Bo-Yee Ngan

Pediatric renal cell carcinoma: clinical, pathologic, and molecular abnormalities associated with the members of the mit transcription factor family.

We describe the clinical features, outcome, pathology, cytogenetics, and molecular aspects of 13 pediatric papillary renal cell carcinomas during a 19-year period. Seven cases (54%) had translocations involving Xp11.2 (TFE3). They were identified by cytogenetic, molecular, and/or immunohistochemical analyses. All Xp11.2+ translocations were TFE3+ by immunostaining. Cytogenetic and/or polymerase chain reaction analyses identified 3 cases with t(X17) and 1 case with t(1;17), and all had additional translocations. Histologic features in common in TFE3+ tumors also were present in some TFE3- tumors. One TFE3- tumor had complex cytogenetic abnormalities, 55XY,+2,del(3)(p14),+7,+8,+12,+13,+16,+17,+20[11 ], and 2 cases had normal karyotypes. None had t(6;11)/TFEB+ immunostaining. Five cases had focal, weak MITF tumor immunostaining. The key clinical findings were as follows: (1) The presence of an Xp11.2 (TFE3) translocation frequently is associated with advanced stage at initial examination. (2) All patients who underwent complete, partial nephrectomy with clear margins (adequate only for stage 1) and resection of metastases were alive and relapse-free at last follow-up. (3) The mean +/- SD event-free survival and overall survival rates at 5 years were both 92% +/- 7.4%. (4) One patients with a TFE3+ and MITF+ tumor and 66-87,XXY,der(1)t(1;8)del(4)(q?) der(11)t(11;15)der17t(X;17 abnormalities died 9 months after diagnosis.

Identification of EpCAM as the Gene for Congenital Tufting Enteropathy

BACKGROUND & AIMS Congenital tufting enteropathy (CTE) is a rare autosomal recessive diarrheal disorder presenting in the neonatal period. CTE is characterized by intestinal epithelial cell dysplasia leading to severe malabsorption and significant morbidity and mortality. The pathogenesis and genetics of this disorder are not well understood. The objective of this study was to identify the gene responsible for CTE. METHODS A family with 2 children affected with CTE was identified. The affected children are double second cousins providing significant statistical power for linkage. Using Affymetrix 50K single nucleotide polymorphism (SNP) chips, genotyping was performed on only 2 patients and 1 unaffected sibling. Direct DNA sequencing of candidate genes, reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blotting were performed on specimens from patients and controls. RESULTS SNP homozygosity mapping identified a unique 6.5-Mbp haplotype of homozygous SNPs on chromosome 2p21 where approximately 40 genes are located. Direct sequencing of genes in this region revealed homozygous G>A substitution at the donor splice site of exon 4 in epithelial cell adhesion molecule (EpCAM) of affected patients. Reverse-transcription polymerase chain reaction of duodenal tissue demonstrated a novel alternative splice form with deletion of exon 4 in affected patients. Immunohistochemistry and Western blot of patient intestinal tissue revealed decreased expression of EpCAM. Direct sequencing of EpCAM from 2 additional unrelated patients revealed novel mutations in the gene. CONCLUSIONS Mutations in the gene for EpCAM are responsible for CTE. This information will be used to gain further insight into the molecular mechanisms of this disease.

Amelioration of the Effects of Citrobacter rodentium Infection in Mice by Pretreatment with Probiotics

BACKGROUND Citrobacter rodentium is a naturally occurring murine pathogen that causes colonic epithelial-cell hyperplasia, disrupts the colonic mucosa, and elicits a predominantly T helper 1 cellular immune response; it thereby serves as a model for the study of mechanisms of disease induced by human attaching-effacing pathogens. We sought to determine whether pretreatment of mice with a mixture of Lactobacillus rhamnosus and L. acidophilus probiotics would attenuate C. rodentium-induced colonic disease in mice. METHODS Mice were administered sterile drinking water, probiotics (10(9) cfu/mL) in sterile drinking water, maltodextrin in sterile drinking water, orogastric C. rodentium (10(7) cfu in 0.1 mL), or maltodextrin in sterile drinking water for 1 week before C. rodentium infection, or they were pretreated with probiotics (10(9) cfu/mL) for 1 week before challenge with C. rodentium. RESULTS Mice that received viable probiotics remained healthy. C. rodentium infection elicited mucosal inflammation, epithelial-cell hyperplasia, apoptosis in the colon, and interferon (IFN)- gamma production by splenocytes. Pretreatment with probiotics decreased levels of all but IFN- gamma production. CONCLUSIONS Pretreatment with probiotics attenuates the effects of C. rodentium infection in mice. Understanding the mechanism of these beneficial effects will aid in determining the efficacy of probiotics in preventing infection with related attaching-effacing enteric pathogens in humans.

Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn’s disease

Background: Patients with Crohn’s disease have defects in intestinal epithelial permeability that are inadequately explained by known inflammatory bowel disease (IBD) susceptibility genes. E-cadherin (CDH1) plays a vital role in maintaining the integrity of the intestinal barrier and its cellular localisation is disrupted in patients with Crohn’s disease. Aim: To determine if polymorphisms in the CDH1 gene are associated with Crohn’s disease and to determine the function associated with these polymorphisms. Methods: The hypothesis was tested using a candidate gene approach using 20 Tag SNPs derived from the HapMap and Crohn’s disease trios. Functional studies were carried out using HapMap cell lines and polarised epithelial cell lines (MDCK-1 and Caco2). Results: Here we show that CDH1 is associated with Crohn’s disease in 327 trios (rs10431923 excess transmission of “TT” genotype; p = 0.0020) and is replicated in the Wellcome Trust Case Control Consortium CD data set (TT risk allele; OR 1.2, p = 0.005). Patients with the Crohn’s disease risk haplotype (rs12597188, rs10431923 and rs9935563; GTC allelic frequency 21%; p = 0.000016) exhibited increased E-cadherin cytoplasmic accumulation in their intestinal epithelium which may be explained by the presence of a novel truncated form of E-cadherin. Accordingly, expression of this truncated E-cadherin in cultured polarised epithelial cells resulted in abnormal intracellular accumulation and impaired plasma membrane localisation of both E-cadherin and β-catenin. Conclusion: The mis-localisation of E-cadherin and β-catenin may explain the increased permeability seen in some patients with Crohn’s disease. Thus, the polymorphisms identified in CDH1 are important for understanding the pathogenesis of Crohn’s disease and point to a defect in barrier defence.

Pediatric Heart Transplantation in Human Leukocyte Antigen–Sensitized Patients Evolving Management and Assessment of Intermediate-Term Outcomes in a High-Risk Population

Background— There is an elevated risk for poor outcomes after heart transplant (HTx) in patients sensitized to human leukocyte antigens including graft dysfunction, acute cellular and antibody-mediated (AMR) rejection, and cardiac allograft vasculopathy. We report our experience with human leukocyte antigens–sensitized pediatric HTx recipients. Methods and Results— We identified pediatric HTx patients with elevated pre-HTx Panel Reactive Antibody (Class I/II; >10%), or a positive T- or B-cell crossmatch. Thirteen patients met criteria (5 female, 39%). The median age at HTx was 7 months (3.5 months to 15.5 years). Nine were infants who had prior palliation for congenital heart disease. Four were older patients (median 7.3 years; 4.8 to 15.5 years): 2 had congenital heart disease (Fontan), 2 were re-HTx. B-cell therapies were used in all patients, guided by assessment of CD19+ and CD20+ cells. Immunosuppression included thymoglobulin induction, and tacrolimus, mycophenolate mofetil, and steroids. Daily plasmapheresis ± intravenous immunoglobulin G was used if there was a positive crossmatch on day 1, with a gradual, biopsy-guided weaning schedule. Rituximab was used when AMR was detected on biopsy: more recently (n=3), used empirically perioperatively. AMR was confirmed in 9 patients within median 0.9 months post-HTx. Seven had early acute cellular rejection (≥ ISHLT Grade 2 R) with no hemodynamic compromise or graft dysfunction. There were 4 deaths post-HTx (range, 11 days to 9 months). The median follow-up of 9 survivors was 1.7 years (0.3 to 3.7 years). Of 7 patients >6 months post-HTx, no AMR or cardiac allograft vasculopathy was observed at a mean of 1.9+1.1 years post-HTx and no cardiac allograft vasculopathy. Conclusions— Despite aggressive management, acute cellular rejection and AMR occurred frequently early post-HTx. An algorithm of B cell–directed strategies can be effective in managing these patients with reasonable intermediate-term outcomes.

Single Nucleotide Polymorphisms That Increase Expression of the Guanosine Triphosphatase RAC1 Are Associated With Ulcerative Colitis

BACKGROUND & AIMS RAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis. METHODS We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium. RESULTS We observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (P(combined UC) = 3.3 × 10(-8), odds ratio = 1.43 [95% confidence interval: 1.26-1.63]) and rs4720672 (P(combined UC) = 4.7 × 10(-6), odds ratio = 1.36 [95% confidence interval: 1.19-1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium-induced colitis. CONCLUSIONS Human studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

Protein-Tyrosine Phosphatase Sigma Is Associated with Ulcerative Colitis

Inflammatory bowel disease (IBD), a relatively common chronic debilitating intestinal illness, is composed of two broadly defined groups, Crohns disease (CD) and ulcerative colitis (UC). Although several susceptibility genes for CD have been recently described, susceptibility genes exclusive for UC have not been forthcoming. Here, we show that receptor protein-tyrosine phosphatase sigma (PTPRS-encoding PTPsigma) knockout mice spontaneously develop mild colitis that becomes severe when challenged with two known inducers of colitis. We also demonstrate that E-cadherin and beta-catenin, two important adherens junction proteins involved in maintenance of barrier defense in the colon, act as colonic substrates for PTPsigma. Furthermore, we show that three SNPs (rs886936, rs17130, and rs8100586) that flank exon 8 in the human PTPRS gene are associated with UC. The presence of these SNPs is associated with novel splicing that removes the third immunoglobulin-like domain (exon 9) from the extracellular portion of PTPsigma, possibly altering dimerization or ligand recognition. We propose that polymorphisms in the human PTPRS gene lead to ulcerative colitis.

Pediatric aggressive fibromatosis of the head and neck: a 20-year retrospective review

UNLABELLED Aggressive fibromatosis in children is a rare, benign condition that is locally infiltrative and destructive. It often presents as a rapidly growing, painless lump in the head and neck region. To date, only small series and case reports have been reported, and the management of the condition remains unclear. Recently, nuclear beta-catenin expression has been suggested as a tumor-specific marker for aggressive fibromatosis (desmoid). AIM The aims of the study were to review our experience of the presentation, management, and treatment outcome of pediatric aggressive fibromatosis in the head and neck and to identify the presence of the desmoid tumor marker beta-catenin within this population. METHOD The study was conducted as a retrospective case review of children diagnosed with aggressive fibromatosis in the head and neck for a period of 20 years and a review of the literature. Pathologic review of the original tumor specimens was undertaken for evidence of positive tumor margins and presence of nuclear beta-catenin expression. RESULTS A total of 10 patients (6 males, 4 females) were identified. The age at presentation ranged from 12 months to 14 years. In total, 8 patients were treated with surgery alone. This included 7 patients with extension of the tumor to the resection margin; all had good long-term outcomes with no disease progression. Two patients received chemoradiotherapy, one as primary treatment, and the other as adjuvant treatment after gross incomplete resection. Both resulted in poor outcomes requiring further treatments. Within our series of pediatric fibromatosis, only 4 cases (40%) had positive results for any nuclear beta-catenin expression, and 6 (60%) of 10 patients had negative results for beta-catenin. CONCLUSION Our experience is that total gross resection and preservation of form and function is of higher priority than achieving a negative resection margin. Pediatric fibromatosis though aggressive is still a benign condition, and careful thought should be taken before considering adjuvant chemoradiotherapy. Nuclear beta-catenin expression should not be considered a specific tumor marker for pediatric aggressive fibromatosis of the head and neck. Pediatric aggressive fibromatosis in this region may be a distinct subtype of desmoid tumor from its adult form.

Post-transplant lymphoproliferative disorder in pediatric heart transplant recipients.

BACKGROUND Post-transplantation lymphoproliferative disorder (PTLD) is a major cause of morbidity and mortality after pediatric heart transplantation. METHODS Heart transplant recipients at The Hospital for Sick Children, Toronto, from 1990 to May 2008, were reviewed. Competing risk hazard analysis was used to model the natural history of the disease. Patients were matched for gender and duration of follow-up to identify potential covariates associated with increased risk of PTLD. RESULTS A total of 173 heart transplant recipients (42% <1 year old) were reviewed. Twenty-three developed PTLD at a median of 4 years post-transplantation. After transplantation, PTLD affected 9%, 15% and 28% at 3, 5 and 10 years, respectively. Freedom from death or PTLD recurrence was 72%, 58% and 50% at 1, 3 and 5 years, respectively, after PTLD diagnosis. Higher maximum Epstein-Barr viral (EBV) load (hazard ratio [HR]: 2.6, p = 0.004) and longer duration of induction therapy (HR: 1.7, p = 0.02) were associated with increased risks of PTLD. Higher cumulative cyclosporine doses over the first year post-transplantation were associated with increased risks of PTLD (HR: 1.2 per 1 mg/kg/day equivalent, p = 0.03), but higher tacrolimus doses were not (p = 0.38). Patients on cyclosporine at 6 months post-transplantation were at higher risk of PTLD than those on tacrolimus (HR: 5.2, p = 0.003). The use of anti-viral prophylaxis in patients with high EBV load may provide some protection (HR: 7.6 vs 15.4 with no anti-viral, p = 0.02). CONCLUSIONS PTLD is a major concern in pediatric heart transplant recipients and is associated with high morbidity/mortality. Exposure to EBV and higher intensity of immunosuppression seems to be associated with increased risk.

Epstein-Barr virus infection in transplant recipients: Summary of a workshop on surveillance, prevention and treatment.

Diseases caused by the Epstein-Barr virus are of great significance among organ transplant recipients. One of these diseases, post-transplant lymphoproliferative disease, is a major complication among organ transplant recipients. Management of this entity is problematic due to the difficulties with laboratory surveillance, diagnosis, prevention and treatment. A group of Canadian and American experts was assembled to discuss these aspects of Epstein-Barr virus diseases in Canadian organ transplant recipients. This report summarizes the relevant background literature and levels of evidence in relation to the outcomes of the deliberations and recommendations by the expert panel.