A 4 year old African American female presented with sun sensitivity related skin lesions and hepatomegaly with elevated liver enzymes after a trip to the family beach home. Diagnostic work up revealed elevated porphyrin levels consistent with erythropoietic protoporphyria with liver involvement. By 8 years of age, she had developed end-stage liver failure with jaundice, coagulopathy, hypoalbuminemia and portal hypertension. Liver transplantation alone was not a curative option for her, as her primary disorder would eventually cause a transplanted organ to fail. Due to her failing hepatic function we felt that she would not survive stem cell transplant alone. We decided to pursue liver transplantation followed by stem cell transplantation. Approximately 11 months after deceased donor liver transplantation, a double cord blood allogeneic transplant was performed. The patient was conditioned with busulfan (0.8 mg/kg/dose q 6 × 16 doses on days -9 to -6), cyclophosphamide 50 mg/kg q 24 hours × 4 doses on days -5 to -2), and equine ATG (15 mg/kg q 24 hours × 3 doses on days -3 to -1). GVH prophylaxis was provided with cyclosporine and mycophenolate. The two cord blood units had the following characteristics: both units were 4/6 matches. The first unit was an allelic mismatch at B and DRB1, while the second unit was a B antigen mismatch and DRB1 allelic mismatch. The second unit was the ultimately engrafting unit. The patient was CMV negative, the engrafting donor was CMV positive. Neutrophil engraftment occured on day +26; platelet engraftment on day +170. Transplant was complicated by several episodes of cholangitis and bacteremia. The patient had grade II acute skin GVH after her transplant, with grade III acute gut GVH, controlled with oral steroids, oral budesonide, mycophenolate, etanercept, and cyclosporine. 89 days post cord blood transplant, zinc protoporphyrin levels have fallen from pre transplantation levels of >800 mcg/dL to 139 micrograms/dL. 197 days post cord blood cell transplantation, the patient has normal hepatic synthetic function, total bilirubin of <0.1 mg/dL, no evidence of hepatic graft rejection, subjective improvement in overall skin coloration, cessation of porphyria related pain, and no current active GVH. This represents to our knowledge the first pediatric serial liver followed by double cord blood transplant for erythropoietic protoporphyria.

363 A life-threatening complication of transplantation is B cell post-transplant lymphoproliferative disorder (BPLD). Pediatric transplants are exquisitely sensitive to this spectrum of EBV-associated (pre)neoplastic disorders because of the higher susceptibility of the EBV-naive child to develop primary EBV infection in the early posttransplant period, a clinical setting associated with the highest risk of developing fulminant disease. The most likely source of EBV is the donor organ. Recently, the EBV load was shown to be clinically significant in the management of BPLD. Purpose-To determine the sensitivity of semi-quantitative dilution (SD-PCR) and competitive (SC-PCR) methods in determining EBV load in organ donor PBMCs. Methods-Sequential plasma specimens obtained from UNOS donors (n=88) were measured for EBV serology by ELISA (Gull Laboratories, USA) resulting in 97% positive for EBV IgG and EBNA 1, while all were negative for EBV IgM. WBCs from parallel specimens (n=51) were used for DNA isolation or further purified by CD19 coated magnetic beads (Miltenyi Biotech); the efficiency of this method was determined to be >95% by FACS analysis. DNAs were then analyzed by SD-PCR and/or SC-PCR using EBV type-specific sequences cloned into pCRII to determine EBV subtype and load. Results/Conclusion-EBV detection in donor PBMCs was enhanced by enriching the B cell population and by increasing the DNA amounts. Using T cell with Namalwa DNA (1-2 copies per genome) or cloned target DNA, we found that a 25-45 fold increase in the EBV target allowed detection. In the donor population, this was accomplished by B cell enrichment (range 6-9 fold) and by increasing the input amount of cells to approximately 6 × 105 B cells. This allowed detection in donor specimens that were ordinarily infected below PCR detection levels. Other viral analyses having a subset of cells as infective targets will benefit from these methods. While detecting EBV in the donor, the question remains whether either the threshold or the competitive method will discriminate between organs at risk for transmitting active infection.

544 Within the EBV infected/susceptible pediatric population, there are subpopulations with a greatly increased risk for cancer. These include immunosuppressed transplant and AIDS infected children, as well as those affected with inherited immune deficiencies. In several, sparse genetic information exists because of tissue related limitations. Hybridization for comparative genomic (CGH) analysis provides as one-step method for screening the genome of tumors for cancer related changes in the DNA. However, results from archival material have been inconsistent. We report the development of a method for the handling, microdissection and labeling of the isolated DNA prior to CGH analysis and the application of this method to EBV-associated cancers. Initially 5 paraffin blocks showing discrete but limited amounts of EBV-associated tumor embedded within normal tissue from pediatric patients[(PTLD, n=3) and (AIDS-associated leiomyoma, n=2)] were analyzed. The PTLDs included: a large cell lymphoma of null cell origin, a diffuse large cell lymphoma with an immunoblastic B-cell phenotype (kappa) and an EBV related hepatic lymphoproliferative disorder. Additionally, two AIDS-associated bronchial and splenic leiomyomas diagnosed were also microdissected. Because of the power of this amplification technique, blocks were handled under stringent conditions. DNA for CGH was extracted from the microdissected tissue (Puregene™, Gentra System Inc.), and subjected to universal PCR using our method that consist of a two enzyme system using slowly increasing temperatures and conditions defined to increase priming (Touch-up PCR). Our results defined 4 recurrent abnormalities in these five tumors including: gains on 1q, 8q and 19q, while a region of 1p was lost. Myc(activation previously defined cytogenetically in a proportion of PTLDs) andp73 (a preliminary characterized apoptotic suppressor gene) remain strong candidates for the gain on 8 and the loss on 1. By expanding this analysis, we will finely map these abnormalities and define candidate genes involved in EBV-associated tumorigenesis.