Pediatric allogeneic stem cell transplant (AlloSCT) patients are at substantial risk of developing kidney injury (KI), and KI contributes to transplant-related morbidity and mortality. We compared the estimated creatinine clearance (eCrCl) at 1, 3, 6, 9, and 12 months post-AlloSCT in 170 patients following reduced toxicity conditioning (RTC) versus myeloablative conditioning (MAC) to baseline. eCrCl was calculated using the Schwartz equation. Patients with ≥ 50% drop in eCrCl from the baseline were considered to have KI. Patients received tacrolimus and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis. The logistic regression model was used for assessing risk factors for KI. Seventy-six patients (median age = 10.6 years) received RTC AlloSCT; 94 patients (median age = 8.5 years) received MAC AlloSCT. The incidence of KI at 1 month post-AlloSCT was significantly higher in MAC versus RTC AlloSCT (43/94 [45.7%] versus 13/76 [17.1%] P < .0001). There was no statistical difference in KI at 3, 6, 9, and 12 months post-AlloSCT between the 2 conditioning groups. On multivariate analysis, only MAC was a significant risk factor for KI (odds radio [OR] 3.44, 95% confidence interval [CI] 1.59-7.42, P = .002). In multivariate analysis for risk factors affecting overall survival (OS), the following were statistically significant: MAC versus RTC (hazard ratio [HR] 2.66, P = .0008), average versus poor-risk disease status (HR 2.09, P = .004), matched sibling donor (MSD) and matched unrelated donor (MUD) versus umbilical cord blood (UCB) (HR 2.31, P = .013), no KI versus KI (HR 2.00, P = .005). In children, MAC is associated with significant risk of KI in the first month after transplant, and KI in the first month post-AlloSCT is associated with a significantly decreased OS.

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Epidermolysis bullosa simplex (EBS) is a heritable skin disorder characterized by skin fragility and blistering. While its most severe variant, dystrophic epidermolysis bullosa (DEB) is associated with squamous cell carcinoma (SCC), the development of extracutaneous neoplasms in EBS is extremely rare. We report a novel case of supratentorial primitive neuroectodermal tumor (sPNET) in a 7-year male with EBS. Experience of radiation therapy and its challenges in children with EBS has rarely been reported.

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4297 Introduction: Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D ligands, including MIC A/B (Crewenka et al, Science, 1998). Tumor cells expressing ligands for NKG2D can become susceptible to NK cell killing despite normal MHC class I expression (Lanier LL, Nat Med, 2001). However, tumor cells may shed MIC A/B and escape immuno-surveillance. HDACi increases the expression of NKG2D ligands MIC A/B. Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, is also an important regulatory enzyme in the expression of MIC A/B in response to romidepsin (RM) (Skov et al, Cancer Res, 2005). Objective: To determine the expression of MIC A/B in response to RM in leukemia and lymphoma cells (LL), its influence on NK cell mediated in vitro and in vivo cytotoxicity in NOD-SCID mice and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. Methods: LL cells (106/ml, RS 4:11 [MLL-ALL], Ramos [Burkitt's lymphoma]) were exposed to RM (10 ng/mL) (generously provided by Gloucester Pharmaceuticals) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B. Peripheral blood NK cells were isolated via magnetic separation followed by 12 hrs incubation with interleukin-2 (IL-2) [3000 IU/ml]. Cytotoxicity assays (europium assay) were performed at effector target (E:T) ratio of 5–10:1. RS4:11 and Ramos cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity. The mammalian expression construct (ffLucZeo-pcDNA [generously provided by Laurence Cooper, MD, PhD]) was transfected to RS4:11 and Ramos cells using lipofectin. The transfected cells were selected by zeocin to make stable transfection cells using lipofectin. Six week old NOD-SCID mice received 5×106 LL cells subcutaneously. Once LL engraftment was established in NOD-SCID mice, the xenografted animals were divided in various groups, 1) Control NOD-SCID mice were injected with PBS, 2) NOD-SCID mice with leukemia or lymphoma, 3) NOD-SCID mice with leukemia or lymphoma + NK cell therapy and 4) NOD-SCID mice with leukemia or lymphoma + RM + NK cell therapy. NOD-SCID xenografted mice in group 3 received weekly injections of purified IL-2 activated adult NK cells (5×106) for 6 weeks and mice in group 4 received weekly injections of RM (4.4mg/kg) followed by an infusion of IL-2 activated NK cells 24 hrs later. Xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size, bioluminescent imaging (Inoue et al, Exp. Hem, 2007) and survival. The survival distribution for each of the groups of mice was estimated using Kaplan-Meier estimates. The log-rank test was used to compare the survival distributions between treatment groups. Results: MIC A/B expression significantly increased in response to RM ([RS4:11 0.2% vs. 82%, p< 0.0001] and [Ramos 0.57% vs. 67%, p=0.0003]). Enhanced expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl RS 4:11 [RM vs RM+LiCl] 82% vs. 5%, p<0.0001; Ramos [RM vs. RM+LiCl] 67% vs. 35%, p<0.0001). In vitro cytotoxicity assays revealed significant increases against both RS 4:11 and Ramos cells at E:T ratio of 5–10:1 (p<0.01). The median survival time for NOD-SCID mice with RS4:11 was 24 days and RS4:11 + NK cell therapy was 34 days and RS4:11 +RM + NK cell therapy was: 46.5 days, respectively; Log-rank test p-value = 0.003. Median survival time for mice with Ramos was 16 days, Ramos + NK cells was 29 days and Ramos + RM + NK cell therapy was 32 days, respectively; Log-rank test p-value <0.001 Background: Expression of MIC A/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D- MIC A/B mediated cytotoxicity by NK cells. NK cells + RM leads to increase in survival of NOD-SCID mice xenograft with LL. Furthermore, up-regulation of MIC A/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. Disclosures: No relevant conflicts of interest to declare.

3027 Poster Board II-1003 Introduction Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B, which are known to be expressed on epithelial tumors, resulting in tumor cytotoxicity (Ayello/Cairo et al, BBMT, 2006). The expression of MIC A/B on these tumors can be induced by in vitro exposure of these cells to HDACi, specifically Romedepsin (RM) (Skov et al, Cancer Res, 2005). Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, has also been shown to be an important regulatory enzyme in the expression of MIC A/B in response to RM (Doble et al, J cell Sci 2003; Frame et al, Biochem J 2001; Skov et al, Cancer Res, 2005). Objective We sought to determine of the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. Methods LL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B antibody to determine surface expression of MIC A/B. Peripheral blood NK cells (CD3-/56+) were isolated via magnetic separation followed by IL-2 activation (3000IU/ml, 18 hrs). LL cells exposed to RM (generously supplied by Gloucester Pharmaceuticals) were subjected to NK cell mediated cytotoxicity assays (using an europium assay) at effector:target (E:T) ratio of 10:1, as we had previously described (Ayello/Cairo et al, BBMT 2006). LL cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity (Davies et al, Biochem J, 2000), to determine the role of this regulatory enzyme in the RM mediated expression of MIC A/B in these LL cells. Finally, blocking studies were also performed with anti-NKG2D receptor blockers to determine the specific role of NKG2D signal transduction pathway in NK cell mediated cytotoxicity. Results MIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 19.2%, p< 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 44.7%, p< 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 33.6%, p=0.0003]). In addition, the expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p<0.0001; RS 4:11 [RM vs RM+LiCl] 82% vs 5%, p<0.0001; Ramos [RM vs RM+LiCl] 67% vs 35%, p<0.0001). Cytotoxicity assays revealed significant increases in-vitro cytotoxicty in RS 4:11, Ramos and REH cells at E:T ratio of 10:1. Mean specific release (MSR)(±SEM) was measured for four conditions in the assay: NK cells (A) vs NK cells with 10 ng/ml RM (C) vs IL-2 activated NK cells (B) IL-2 activated NK cells +10 ng/ml of RM (D). MSR for RS 4:11 10%±3.6% vs 108%±47% and 12%±4% vs 145%±45% p<0.05(A vs C, B vs D) for RS 4;11. MSR for REH was 13.2%±14.4% vs 50%±17.6% and 22%±18% vs 132%±29% p<0.01(A vs C, B vs D). MSR for Ramos was 7.6%±7.6% vs 79%±15.6% and 35%±13% vs 90%±22% p<0.05(A vs C, B vs D). NKG2D receptor-blocking experiments resulted in significant decrease in NK cell mediated cytotoxicity in REH (p<0.03) and Ramos cells (p<0.001). Conclusion Our data suggests that the surface expression of MICA/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D-mediated cytotoxicity by NK cells. Furthermore, up-regulation of MICA/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. Disclosures No relevant conflicts of interest to declare.

process of daily bathing using wipes, documentation, patient/family education, and ordering of discharge bath supplies. Outpatient staff required instruction in the process for protocol consent, skin swab collection, review of a daily bath diary, and a patient satisfaction survey. Findings & Interpretation: Evaluation of the protocol's implementation process included monitoring of 12 study inpatients. Evaluation included 4 criteria: correct selection of bath wipe, maintaining blinding, correct process for bath administration, and proper documentation. Of the 12 patients, 7 met all 4 evaluation criteria. Of the 5 who did not, education was provided on site. Discussion & Implications: A critical component of research and maintaining internal validity of a study is ongoing monitoring to determine if the protocol is being implemented correctly. We plan to continue our evaluation on an ongoing basis and provide education as needed. We also plan to add the outpatient evaluation component.