS9)) that has a mutation in the alternatively spliced region of exon 9. APC(AS9) was found to down-regulate beta-catenin-regulated transcription, the major tumor-suppressor function of APC, as did the wild-type APC. Mutation analysis showed that both APC(AS9) and the wild-type APC alleles were somatically mutated in most colorectal tumors from these patients. Functional analysis showed that 4666insA, a common somatic mutation in APC(AS9) in these tumors, did not inactivate the wild-type APC. Our results indicate that carriers of APC(AS9) develop fewer colorectal tumors than do typical patients with FAP because somatic inactivation of both APC alleles is necessary for colorectal tumorigenesis. However, these patients develop colorectal tumors more frequently than does the general population because APC(AS9) is inactivated by mutations that do not inactivate the wild-type APC.

Effect of calcium supplementation on rectal epithelial hyperproliferation in intestinal bypass subjects

BACKGROUND/AIMS Fatty acids and bile acids are tumor promoters of experimental colon cancer in rats. Calcium can inhibit their effects. After intestinal bypass (IB), fecal bile acid and lipid levels increase markedly. In rats, IB increases colonic cell proliferation and carcinogen-induced colon tumor incidence. Whether fecal bile acids and lipids influence rectal epithelial proliferation in humans is uncertain. This study compared rectal epithelial proliferation in IB subjects and in controls matched for age, sex, and body mass index and investigated the effects of calcium carbonate supplementation (2400 or 3600 mg Ca2+/day for 12 weeks) on proliferation indices in IB subjects. METHODS Epithelial proliferation was studied by in vitro incubation of rectal biopsy specimens with [3H] thymidine. Twenty-four-hour stool collections were assayed for bile acids, lipids, and calcium. RESULTS Whole crypt labeling index (LI) and upper crypt LI were increased in IB subjects compared with controls (P < 0.005). Calcium reduced whole crypt LI by 38% (P < 0.001) and upper crypt LI by 56% (P < 0.05). Levels of fecal bile acids (4.5 mmol/day) and lipids (131.9 g/day) were markedly elevated in IB subjects (P < 0.005). CONCLUSIONS IB induces rectal hyperproliferation and expansion of the proliferative zone in association with excessive output of fecal bile acids and lipids. Oral calcium reverses the proliferative changes.

Blood and gastric FOXP3+ T cells are not decreased in human gastric graft-versus-host disease.

Previous studies suggest regulatory T cells (Tregs) inhibit graft-versus-host disease (GVHD) in mouse and human hematopoietic cell transplant (HCT) recipients. As the gastrointestinal tract represents one of the most common and severe sites of GVHD-related tissue damage, we sought to determine whether a deficit in circulating or gastric mucosal Treg numbers correlates with the clinical onset of gastric GVHD. We used the marker FOXP3 to quantify Tregs in blood and in gastric antral biopsies in a cohort of 60 allogeneic HCT recipients undergoing endoscopy at a single center to evaluate symptoms suspicious for gastrointestinal GVHD. We show for the first time in the gastric mucosa and, contrary to existing reports, in the blood, that the percent of T cells expressing FOXP3 is at least as high in the presence as in the absence of GVHD involving the upper gut. There was no correlation of Treg frequency with the histologic or clinical severity of gastrointestinal GVHD. We conclude that Treg depletion is not a central feature in the pathogenesis of gastric GVHD in humans.