ATG Use Research Articles

High-Resolution Donor-Recipient HLA Matching Does Not Influence Acute or Chronic Gvhd in Umbilical Cord Blood Transplantation

The degree of antigen-level (HLA-A, -B) and allele-level HLA-DRB1 matching has been correlated with the risk of acute GVHD (aGVHD) in prior studies of umbilical cord blood transplantation (UCBT), however no impact of HLA matching on development of chronic GVHD (cGVHD) has been reported for ≥4/6-matched UCBT. To understand the impact of high-resolution HLA allele matching at -A, -B, -C, -DRB1 and -DQ loci on GVHD we studied 404 recipients of single- (n=129) and double-unit (n=275, based on the predominating unit) UCBT from 2000 to 2010. Overall cumulative incidence of grade II-IV aGVHD, grade III-IV aGVHD, and cGVHD was 47%, 18%, and 20%, respectively. HLA allele-matched recipients, grouped as 9-10/10 (17%), 6-8/10 (61%), and <6/10 (22%), had a similar cumulative incidence of grade II-IV (43% vs. 47% vs. 52%, p=0.5) or grade III-IV (17% vs. 16% vs. 22%, p=0.4) aGVHD, respectively. In multivariable analysis HLA matching did not influence grade II-IV and III-IV aGVHD (all p>0.5) after adjustment for the number of units, CD3, CD34 dose, age, gender match, conditioning, recipient CMV status, ATG use, and the year of transplant (Table). Use of ATG was independently associated with a lower risk of grade II-IV aGVHD (relative risk [RR]=0.5, p<0.01). Univariate difference in cumulative incidence of cGVHD for 9-10/10 (17%), 6-8/10 (17%), and <6/10 (29%) HLA match was confounded by age (RR=3.4 for ≥18 years, p<0.01) and prior aGVHD (RR=2.3, p<0.01) in multivariable analysis adjusted for CD34, CD3 dose, and the number of cord units. No other factors influenced cGVHD or modified the risk of HLA matching on cGVHD. Similar results were found in a multivariable analysis for stratified single- and double-unit UCBT. We conclude that stringent high-resolution HLA matching at -A, -B, -C, -DRB1 and -DQ loci does not influence the incidence of acute or chronic GVHD among recipients of UCBT.Tabled 1Relative risk* of acute and chronic GVHD based on degree of high-resolution HLA matchingFactorRR of aGVHD (95% CI)RR of cGVHD (95% CI) (n=79)Grade II-IV (n=191)Grade III-IV (n=72)HLA match**9-10/10 (n=69)1.01.01.06-8/10 (n=246)1.1 (0.7-1.8)1.1 (0.5-2.5)0.7 (0.3-1.5)<6/10 (n=89)1.1 (0.7-2.0)1.4 (0.6-3.3)1.1 (0.1-2.5)*Adjusted for age, gender match, year of transplant, number of units, CD3, CD34 dose, ATG use, conditioning, recipient CMV status, prior aGVHD as appropriate; **High-resolution allele match at HLA-A, -B, -C, -DRB1, and -DQ loci; RR, relative risk; CI, confidence interval Open table in a new tab *Adjusted for age, gender match, year of transplant, number of units, CD3, CD34 dose, ATG use, conditioning, recipient CMV status, prior aGVHD as appropriate; **High-resolution allele match at HLA-A, -B, -C, -DRB1, and -DQ loci; RR, relative risk; CI, confidence interval

Ethnic Variation in Chronic Graft-Versus-Host Disease (cGVHD) Manifestations

s / Biol Blood Marrow Transplant 19 (2013) S313eS341 S321 while related vs. unrelated donor, complete HLA match, ATG use, and recipient age are not. Multivariable analysis was challenging given the correlation among covariates. However, male donor is significantly associated with improved OS for pts with grade 3-4 aGVHD in all models, while time pd is not significant when adjusting for other factors. Conclusions: While OS for pts with grade 3-4 aGVHD has improved, it continues to impact >13% of pts and has poor outcomes. The use of a male donor may predict improved OS for pts with grade 3-4 aGVHD independent of time pd. Additional study is warranted to validate these findings and improve therapies.

Outcomes After Double Cord Blood Transplantation Compared to Single Cord Blood Transplantation in Adults with Acute Leukemia Given a Reduced Intensity Conditioning Regimen

Abstract Abstract 232 Unrelated cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cell transplantation (HSCT) for adults with acute leukemia lacking a HLA matched donor. Double cord blood unit (dUCBT) has been increasingly used over single CB unit (sUCBT) after reduced intensity conditioning regimen (RIC). Since there is an increased relapse incidence (RI) using RIC-HSCT compared to myeloablative conditioning regimen, we have driven the hypothesis that RI may be decreased and leukemia-free survival (LFS) rate increased after dUCBT compared to sUCBT, due to probably increased graft-versus leukemia (GVL) effect. With this aim, we analyzed 360 adults (>18 years) with ALL (n= 77) or AML (n=238) in CR1 (n=212) and in CR2 (n=148) transplanted with a sUCBT (n=131) or a dUCBT (n=229) after a RIC. Only patients transplanted with a single unit containing more than 2.5×107/kg total nucleated cells (TNC) were included. Patients were transplanted from 2005–2011 in EBMT centers. Comparing the two groups of patients receiving a sUCBT or a dUCBT in CR1, there were no statistical differences according to age, diagnosis (AML or ALL), weight, CMV serostatus, cytogenetics risk, number of HLA incompatibilities. However, dUCBT were performed more recently (2009 vs 2008), the time from CR1 to transplantation was longer (142 days vs 121 days), more frequently transplanted with CY+FLU+TBI2Gy (87% vs 68%), lower frequency of ATG use (21% vs 35%) and finally, dUCBT recipients received higher number of TNC collected (5×107/kg vs 3.9×107/kg) or infused (4×107/kg vs 3.1×107/kg). Median follow-up was 23 months in both groups. Cumulative incidence (CI) of 60 days neutrophil recovery was 82±3% after dUCBT and 76±2% after sUCBT (p=0.86) and frequency of full donor chimerism at day 100, was not statistically different between dUCBT (81%) and sUCBT (86%). At day 100, CI of acute GVHD (grade II-IV) was 35% in both groups, however there was a trend of increased incidence of grade III-IV after sUCBT (19%) compared to dUCBT (10%, p=0.06) but increased incidence of grade II aGVHD after dUCBT (28%) compared to 17% after sUCBT (p=0.05). CI of chronic GvHD at 2 years was 21±4% after dUCBT and it was 12±5% after sUCBT (p=0.15). At 2 years, CI of non relapse mortality (NRM) after dUCBT was 28±4% and it was 30±6% after sUCBT (p=0.87). However, CI of 2y relapse was 21±4% after dUCBT whereas it was 38±2% after sUCBT (p=0.03). In a multivariate analysis adjusting for the differences between the 2 groups, dUCBT was associated with lower incidence of relapse compared to sUCBT (HR=0.74, p=0.01). Therefore, there was an improved 2-y LFS after dUCBT (51±5%) compared to sUCBT (32±3%; p=0.03). This was confirmed in a multivariate analysis (HR=0.64, p=0.04). Concerning patients transplanted in CR2 (n=148), there were no statistically differences of outcomes after dUCBT (n=93) or sUCBT (n=55). At 2y, LFS was 40±6% after dUCBT and 48±3% after sUCBT (p=0.32). In a subgroup analysis of dUCBT (n=118) and sUCBT (n=51) recipients using the same conditioning regimen (CY+FLU+TBI2Gy), 2 y LFS were 54±5% and 33±7% respectively (p=0.05). Conclusion: In this retrospective comparative based registry analysis, in AL patients transplanted in CR1, neutrophil recovery, GVHD and NRM were not statistically different after RIC-dUCBT or RIC-sUCBT, however, dUCBT recipients had decreased relapse incidence and improved LFS. For AL patients transplanted in CR2, there was no benefit of using dUCBT when compared to sUCBT recipients. Disclosures: No relevant conflicts of interest to declare.

Low Dose Busulfan Is Associated with Bronchiolitis Obliterans Syndrome Following Allogeneic Hematopoietic Stem Cell Transplantation.

AbstractAbstract 3128 Rationale:Bronchiolitis obliterans syndrome (BOS) is the most common late non-infectious pulmonary complication following hematopoietic stem cell transplantation (HSCT). The prevalence is estimated to be between 5–10% after allogeneic HSCT with a significant increase in morbidity and mortality. We conducted a retrospective case control study to evaluate the associations between patients with BOS and those who did not develop airflow obstruction or symptoms. Methods:Between January 1st 2000 and June 30th 2010 all patients who underwent an allogeneic HSCT at the Dana-Farber Cancer Institute/Brigham and Women’s Hospital (n=1854) were screened for the development of BOS, using a modified NIH diagnostic criteria. BOS was defined as 1) new onset airflow obstruction, FEV1/FVC ratio ≤0.7 and FEV1 <80% predicted; 2) irreversible obstruction defined as no response to bronchodilator per ATS criteria; 3) if airflow obstruction was noted prior to HSCT, a ≥15% decline in FEV1 from baseline; 4) BO confirmed by pathology irrespective of meeting the spirometric definition of BOS. We matched the BOS cases, based on date of HSCT (±90 days), with 2 groups of control subjects: 1) subjects who developed chronic graft vs. host disease (cGVHD) without BOS and 2) subjects who developed neither cGVHD nor BOS. Comparisons between BOS subjects and controls were conducted using t-test or Fisher’s exact tests. We also performed a multivariate regression analysis predicting the development of BOS; all variables with a p <0.1 on univariate analysis were included in the model. All statistical analyses were performed using R. Results:We identified 89 subjects meeting our diagnostic criteria for BOS with a prevalence of 4.8%. The median time from transplantation to meeting criteria for BOS was 491 days (range: 48–2067). Univariate analysis demonstrated that BOS was significantly associated with previously reported risk factors including age, pre-transplantation PFT’s, high risk disease, source of peripheral blood stem cells, unrelated donor, history of respiratory infection prior to day 100, and busulfan based conditioning regimens. (all p<0.05, table 1). Upon further stratification of regimens, univariate analysis demonstrated that reduced intensity conditioning busulfan was associated with the development of BOS (p<0.001). After analyzing our univariate associations in a multivariate model, busulfan-based conditioning and unrelated donors were independent factors associated with BOS (p <0.001), while, ATG use conferred lower odds of BOS (p<0.001). Conclusion:Our findings extend those from prior reports of risk factors associated with BOS. Our results demonstrate that busulfan, even when used at lower doses in reduced intensity conditioning transplantation, confers an increased risk for BOS. Future studies in prospective cohorts will be important to confirm these findings. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pre-Transplant Rituximab Is Associated with a Greatly Reduced Risk of Epstein-Barr Virus Reactivation Following Allogeneic Stem Cell Transplantation

Abstract 460Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic stem cell transplantation (alloSCT). Monitoring of EBV genomes in blood using quantitative PCR (EBV qPCR) coupled with pre-emptive administration of Rituximab in response to high-level EBV reactivation has emerged as a strategy to reduce mortality from PTLD. However, the effect of pre-transplant Rituximab therapy on the risk of EBV reactivation and survival post-alloSCT is unknown.This retrospective study examined 193 consecutive adult patients undergoing T cell depleted or cord blood alloSCT at University Hospital Birmingham, UK (UHB) and Nottingham University Hospital, UK (NUH) between May 2009 and April 2011. Median age at transplant was 54 years (range 16–73 years). Conditioning was reduced intensity in 84% and myeloablative in 16%. Stem cell source was matched unrelated donor in 70%, sibling in 24% and cord blood in 6%. T cell depletion was with in vivo Alemtuzumab in 89% and ATG in 6%. Patients were monitored by EBV qPCR whole blood assay, performed every 1–2 weeks post-transplant. EBV reactivation was defined as a single positive EBV qPCR result, whilst high-level EBV reactivation was defined according to institutional thresholds; 30,000 and 10,000 EBV genomes/ml for UHB and NUH respectively. All patients with high-level reactivation were pre-emptively treated with Rituximab.Median follow-up was 23 months (interquartile range [IQR] 18–30 months), with EBV qPCR testing for a median of 8 months (IQR 4–13 months) post-transplant. The cumulative incidence of EBV reactivation, adjusting for the competing risk of death, was 41% at 2 years post-transplant. Amongst those reactivating, the median time to EBV qPCR positivity was 120 days (IQR 77–198 days). High-level EBV reactivation was observed in 34/193 (18%) patients, accompanied by PTLD in 10 patients (4 biopsy-proven and 6 probable cases). Of patients developing high-level EBV reactivation, in 30/34 (88%) the interval from first EBV qPCR positivity to high-level reactivation was less than 4 weeks, with 15/34 (44%) exhibiting high-level reactivation at first qPCR positivity.In univariate analysis, significant predictors for EBV reactivation were older age (hazard ratio [HR] 1.02 per year; p=0.04), male sex (HR 1.75; p=0.03) and T depletion with ATG (HR 4.8; p<0.0001). A primary diagnosis of non-Hodgkin lymphoma (NHL) carried a significantly reduced risk of EBV reactivation (HR 0.04; p=0.0018). Reduced intensity conditioning carried an increased risk of reactivation with borderline significance (HR 2.04; p=0.07). Donor type and HLA-mismatch were not significant risk factors. Acute graft-versus-host disease (GvHD) and cytomegalovirus (CMV) reactivation were also not significantly associated with EBV reactivation.Twenty-nine patients received Rituximab in the year preceding alloSCT, of whom 25 had NHL, 3 had chronic lymphocytic leukaemia (CLL) and 1 had acute lymphocytic leukaemia. Only one of these patients developed EBV reactivation by 12 months post-transplant - a patient with high-level reactivation associated with PTLD. Two other patients developed low-level EBV reactivation beyond 12 months. In univariate analysis, pre-transplant Rituximab was highly predictive for (lack of) EBV reactivation (HR 0.15, 95% confidence interval [CI] 0.05–0.48; p=0.0002; see figure). Applying a multivariate model including age, sex and ATG use, pre-transplant Rituximab remained highly predictive (HR 0.15, CI 0.05–0.47; p=0.0012). As expected, strong confounding between NHL and pre-transplant Rituximab made a model including both uninformative. There was no significant association between pre-transplant Rituximab and the risk of relapse, acute GvHD or CMV reactivation.Overall mortality was 50% at 2 years, with 4 deaths due to PTLD. There was no evidence of a link between EBV reactivation and survival (p=0.33). Pre-transplant Rituximab was associated with a significantly reduced risk of mortality (HR 0.49, CI 0.23–1.00; p=0.05) although the aforementioned confounding with NHL should be noted.We report the novel and clinically important finding that pre-transplant Rituximab is associated with a markedly reduced risk of EBV reactivation and a possible survival benefit after alloSCT. Our data make a strong case for prospectively evaluating the role of Rituximab in allograft conditioning. [Display omitted] Disclosures:Off Label Use: Rituximab for prevention of Epstein-Barr virus reactivation after allogeneic stem cell transplantation. Fox:Roche: Honoraria.

Outcomes After Double Cord Blood Transplantation Compared to Single Cord Blood Transplantation in Adults with Acute Leukemia Given a Reduced Intensity Conditioning Regimen

AbstractAbstract 232▪▪This icon denotes a clinically relevant abstractUnrelated cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cell transplantation (HSCT) for adults with acute leukemia lacking a HLA matched donor. Double cord blood unit (dUCBT) has been increasingly used over single CB unit (sUCBT) after reduced intensity conditioning regimen (RIC). Since there is an increased relapse incidence (RI) using RIC-HSCT compared to myeloablative conditioning regimen, we have driven the hypothesis that RI may be decreased and leukemia-free survival (LFS) rate increased after dUCBT compared to sUCBT, due to probably increased graft-versus leukemia (GVL) effect. With this aim, we analyzed 360 adults (>18 years) with ALL (n= 77) or AML (n=238) in CR1 (n=212) and in CR2 (n=148) transplanted with a sUCBT (n=131) or a dUCBT (n=229) after a RIC. Only patients transplanted with a single unit containing more than 2.5×107/kg total nucleated cells (TNC) were included. Patients were transplanted from 2005–2011 in EBMT centers. Comparing the two groups of patients receiving a sUCBT or a dUCBT in CR1, there were no statistical differences according to age, diagnosis (AML or ALL), weight, CMV serostatus, cytogenetics risk, number of HLA incompatibilities. However, dUCBT were performed more recently (2009 vs 2008), the time from CR1 to transplantation was longer (142 days vs 121 days), more frequently transplanted with CY+FLU+TBI2Gy (87% vs 68%), lower frequency of ATG use (21% vs 35%) and finally, dUCBT recipients received higher number of TNC collected (5×107/kg vs 3.9×107/kg) or infused (4×107/kg vs 3.1×107/kg). Median follow-up was 23 months in both groups.Cumulative incidence (CI) of 60 days neutrophil recovery was 82±3% after dUCBT and 76±2% after sUCBT (p=0.86) and frequency of full donor chimerism at day 100, was not statistically different between dUCBT (81%) and sUCBT (86%). At day 100, CI of acute GVHD (grade II-IV) was 35% in both groups, however there was a trend of increased incidence of grade III-IV after sUCBT (19%) compared to dUCBT (10%, p=0.06) but increased incidence of grade II aGVHD after dUCBT (28%) compared to 17% after sUCBT (p=0.05). CI of chronic GvHD at 2 years was 21±4% after dUCBT and it was 12±5% after sUCBT (p=0.15). At 2 years, CI of non relapse mortality (NRM) after dUCBT was 28±4% and it was 30±6% after sUCBT (p=0.87). However, CI of 2y relapse was 21±4% after dUCBT whereas it was 38±2% after sUCBT (p=0.03). In a multivariate analysis adjusting for the differences between the 2 groups, dUCBT was associated with lower incidence of relapse compared to sUCBT (HR=0.74, p=0.01). Therefore, there was an improved 2-y LFS after dUCBT (51±5%) compared to sUCBT (32±3%; p=0.03). This was confirmed in a multivariate analysis (HR=0.64, p=0.04).Concerning patients transplanted in CR2 (n=148), there were no statistically differences of outcomes after dUCBT (n=93) or sUCBT (n=55). At 2y, LFS was 40±6% after dUCBT and 48±3% after sUCBT (p=0.32). In a subgroup analysis of dUCBT (n=118) and sUCBT (n=51) recipients using the same conditioning regimen (CY+FLU+TBI2Gy), 2 y LFS were 54±5% and 33±7% respectively (p=0.05). Conclusion:In this retrospective comparative based registry analysis, in AL patients transplanted in CR1, neutrophil recovery, GVHD and NRM were not statistically different after RIC-dUCBT or RIC-sUCBT, however, dUCBT recipients had decreased relapse incidence and improved LFS. For AL patients transplanted in CR2, there was no benefit of using dUCBT when compared to sUCBT recipients. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease in Acute Myeloid Leukemia (AML) and Hematopoietic Stem Cell Transplantation (HSCT) - A Retrospective Study

Abstract 4469Minimal Residual Disease (MRD) is an important criterion to define risk of relapse in Acute Leukemia (AL). The most powerful methods for MRD characterization are PCR, and flow cytometry (FCM). Although PCR is more sensible that FCM, respectively 10−4 and 10−3 logs, FCM is more applicable than PCR, usually more than 80% versus less than 50% to PCR. In patients eligible for HSTC, the value of MRD is being studied and some results showed implications both in overall survival (OS) as in event free survival (EFS). The RCI seems to be influenced by the presence or absence of MRD, as well. Nowadays the most important factor that influences survival is the relapse. Then, the earlier relapse detection has a potential to improve the therapeutic results.We presented the results of MRD before and after HSTC and its prognostic significance. Other evaluated factors were: age, acute and chronic GVHD (aGVHD and cGVHD), cell source (bone marrow, peripheral blood, cord blood), ATG use, myeloablative conditioning or not, related (RD) and unrelated donor (UD), donor gender and disease status.Bone marrow of 112 AML patients were analyzed by FCM, since January/ 2008 until April / 2011. The evaluation was made in irregular period of time with variable number of samples by patients. They were from 1 to 61 years old. It was considered for analysis the patients that had MRD > 0,1% and < 5%. It was considered as negative result MRD < 0,1%. A dual laser FACSCalibur™ flow cytometer was used to data acquisition and analysis. Four color panels of monoclonal antibodies were chosen according the initial disease phenotype. Cell Quest Software was used for acquisition data and PAINT-A-GATE PRO and Infinicyt ™ for FMC analysis. It was done a live gate in reference markers and it was acquired a minimum of 500000 total events in each tube. Statistical analysis were performed by Kaplan Meier curves for OS e EFS and Cox regression.It was observed statistical significance in univariate analysis related to OS the following factors: RD (64%) × UR (42%) (p = 0,002), no ATG use (64%) × ATG use (44%) × (p = 0,003%), no aGVHD (63%) (p = 0,016) × aGVHD II to IV (50%), age > 40 years old (yo) (70%), 20 to 40 yo (59%) and < 20 yo (46%) (p = 0,048), MRD after HSCT <0,1% (77%) and > 0,1% (36%) (p=0,001) (fig 1). The other factors did not show statistical significance with a negative tendency to cord blood use (p = 0,079).To RCI, the significance was observed in: patient age < 21 yo (52%), 21 to 40 yo (30%) and > 40 yo (21%) (p=0,015); and MRD after TCTH > 0,1% (73%) × MRD < 0,1% (15%) (p = 0,000). Exerted statistical tendency: MRD < 0,1% before HSCT (p = 0,06), 1st remission (p = 0,07) and cGVHD (p = 0,08). The group of 44 patients that did not has MRD neither before or after HSCT showed OS of 85% in three years and 11% of RCI. The five morphologic relapses observed in this group suggest that a regular monitorization of MRD must be done.The only factor that kept statistical significance in multivariate analysis was MRD > 0,1% after TCTH in RCI (p = 0,001 HR = 9,93) and for OS (p = 0,001 HR = 3,77) (Tables 1 and 2).In conclusion, the presence of MDR > 0,1% was the only independent prognostic factor for survival and relapse in AML patients after HSCT. Disclosures:No relevant conflicts of interest to declare.Table 1:OS multivariate analysisTable 2:RCI multivariate analysisFigure 1: OS of patients after HSCT P = 0,000

ATG Use Is Associated with Frequent Occurrence of Positive Blood Cultures in Patients in the Early Phase After Hematopoietic Stem Cell Transplantation

Abstract 4564 Background:Infectious complications remain an important cause of morbidity and mortality in the early phase after hematopoietic stem cell transplantation (HSCT). Aim:The aim of this study was to assess the frequency of positive blood cultures and its potential correlation with different studied parameters in large patient population studied in the first 30 days after HSCT. Material and methods:431 patients at median age of 47 years (range 18–85) transplanted between 2009–2011 for hematological and non-hematological malignancies were included in our analysis. There were 242 males and 189 females. Results:The indications for autologous and allogeneic HSCT were following: AML – 105 (24%), NHL – 86 (20%), MM – 75 (17,5%), HL – 48 (11%), ALL – 40 (9%), MDS – 17 (4%), AA – 15 (3,5%), CML – 12 (2,8%), PNH – 11 (2,6%), connective tissue diseases – 5 (1,2%), CLL – 3 (0,7%) and other – 14 (3,2%). The following transplant procedures were performed: ABCT – 213 (49%), ABMT – 3 (0,7%), alloBCT – 56 (13%), alloBMT – 21 (5%), URDBCT – 87 (20%), URDBMT – 51 (12%). Pre-transplant ATG and anti-CD52 antibody were used in 142 (33%) and 5 (1.2%) patients, respectively. Amongst 431 transplanted patients, 495 blood cultures were collected; range 0–8 (median 1). Eighty seven blood samples were positive (17,6%). The following pathogens were detected: gram-positive bacteria in 48% (n=42), gram-negative bacteria in 38% (n=33), fungi in 1% (n=1) and both G(+) and G(−) bacteria in 13%(n=11). The gram-positive bacteria included: Staphylococcus epidermidis: 21 (50%), Micrococcus spp: 4 (9%), Enterococcus faecium: 3 (7%), Enterococcus faecalis: 3 (7%), Streptococcus haemolyticus: 3 (7%). The following gram-negative bacteria were found: Enterobacter cloacae: 10 (30%), Escherichia coli: 7 (21%), Pseudomonas aeruginosa: 5 (15%), Klebsiella pneumonia: 5 (15%). Candida albicans was detected only in one case. The use of ATG was associated with higher number of total blood draw and positive blood cultures. No significant correlation was found between the specific pathogen and the use of ATG. Male gender was associated with significantly higher number of blood sampling and with tendency to higher number of positive blood cultures. The type of conditioning regimen, the source of stem cell and the donor origin (auto vs sibling vs unrelated) did not influence the number of positive blood culture. There was tendency to higher number of blood intake, but not positive blood culture in patients transplanted in NR if compared to PR or CR. Conclusions:Positive blood cultures were positive in about 20% of patients after HSCT. Only pre-transplant ATG use was associated with the higher number of positive blood culture. Disclosures:No relevant conflicts of interest to declare.

Quantitative Monitoring of EBV Viral Load in 222 Hematopoietic Stem Cell Transplant Patients: Risk Analysis and Development of EBV-Associated Post-Transplant Lymphoproliferative Diseases (PTLD)

Abstract 3018Background and Purposes Epstein-Barr virus (EBV)-associated PTLD is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Independent risk factors include use of ATG, acute GVHD, CMV antigenemia, T-depleted graft, and unrelated donor in our previous study. Quantitative real-time polymerase chain reaction (Q-PCR) was developed for early detection and intervention of EBV reactivation to prevent PTLD-related mortality.Patients and Methods Between Apr 2004 and Oct 2010, EBV viral load in plasma was monitored by Q-PCR in 222 HSCT patients (total 2945 samples) in NTUH. EBV reactivation was defined as > 500 copies/mL in two consecutive assays or > 10-fold elevation than baseline level.Results EBV reactivation occurred in 50 (22%) patients. The cumulated incidence of EBV reactivation was 28% at 1-year and 32% at 2-year. Median time to EBV reactivation was 40 days (ranges, 26–406) after SCT and median peak EBV-viral load, 10888 copies/ml (ranges, 948–3×107). The risk of EBV reactivation was significantly higher in patients receiving ATG (52% vs. 13%, p< 0.001), use of TBI (58% vs. 26%, p<0.001), mismatched donor (69% vs. 27%, p<0.001), CMV reactivation (44% vs. 19%, p<0.001) in univariate analysis. In multivariate analysis, independent risk factors include: ATG use (HR 5.44, p<0.001), TBI (HR 3.52, p<0.001), and Fludara-based conditioning (HR 3.16, P=0.01). PTLD developed in 8 patients (5%) who didn’t monitor EBV viral load regularly, and two were dead.Conclusions Monitoring of EBV viral load is a sensitive and useful tool in the surveillance of EBV-reactivation. Frequent monitoring in high-risk patients is important to prevent occurrence of EBV-PTLD. Disclosures:No relevant conflicts of interest to declare.

Prevention of mucositis with KGF in patients undergoing allogeneic stem cell transplantation.

6543 Background: Prospective studies have shown that palifermin (keratinocyte growth factor, KGF) reduces the duration and severity of oral mucositis after auto hematopoietic stem cell transplant (HSCT). Methods: To test the hypothesis that KGF reduces mucositis following ablative allogeneic (allo)-HSCT, we conducted a retrospective study on 251 consecutive adult patients with hematologic malignancies who had a T cell depleted allo-HSCT from 2004-2009. 154 patients received KGF (TBI=77, chemo=77) and 97 patients did not receive KGF (TBI=44, chemo=53). The number of days of PCA and TPN use and length of hospital stay (LOS) were evaluated as clinical surrogates for mucositis severity. Outcomes were compared between KGF and non-KGF recipients using the Wilcoxon rank-sum test. Adjusting for age, ATG use and conditioning regimen, linear regression models evaluated the effect of KGF on each of these outcomes. Results: In the entire cohort, KGF was significantly associated in univariate and multivariate analysis with fewer days on PCA and TPN, and shorter LOS (Table). While the same significant association between KGF and outcomes was noted in recipients of TBI-based allo-HSCT, KGF did not affect mucositis severity in recipients of a chemo-based allo-HSCT. KGF did not affect overall survival, event-free survival, engraftment or acute graft-versus-host disease. Conclusions: Our data demonstrate that KGF decreases mucositis severity in recipients of TBI-based but not chemo-based allo-HSCT. We cannot exclude a benefit of KGF in other chemotherapy regimens. Prospective studies evaluating KGF’s impact on mucositis following allo-HSCT should preferentially focus on patients who receive TBI-based transplants. KGF P value Yes Median (range) No Median (range) Wilcoxon rank sum Adjusted test Entire cohort PCA days 2 (0-78) 7 (0-106) 0.002 0.023 TPN days 12 (0-53) 15 (0-63) 0.003 0.006 LOS 31 (11-89) 33 (15-128) 0.001 0.014 TBI regimens PCA days 5 (0-45) 11 (0-106) 0.011 0.033 TPN days 11 (0-51) 17 (0-32) <0.001 <0.001 LOS 31 (24-72) 37 (16-106) <0.001 0.001 Chemo regimen (busulfan/melphalan/fludarabine) PCA days 0 (0-78) 5 (0-34) 0.032 0.288 TPN days 12 (0-53) 13 (0-63) 0.573 0.490 LOS 31 (11-89) 32 (15-128) 0.696 0.459

Antithymocyte Globulin (ATG) for Graft-Versus-Host-Disease (GvHD) Prophylaxis In Patients Undergoing Allogeneic Hematopoietic Cell Transplantation (allo-HCT): a Systematic Review and Meta-Analysis.

AbstractAbstract 1513 Introduction:Despite advances in high resolution HLA typing, GvHD remains a major complication after allo-HCT and results in significant mortality and morbidity. Transplant related mortality (TRM) in patients with severe acute GvHD has been estimated to be in the range of 28 to 92%. The 3-year risk for TRM in patients with chronic GVHD ranges from 28 to 48% depending on the extent of GVHD (limited versus extensive). Effective GvHD prophylaxis is key to prevention of GvHD. ATG is one of several immunosuppressive agents widely used for GvHD prophylaxis following allo-HCT. The effect of ATG for the prevention of GVHD has been evaluated in several randomized controlled trials (RCTs). However, data on the efficacy of ATG for the prevention of GvHD has been conflicting across RCTs. Informed clinical decision-making requires synthesis of all research evidence. Therefore, we have performed a systematic review to assess effect of ATG for GvHD prophylaxis in patients undergoing allo-HCT for various hematologic diseases. Methods:All completed phase III RCTs comparing use of ATG versus control for the prevention of GVHD in patients undergoing allo-HCT were eligible for inclusion. Medline (PubMed), and Cochrane database of RCTs published through April 2010 were searched to identify potentially relevant studies. Abstracts from the annual proceedings of the American Society of Hematology (2008-2009), American Society for Clinical Oncology (2008-2009) were also searched. Data were extracted on benefits and harms associated with compared treatments (ATG versus “no ATG”). Outcomes analyzed included: (i) GvHD incidence (ii) overall survival and (iii) TRM. Data on the methodological quality of included RCTs were also extracted to assess the robustness of the findings. Any discrepancies in data extraction were resolved by consensus. Data were pooled under a random effects model. A formal statistical test for heterogeneity using the chi square and I2 test was performed. The meta-analysis was performed using Review Manager Software. Results:Initial search of Pubmed, Cochrane database of RCTs and ASH and ASCO meeting abstracts yielded 141 citations of which 7 RCTs met the pre-defined inclusion criteria. Altogether these RCTs enrolled a total of 733 patients. The pooled results showed a statistically non-significant benefit for overall survival (7 RCT, 733 patients) with use of ATG for the prevention of GVHD. The combined hazard ratio (HR) for the comparison of ATG versus no ATG was 0.91 (95%CI, 0.75 to 1.10; p=0.32). There was no heterogeneity among included studies. The results did not change when data was analyzed according to the source of ATG (horse versus rabbit). There was a statistically significant benefit associated with ATG use for the prevention of severe grade III-IV acute GVHD (5 RCTs, 609 patients). The risk ratio (RR) was 0.56 (95%CI, 0.38 to 0.82; p=0.003). There was a statistically significant heterogeneity among included trials. However, there was no benefit with use of ATG for the prevention of either grade II (3 RCTs, 238patients; RR=0.78; 95%CI, 0.47 to 1.29; p=0.33) or grade I acute GVHD (2 RCTs, 185 patients; RR=1.42; 95%CI, 0.75 to 2.69; p=0.28). Use of ATG was not associated with any significant effect on TRM. The risk ratio was 0.74 (95%CI, 0.53 to 1.03; p=0.08). The results were not affected by methodological quality of studies. Conclusion:This comprehensive assessment of the effect of ATG in preventing GvHD in patients undergoing allo-HCT suggests that ATG has a beneficial effect on the prevention of severe grade III-IV acute GvHD. However, the available research evidence does not show a significant impact on overall survival. Additional prospective trials with adequate sample size are still needed to provide a more definitive answer. Disclosures:No relevant conflicts of interest to declare.

Surgical Complications in Early Period after Renal Transplantation

Surgical Complications in Early Period after Renal Transplantation Introduction. Growing number of elderly transplant recipients and expansion of the criteria for organ donation may increase the risk of post-transplant complications and impact outcomes. Aim of the Study. The aim of this study was to define the rate of surgical complications needing re-operations and their impact on posttransplant outcomes. Materials and methods. Study includes 202 consecutive deceased donor renal transplantations performed from 01.01.2004 till 31.12.2006. with further follow-up for 3 years. We analyzed the rate of re-operations, associated donor, recipient and transplantation factors and impact on post-transplant outcomes. Results. Reasons for re-operations were bleeding and hematoma formation (n=27), urological complications (n=18) and lymphocele (n=26). Hematomas were associated increased donor body mass index (BMI, p=0,067), presence of glomerular sclerosis at zero-time biopsy (p=0,034) and with development of urological complications (p=0,004) and delayed graft function (p=0,012). Urological complications were not associated with donor, recipient and transplant factors. Lymphocele were associated with donor factors (non-traumatic brain death, p=0,034, asystoly and hypotension, p=0,077, BMI, p=0,061, presence of glomerular sclerosis at zero-time biopsy, p=0,030), re-transplantations (p=0,092) and ATG use (p=0,094). Graft loss and patient survival during the follow-up period were not associated with mentioned surgical complications. Conclusions. Post-transplant surgical complications are associated with donor condition but without impact on three-year graft and patient survival.

Comparison of Outcomes for Non-Myeloablative (NMA) and Myeloablative (MA) Conditioning for Adults with Acute Lymphoblastic Leukaemia (ALL) in First and Second Complete Remission (CR): a Center for International Blood and Marrow Transplant Research (CIBMTR) Analyisis.

Abstract 872The efficacy of reduced intensity or NMA conditioning for allogeneic hematopoietic stem cell transplantation (HCT) for adults with ALL is uncertain. Using CIBMTR data we compared the outcomes of 92 patients ≥16 years who had NMA conditioning with 1421 patients who had myeloablative conditioning (MC) for allografts using sibling and unrelated donors for ALL in CR1 or CR2. Conditioning in the NMA group included regimens containing busulfan ≤ 9 mg/kg (27), melphalan ≤ 150 mg/m2 (23) or low-dose total body irradiation (36) and others (7). The NMA conditioning group were older (median 45 vs. 28 years, p<0.001) and more received peripheral blood grafts (73% vs. 43%, p<0.001). Other major potential prognostic factors were similar in the two groups. After a median follow-up of 54 vs. 38 months respectively, the NMA vs. MA conditioning groups had slightly less acute grade 2-4 graft-vs-host-disease (GVHD), less chronic GVHD but similar transplant-related mortality (TRM). However the NMA conditioning group experienced slightly more relapse (35% vs. 26%, p=0.08) yet similar overall survival (OS) (Figure):Outcome:MANMAP-valueAcute GVHD @ 100 days, grades (2-4)46 (43-49)39 (29-49)0.16Chronic GVHD @ 3 years42 (39-44)34 (24-44)0.16TRM @ 3 years, %33 (31-36)32 (23-43)0.86Relapse @ 3 years, %26 (23-38)35 (25-46)0.08Leukemia-free survival (LFS) @ 3 years, %41 (38-44)32 (22-43)0.12OS @ 3 years, %43 (40-46)38 (28-49)0.39Multivariate analysis showed that a low Karnofsky score (KPS) and T cell depletion were associated with higher TRM but conditioning intensity had no impact on TRM (RR with NMA 0.97, P=0.89). Relapse risk with NMA conditioning was slightly, but not significantly higher ( (RR)=1.34, p=0.15) as was a CR2, particularly with a short (<12 months) initial CR (RR=2.74; longer remission (12 months) RR1.51, P<0.0001). Multivariate analysis demonstrated significantly improved OS with: KPS>80, CR1, lower WBC, no extramedullary disease, a well matched unrelated or a sibling donor, transplant since 2001, in younger patients (<30y), conditioning without TBI and GVHD prophylaxis without T-cell depletion. However ATG use did not affect survival.. The most common cause of death was relapse; which was similar in MA and NMA HCT (46% vs. 35%). Despite the older age in the NMA group, OS and LFS at 3 years was similar to those receiving MA HCT. In comparing the outcomes of NMA and MA conditioning in sibling vs. unrelated donor transplant recipients we found that there was slightly, but not significantly more relapse with NMA [34 (18-52)% vs. 26 (23-30)%, p=NS and 36 (24-49)% vs. 25 (22-28)%, p=NS respectively]. This was associated with similar OS of 40 (23-59)% vs. 50 (45-54)% and 37 (25-50) vs. 38 (34-41)% in the sibling and unrelated donor groups. Conclusions:These data suggest that NMA conditioning is worthy of investigation in prospective clinical trials of adult ALL. These trials should include both well matched unrelated and related donors, but importantly, NMA conditioning may not fully overcome the adverse impact of poor pre-HCT KPS on outcome. [Display omitted] >Disclosures:No relevant conflicts of interest to declare.

Detection of Circulating Antigen-Specific CD8+ Lymphocytes against Leukemia Associated Antigens WT1, PR1 and Survivin Using HLA 0201 Pentamers After Allogeneic Stem Cell Transplantation.

Abstract 5115 INTRODUCTIONAllogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment option for patients with hematological disorders. Alloreactive donor-derived T lymphocytes exert a beneficial graft-versus-leukemia (GVL) effect through the recognition of leukemia-restricted (or preferentially expressed) antigens as Wilms tumor protein (WT1), survivin (SURV) or proteinase (PR1). Currently research in transplant immunology focuses in enhancing GVL while preventing the deleterious graft-versus-host disease (GVHD) that could be achieved by manipulating donor-derived antigen-specific T-populations. In this study we tested the presence of peripheral blood leukemia-associated antigen-specific CD8+ T-lymphocytes during post allo-SCT follow-up. PATIENTS AND METHODSForty-three consecutive HLA*0201 patients (homo or heterozygotous) undergoing conventional myeloablative (n=24) or non-myeloablative (n=19) allo-SCT as treatment of hematological disorders were included. Allogeneic donor was an HLA-identical sibling in 26 cases (60.5%) and unrelated in 17 cases (39.5%). Hematopoietic stem cell source included mobilized peripheral blood (n=20), bone marrow (n=18) and umbilical cord blood (n=5). As GVHD prophylaxis regimens Cyclosporine plus Methotrexate (n=20) or Cyclosporine plus Mofetil micofenolate (n=23) were employed. In addition, 22 patients received rabbit antithymocyte globulin at 6-8mg/kg. At last follow-up four patients had relapsed 9-14 months after allo-SCT. We sought for leukemia-antigen specific CD8 lymphocytes in peripheral blood samples drawn within a median of 7 months (range 2-38) when lymphocyte recovery had occurred and complete donor chimerism was achieved. We used four color multiparametric flow cytometry in a FACSCanto II acquiring at least 5 ×105 viable (Propidium Iodide low) lymphoid gated events, stained with MnAbs: CD8-FITC and CD3PE/APC MnAb. To identify leukemia-antigen specific CD8 lymphocytes we used class I HLA pentamers 0201 APC or PE conjugated (Proimmune, London, UK) against the following nonapeptides: Proteinase 1: VLQELNVTV (169-177) WT1: RMFPNAPYL (126-134) and SURV: ELTLGEFLKL (95-104). As positive staining control we used CMV pp65: NLVPMVATV (495-503) and as negative controls we used irrelevant nonapeptide and peripheral blood samples from patients lacking HLA* 0201 genotype. RESULTSDetection of donor-derived CD8+ lymphocytes against CMV pp65 occurred in 61% of recruited patients with a median percentage of 0.1% (range 0.03-13 over CD3+CD8+ events). Likewise, it was possible to detect CD8+ lymphocytes specific for PR1, WT1 and SURV in 65.2%, 47.8% y 39.1% of recruited patients respectively. Median percentage of PR1 and WT1 leukemia-antigen specific lymphocytes was 0.1% (range 0.04-1% over CD3+CD8+ events) and for WT1 0.1% (range: 0.01-0.2%). Detection of leukemia-antigen specific CD8+ lymphocytes was not significantly associated with clinical variables such as conditioning regimen (conventional or non-myeloablative), Disease status at transplant, donor type (sibling or unrelated), ATG use or HLA-disparity degree. The presence of WT1 specific CD8+ lymphocytes was significantly more frequent in patients undergoing allo-SCT for lymphoid hematological malignancy (p=0.04). By contrast, the presence of circulating anti-PR1 specific CD8+ lymphocytes was not more frequently found in patients undergoing allo-SCT for myeloid malignancies. Of note, none of the four patients who eventually relapsed harbored circulating leukemia-specific CD8+ lymphocytes. CONCLUSIONSMultiparametric flow cytometry is a useful tool to detect and quantify rare donor-derived CD8+ lymphocytes specific for leukemia-associated antigens as PR1, WT1 or SURV. The presence of these populations in peripheral blood is not associated to conventional clinical variables and in our series anti-WT1 CD8+ lymphocytes were more frequently detected in patients receiving allo-SCT for lymphoid malingnacies. By contrast, larger series are needed to assess if the lack of these leukemia-associated antigen-specific CD8 lymphocytes in peripheral blood could identify patients in a higher risk of relapse. Financial supportThis study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematología y Hemoterapia. DisclosuresNo relevant conflicts of interest to declare.

Adequate Immune Reconstitution in Recipients of Unrelated Donor Hematopoietic Cell Transplants Given Rabbit Anti-Thymocyte Globulin During Conditioning.

Abstract 2234Poster Board II-211Anti-thymocyte globulin (ATG) is known to reduce the risk of developing acute graft vs host disease following allogeneic hematopoietic cell transplant (HCT). Its effects on long-term immune reconstitution are less well defined, particularly in the adult population undergoing unrelated donor (URD) HCT. Since 2004, rabbit ATG (Thymoglobulin, Genzyme Inc, Cambridge, MA) has been used at our institution during the conditioning of patients undergoing URD HCT. We performed a retrospective landmark analysis to compare immune reconstitution in patients who received ATG during conditioning vs. those who did not. Patients had to have completed at least 6 months of follow-up post transplant. Eighty six patients were eligible, and underwent analysis of immune reconstitution in the first year post transplant. Fifty six patients underwent matched related donor HCT and did not receive ATG (no ATG cohort); 30 patients received an URD HCT (ATG cohort). The median age for no ATG cohort was 49 years and for the ATG cohort was 48. There were 40 females in the combined cohorts. The no ATG cohort included patients with the diagnosis of AML (34%), NHL (21%), MM (16%), ALL (9%), along with CML, CLL, MDS, MF and HD (20%). The ATG cohort was comprised of AML (43%), MDS (23%), ALL (13%), CML (13%), along with NHL & SAA (8%). Conditioning regimens used in the no ATG vs. ATG cohorts were 12-Gy TBI-Cy in 18% vs. 47%, Bu-Cy in 42% vs. 40%, and others in 40% vs. 13% (Flu-Mel, Bu-Flu, Flu-Cy, 2-Gy TBI,TBI-VP16). Stem cells were GCSF mobilized PBSC in 95% of the no ATG cohort and in 44% of the ATG cohort. The ATG dose administered was either 7.5 or 10 mg/kg in 3 divided doses, given from day -3 to day -1. With a median follow up of 727 days in the no ATG cohort and 480 days in the ATG cohort, 82% of the patients survived in the no ATG cohort compared to 73% in the ATG cohort (Fisher's exact test P=0.41). Absolute lymphocyte counts at 6, 9 and 12 months following transplantation were (mean ± SD) 1.2 ± 0.6×10 3 /μL vs. 1.0 ± 0.8 (T-Test, P=0.44), 1.5 ± 0.9 vs. 1.3 ± 1.0 (P=0.51) and 1.6 ± 0.9 vs. 1.3 ± 0.9 (P=0.23) respectively in the no ATG cohort vs. ATG cohort. Lymphocyte subset enumeration data was obtained during the first year following HCT at the time of cessation of immunosuppression and was available for 32 and 12 patients in the no ATG and ATG cohorts respectively. Absolute CD3+ cell counts measured at a median of 278 days were 1226 ± 773 vs. 981 ± 442 /μL in the no ATG vs. ATG cohorts (P=0.52). Simultaneously measured absolute CD3+/4+ cell counts were 483 ± 231 vs. 242 ± 122 (P=0.001), CD3+/8+ were 717 ± 627 vs. 701 ± 444 (P=0.94), CD19+ were 250 ± 239 vs. 351 ± 233 (P=0.25) and CD56+ were 181 ± 97 vs. 178 ± 67 (P=0.75) in the no ATG vs. ATG cohorts. Surveillance for EBV and CMV reactivation was performed using PCR. No statistically significant difference was noted in rate of CMV reactivation between the two cohorts in the 6-12 month post-transplant period indicating equivalent functional cellular immune reconstitution. EBV reactivation did not occur in either cohort. During the same time period the incidence of culture proven fungal infections and viral infections was equivalent between the two groups, however there was a significantly higher number of patients who experienced bacterial infection episodes in the ATG group. We are investigating the impact of ATG administration on the relative rate of relapse in these two cohorts. We conclude that ATG administered during conditioning did not adversely impact cellular immune reconstitution in this cohort of patients, even though these high-risk patients had undergone URD HCT with bone marrow as the stem cell source in the majority. This effect may be explained by a reduction in the incidence of acute GVHD secondary to ATG use, which in turn reduces the overall immunosuppressive exposure these patients experience following transplantation. T helper cell reconstitution appears to be delayed and may contribute to the higher number of patients experiencing bacterial infections in the ATG cohort. Disclosures:Off Label Use: Thymoglobulin for GVHD prophylaxis. McCarty:Celgene: Honoraria; Genzyme: Honoraria.

15 NF-κB can be activated by IKK-2 independent pathways in the liver

with pre, peri and postoperative variables, for survival and grade 4 fibrosis did not have donor age as a significant factor. Fibrosis was associated with maintenance steroids (p=0.004), ATG use (p=0.05), concomitant alcohol aetiology (p=0.01) and biochemical with histological hepatitis flare (p=0.005). Conclusions: In this large cohort, donor age did not influence late mortality in HCV transplanted cirrhotics, nor was it related to the development of severe fibrosis, which was however related to immunosuppressive regimens and concomitant alcoholic aetiology.

The effect of ATG on cytokine and cytotoxic T-lymphocyte gene expression in renal allograft recipients during the early post-transplant period.

Despite the long history of ATG use, the exact in vivo mechanism of action remains unclear. In the present study, we analyzed the effect of ATG-induction therapy on expression of 10 immunologically relevant genes in the early post-transplant period. Eight renal allograft recipients received post-transplant prophylactic ATG treatment on 10 consecutive days and an additional three patients received treatment on 5, 6, or 7 consecutive days, respectively. Gene expression was measured at the beginning and the end of therapy and normalized to a control gene using Taqman real-time PCR methodology. Results were compared with those of matched control patients. No patients were diagnosed with rejection. ATG-treated patients showed decreases in the expression of cytotoxic T cell genes perforin (-56%, p = 0.03) and granzyme B (-45%, p = 0.01) and cytokine gene IFN-gamma (-75%, p = 0.005), and significant increases in the expression of cytokine genes IL-7 (550%, p = 0.04), IL-10 (275%, p = 0.01), IL-15 (417%, p = 0.03), TNF-alpha (615%, p = 0.01), and TGF-beta (235%, p = 0.02). No significant changes were observed in the control group, with the exception of a decrease in IL-10 expression (-42%, p = 0.01). There were no significant changes in IL-12 or Fas-L expression in either group. ATG-induced decreases in the expression of IFN-gamma, perforin, and granzyme B and increases in IL-10 and TGF-beta might be considered beneficial to the recipient, whereas increases in the expression of IL-7, IL-15, and TNF-alpha genes might be involved in immunological processes not effected by ATG that may harm the transplant in the long term.