frequency more in severe patients (51.2 +/- 39.2) when compared to mild or negative patients (3.08 +/- 2.8; see. Figure 1). Additionally, patients in both groups ...
Abstracts / Biol Blood Marrow Transplant 19 (2013) S313eS341
clones in peripheral blood could provide a way to risk stratify patients. We collected endoscopic GI tract biopsy samples and matched blood for fourteen patients undergoing myeloblative HCT with suspected GI GVHD within one day of corticosteroid therapy. We also collected peripheral blood approximately thirty days after biopsy. Seven of these patients were negative for GVHD or had mild steroid responsive GI GVHD and seven had severe steroid refractory GI GVHD. We extracted genomic DNA and performed TCR beta CDR3 repertoire sequencing at Stanford and with Gigagen GigaMune Rep-Seq, using the Illumina next generation sequencing MiSeq and HiSeq platforms. For each patient, between 1,000-5,000 unique T cell sequences were identified in the endoscopic tissue. We bracketed the most frequent clones in the GI tissue samples by rank order cut-offs and a floating measure of skewness and followed these clones in the blood. Both methods showed that, on average, the GI identified clones increased in frequency more in severe patients (51.2 +/- 39.2) when compared to mild or negative patients (3.08 +/- 2.8; see Figure 1). Additionally, patients in both groups who received more steroids (mg/kg/day) showed a correlated reduction in GI identified T cell expansion in the blood over time. These results support the use of T cell repertoire sequencing and associated approaches in human patients to both clarify the pathophysiology of GVHD and may provide an independent immune biomarker that could guide GVHD therapy.
Figure 1. GI identified TCR sequences become much more frequent in the blood in an analysis of patients with severe steroid refractory GI GVHD as compared to a patients with steroid responsive GVHD or without GVHD. (a) Graphical depiction of the most frequent TCR sequence identified in the colon and blood. Top panel shows the increasing frequency of the topped rank clone (red), in contrast to the lower panel which shows the decreasing frequency of the top ranked clone (green). (b) The mean fold change of TCR sequences identified in the GI tract of patients tracked in the blood at day 30 to day of diagnosis of 7 patients with severe GVHD and 7 patients with mild or no GVHD (Wilcoxin Test, P < .002). GI sequences were identified as the top 100 by rank order.
453 Identification of Y-Chromosomally Encoded Minor Histocompatibility Antigens Using a Reverse Immunology Approach Bo Kok Mortensen 1, Peter Brændstrup 1, Malene Erup Larsen 2, Mette Voldby Larsen 2, Ole Lund 2, Michael Rasmussen 3, Søren Buus 3, Anette Stryhn 3, Lars Vindeløv 1. 1 Allogeneic Hematopoietic Cell Transplantation Laboratory, Rigshospitalet, Copenhagen Ø, Denmark; 2 Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Lyngby, Denmark; 3 Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen N, Denmark Introduction: In allogeneic hematopoietic cell transplantation (HCT), minor histocompatibility antigens (mHags) are known to play an important role in generating immune responses leading to graft-versus-leukaemia (GVL) effects
S335
and graft-versus-host-disease (GVHD). mHags are results of polymorphisms in the recipients genome, which cause expression of peptides that can be recognised by donor Tcells. Y-chromosomally encoded proteins constitute a constant source of mHags relevant in allogeneic HCTs with female donor and male recipient due to the disparities between these and their homologue X-chromosomally encoded counterparts. Methods: A panel containing 8-11 mer peptides encompassing multiple putative and known mHags encoded by the Y-chromosome was designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were synthesized and used to screen for peptidespecific T-cell responses in peripheral blood mononuclear cells (PBMCs) obtained post-HCT from male recipients of female donor grafts. Following in vitro stimulation, PBMCs were analysed with an inteferon-g ELISpot assay. When a response was found, the T-cells were further analyzed with intracellular cytokine staining (ICS) and flow cytometry to determine whether it was a CD4- or a CD8-response. The optimal epitope and the HLA-restriction was determined by tetramer staining. Results: In one male recipient of a female donor graft a T-cell response was observed with ELISpot against the peptides YFYYNAFHWAI and RESEEESVSL. ICS and flow cytometry revealed that both were CD8 responses. Both peptides were earlier described mHags restricted by HLA-A*24:02 and HLAB60, respectively. Tetramer staining confirmed that the optimal epitopes were YYNAFHWAI and RESEEESVSL presented on HLA-A*24:02 and HLA-B*40:01 (a member of the previously designated HLA-B60 specificity), respectively. PBMCs obtained post HCT from five other male recipients of female donor grafts have been analysed for T-cell responses with ELISpot. Responses have been observed and further analysis is ongoing. Conclusion: Using a HLA-tetramer approach to identify the optimal epitopes of two known mHags encoded by the Ychromosome as well as the presenting HLA restriction elements at high resolution, we have demonstrated the feasibility of a reverse immunology approach in mHag discovery.
454 Decreased Pulmonary Function in Asymptomatic Long Term Survivors After Busulfan-Based Myeloablative Allogeneic Hematopoietic Stem Cell Transplant Annie Oh 1, Pritesh Patel 1, Santosh Saraf 1, Karen Sweiss 2, David Peace 1, John Quigley 1, Nadim Mahmud 1, Steven Dudek 3, Damiano Rondelli 1. 1 Section of Hematology/Oncology, University of Illinois Hospital & Health Sciences System, Chicago, IL; 2 Pharmacy, University of Illinois Hospital & Health Sciences System, Chicago, IL; 3 Section of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois Hospital & Health Sciences System, Chicago, IL Pulmonary function post allogeneic hematopoietic stem cell transplant (HSCT) can be impaired by previous exposure to chemotherapy, infection, or graft versus host disease (GVHD). In this retrospective study, we analyzed 21 patients with hematologic malignancies who are long term transplant survivors with a median follow-up of 48 months who had received a related (60%) or unrelated (40%) HSCT conditioned with a busulfan-based regimen. Of 21 patients, 7 are of Caucasian and 14 non-Caucasian ethnicity. All patients had routine pulmonary function tests (PFTs) repeated within two years from transplant and none had underlying lung disease. To eliminate inter-lab variability, all PFTs were
S336
Abstracts / Biol Blood Marrow Transplant 19 (2013) S313eS341
performed in the same laboratory. Survival rate at the time of analysis was 90%. We initially compared single parameters of PFTs including corrected diffusion limiting capacity of oxygen (DLCO), forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and total lung capacity (TLC) obtained before and after transplant. Post transplant, all PFT parameters showed some degree of reduction compared to pretransplant values. FEV1 and FEV1/FVC were significantly decreased (108 vs 92, P ¼ .04 and 86 vs 80%, P ¼ .04, respectively), with no difference between Caucasian and nonCaucasians. Overall, median values of corrected DLCO and TLC prior to and after transplant were only slightly reduced. However, in non-Caucasian patients a significant reduction of DLCO was observed (93%31 vs 73%11, P ¼ .05). We analyzed intra-patient changes in DLCO and FEV1 values and no correlation was found between these parameters and the development of chronic GVHD. This study shows an overall decrease in pulmonary function in patients without apparent lung disease following a myeloablative busulfan-based HSCT. However, a higher risk of a reduced DLCO was noted only among non-Caucasian patients. At this time it is not known if this observation is due to genetic, biologic or environmental causes.
455 Inonilomab for Children with Steroid-Refractory Acute Graft-Versus-Host Disease : A Nationale Multicenter Phase II Retrospective Analysis Marie Ouachee-Chardin Sr. 1, Benedicte Neven 2, Alain Fischer 3, Isabelle Hirsch 4, Karima Yakouben 5, Mony Fahd 5, Sandrine Leveille 1, Valérie Mialou 6, Yves Bertrand 7, Marie-Pierre Goutagny 6, Claire Galambrun 8, Gerard Michel 9, Geneviève Plat 10, Virginie Gandemer 11. 1 Pediatric ImmunoHematology, Robert Debré Hospital, PARIS, France; 2 Hopital Necker, Paris, France; 3 Unite d'Immunologie et d'Hematologie and INSERUM U 29, Hopital Necker, Paris, Cedex 15, France; 4 Hopital Necker, Paris, France; 5 Robert Debré hospital; 6 Hemato-pediatric, Lyon Hospital, LYON, France; 7 Service d'Hematologie Pediatrique, Lyon Hospital, Lyon, France; 8 Hematology, La Timone Hospital, MARSEILLE, France; 9 Hemato-pediatric, La Timone Hospital, MARSEILLE, France; 10 Hemato-pediatric, Children Hospital, TOULOUSE, France; 11 Hemato-pediatric, Rennes Hospital, RENNES, France One hundred twenty-three children from 6 French centers were evaluated retrospectively for the treatment of steroidrefractory acute graft-versus-host disease (aGVHD) with Inolimomab, a murine anti-IL2R.
Hematopoietic stem cell transplantations (HSCT) were performed between 1995 and 2012. Diseases were non malignant inherited diseases (63%) and hematological malignancies (37%). Median age was 4, 3 years. Donors were MRD (27.5%), MUD (32%), mismatch-MUD (13%), Haploidentical (20.5%) and Cord Blood (CB) (7%). The graft source was BM (80%), PBSC (12%) and CB (8%). 37% patients had a myeloablative conditioning. The median time to aGVHD was 13 days after HSCT. The baseline grade of aGVHD was I in 19%, II 45.5%, III 25% and IV 10.5%. The median delay between aGVHD and Inolimomab was 16 days. The median dose was 0.4mg/kg and the median treatment duration of 27 days. We observe 55% complete responses and 14% partial responses for a total response rate of 69% and with no side effect. Among the responders, 26% relapse. Incidence of infection during treatment was 19.5%. A logistic model on complete response provide evidence of the high predictive negative effect of the baseline grade [Odds Ratio (OR): 5.24]. No particular target organ was a significant predictive factor for treatment response or survival. However, multi-organ involvement predicted worse survival with 34%, 45% and 52% of death for patients with one, two, and three or more organs involved, respectively (P ¼ .234). The observed overall survival probability was 59%, with a median survival time of 463 days. The Overall unadjusted survivals were 87%, 77 %, and 61% at day 100, 200, and one year. Survival was also significantly improved for responders (Relative Risk: 0.413; P ¼ .002). As shown by multivariate Cox model, the survival was significantly lower for patients with non malignant inherited diseases [Hazard Ratio (HR) ¼2.88, P < .001] compared with hematological malignancies, and female patients were associated with higher mortality (HR¼1.72 , P¼ .049). For both response and survival, the success rates were significantly increasing with year of transplantation, and nonsignificant differences were found among centers. Chronic GVHD (cGVHD) occurred in 56% patients, extensive in 42%. cGVHD incidence was 78%, 72% , 42% for Non Responders, Partial Responders and Complete Responders (Anova, P¼ .017, difference only significant between Complete Responders and the others). Patients with hematological malignancies were observed with significantly less cGHVD (OR¼.29, P¼ .012). Fifty-one patients died, 17(33%) before day 100 and 34(67%) after day 100, mostly of infections (55%) and GVHD (51%). In conclusion, Inolimomab is well tolerated and effective for steroid-refractory aGVHD in pediatrics. Further randomized studies are requires to define the optimum regimen.
456 Pulmonary Symptoms Measured by the NIH Lung Score Predict Overall Survival and Non-Relapse Mortality in Chronic GVHD Jeanne Palmer 1, Xiaoyu Chai 2, Paul J. Martin 3, Brenda F. Kurland 4, Steven Z. Pavletic 5, Yoshihiro Inamoto 6, Mukta Arora 7, Corey Cutler 8, Daniel J. Weisdorf 9, Mary E.D. Flowers 10, Madan H. Jagasia 11, Sally Arai 12, Joseph Pidala 13, Stephanie J. Lee 14. 1 Department of Hematology and Oncology, Medical College of Wisconsin, Milwaukee, WI; 2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 3 Fred Hutchinson Cancer Research Center, Seattle, WA; 4 Clinical Research, Fred Hutchinson Cancer Research Center; 5 NCI Experimental Transplantation and Immunology Branch, National Institute of Health NIH, Bethesda, MD; 6 Division of Clinical Research, Fred
