Donor Lymphoid Chimerism Research Articles

Functional Immune Tolerance Induced By Sequential Hematopoietic Stem Cell-Solid Organ Transplantation

Solid organ transplantation (SOT) treats over 1500 children/year in the US. While short-term outcomes have improved, long-term outcomes still remain poor. With the rare exception of monozygotic twins as donors, recipients require lifelong immunosuppression with its accompanying toxicities, the risk of nonadherence, and chronic rejection. Further, up to half of recipients require a second transplant.We have pioneered translational approaches that abrogate rejection and the need for post-transplant immunosuppression. Previous attempts in adult patients utilizing allogeneic hematopoietic stem cell transplantation (HSCT) to eliminate the need for post-SOT immunosuppression in non-histocompatible kidney transplant (KT) recipients have failed(Lowsky R, BMT 2019). The establishment of mixed lymphoid chimerism has permitted a reduction in the number of immunosuppressive drugs needed, but not their discontinuation, whereas complete donor engraftment after HSCT has resulted in fatal graft-versus-host disease in some patients (Leventhal JR, Eur J Pediatr 2000). The use of HSCT to eliminate the need for immunosuppression following pediatric KT has not been evaluated. We have pioneered sequential haploidentical HSCT followed by KT in three patients with Schimke immuno-osseous dysplasia (SIOD) to abrogate kidney rejection and the need for post-transplant immunosuppression.SIOD is a monogenic disorder with progressive nephropathy correctable by KT and a T-cell immunodeficiency curable by HSCT. All three patients received αβT-cell/CD19 B-cell depleted HSCT (αβhaplo-HSCT) from a parent prior to a KT from the same parental donor. The pre-HSCT reduced intensity preparative regimen consisted of fludarabine (a starting dose of 1 mg/kg x 4 days, which was adjusted based on AUC), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m 2, anti-thymocyte globulin (ATG) thymoglobulin® 7.5 mg/kg, and rituximab 200 mg/m 2. Immunosuppressive drugs were not prophylactically administered after HSCT. After confirmation of full donor lymphoid chimerism post-HSCT, the patients received a living donor KT from their parental HSCT donor. Post-KT immunosuppression included intraoperative methylprednisolone and post-operative low-dose oral prednisone (0.5 mg/kg/day with taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce potential reperfusion inflammation. All immunosuppressive drugs were tapered off by Day +30 post-KT. To demonstrate functional tolerance after KT, Mixed Lymphocyte Cultures (MLC) were performed between peripheral blood mononuclear cells (PBMC) and patient/parent-derived irradiated EBV transformed lymphoblastoid cells lines (EBV-LCL) as stimulators (Figure 1). PBMC were isolated from SIOD patients (n=3) by Ficoll Hypaque separation and were labelled with Invitrogen™ CellTrace Violet Proliferation Kit (CTV). EBV-LCL were established from SIOD patients, parents and healthy donors. The assay was performed in round bottom microtiter plates with 100,000 labelled PBMC as responder cells (R) and 50,000 irradiated (30gy) EBV-LCL in RPMI medium with 10% fetal calf serum. Cells were harvested on Day 6, and T-cell proliferation determined by CTV expression of CD3+ T cells using a Novocyte Penteon flow cytometer (Agilent).Donor-derived T cells isolated from the peripheral blood of all three recipients > 1-year post-HSCT/KT, did not respond to the donor cells, while they did proliferate to the non-donor parental and control cells (Fig. 1). These results demonstrate that post-HSCT/KT the circulating donor-derived T cells are functionally tolerant to the transplanted kidney and, therefore, are unable to mediate graft rejection even in the absence of immune suppression. We have achieved two goals: first, ablating the recipient immune system before HSCT we safely established a new donor-derived immune system - that could also, if required, cure a patient's underlying disease- and, second, functional tolerance to the transplanted kidney.At 12 to 24 months after KT, all patients have full donor lymphoid and myeloid chimerism, normal renal function without immunosuppression, MLC demonstrated tolerance towards donor cells, and normal proliferative responses to non-donor parental cells. These findings suggest that the combination of αβhaplo-HSCT and SOT could be extended to other solid transplantation like liver and small intestine. [Display omitted] DisclosuresBertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees. Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Parkman: Jasper Biotech: Consultancy.

COMBINED BILATERAL LUNG TRANSPLANTATION AND BONE MARROW TRANSPLANTATION IN SELECT PATIENTS WITH END-STAGE LUNG DISEASE: A POTENTIAL FOR LONG-TERM ALLOGRAFT TOLERANCE INDUCTION

SESSION TITLE: Tuesday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Lung transplantation is the only life-saving therapy for select patients with end-stage lung disease. However, certain end-stage lung patients are ineligible for conventional lung transplant with underlying diseases such as primary immunodeficiencies (PID) or those with bone marrow failure (e.g., telomere-mediated diseases), as these patients are poor candidates for life-long immunosuppression. METHODS: We have established a single institution clinical trial protocol and program for salvage transplant, using combined bilateral lung transplant followed by bone marrow transplant (Lung/BMTxp) from the cadaveric lung donor. This adult/pediatric protocol employs partial MHC-matching in addition to ABO/size compatibility, a non-myeloablative condition regimen, and procurement of vertebral bodies for stem cell isolation followed by CD3+/CD19+ lymphocyte depletion. RESULTS: To date, our institution has performed five combined, tandem Lung/BMTxp procedures in patients with either PID (n=4) or telomere-mediated disease (n=1) and end-stage lung disease. All patients were consented to an Institutional Review Board study protocol and protocol approved by the FDA. Of these 5 Lung/BMTxp recipients, 3 patients engrafted BM, one patient transiently engrafted but subsequently rejected BM, and another failed to engraft BM. Notably, the first patient, a 14 y/o F with an underlying form of Severe Combined Immunodeficiency (SCID; IL-7R deficiency) underwent successful BMTxp four months after Lung Txp and has maintained high lymphocyte donor chimerism (>99%) after donor lymphocyte infusion. She has been successfully weaned off all immunosuppression for over two years now and has had no allograft rejection or infections. Lung allograft tolerance has persisted despite a fall in myeloid chimerism to a stable 20% level that has been maintained long-term. The fourth patient, a 20 y/o M with interstitial lung disease and myelodysplastic syndrome with monosomy 7 underwent Lung Txp and successfully engrafted donor BM four months later. He has maintained >99% donor chimerism for 10 months now and is in the final stages of weaning off low dose tacrolimus and prednisone, with stable lung function and no evidence of rejection. Currently, two additional patients with PID and end-stage lung disease (a 30 y/o M with Chronic Granulomatous Disease and a 20 y/o M with SCID) are listed for Lung/BMTxp. CONCLUSIONS: Together, our early single center experience supports combined Lung/BMTxp as an effective salvage transplant strategy for select patients with end-stage lung disease who are not candidates for conventional lung transplant. The establishment of high-level donor lymphoid chimerism provides the potential to wean Lung/BMTxp recipients off all immunosuppression and induce long-term lung allograft tolerance. CLINICAL IMPLICATIONS: Our study describes a novel salvage combined transplant approach with the potential for long-term tolerance. DISCLOSURES: No relevant relationships by Jessie Barnum, source=Web Response No relevant relationships by XIAOHUA CHEN, source=Web Response no disclosure on file for Jonathan D'Cunha; No relevant relationships by Stefanie Hannan, source=Web Response No relevant relationships by Ritchie Koshy, source=Web Response no disclosure on file for Geoffrey Kurland; No relevant relationships by John McDyer, source=Web Response no disclosure on file for Shawna McIntyre; No relevant relationships by Matthew Morrell, source=Web Response No relevant relationships by Joseph Pilewski, source=Web Response No relevant relationships by Iulia Popescu, source=Web Response No relevant relationships by Pablo Sanchez, source=Web Response No relevant relationships by Heather Stanczak, source=Web Response no disclosure on file for Paul Szabolcs

HSCT Engraftment Kinetics: Differences in Chimerism between Haploidentical (HI) and Matched Related (MR) Transplant Recipients (HSCT) after the 2 Step Approach

Cyclophosphamide (CY) toleration has been associated with low rates of graft versus host disease (GVHD) and non-relapse mortality after both HI and MR HSCT. At Jefferson, both recipient types undergo a 2 step approach to HSCT in which after myeloablative (MYA) or reduced intensity (RIC) conditioning, patients receive a fixed dose of 2 x 108/kg donor CD3+ T cells (DLI), followed 2 days later by CY daily x 2 for bidirectional tolerization. A CD34-selected stem cell product containing < 5 x 104/kg untolerized CD3 cells is infused 24 hours after CY. Mycophenolate mofetil and tacrolimus are started on day-1 for GVHD prophylaxis. The consistency of cell doses, conditioning, and GVHD prophylaxis in the approach provides an optimal platform for comparing patient outcomes. Because tolerance induction and sustained engraftment in CY-based HSCT approaches depends on elimination of donor and host alloreactive T cells, we hypothesized that engraftment in this model would be different between HI and MR recipients based on the lower proliferation of alloreactive T cells in major histocompatibility (MHC) MR versus HI donor-recipient pairs.Data for 292 consecutive patients (232 HI, 60 MR) treated on a 2-step trial were evaluated for incorporation into this analysis. To be included, patients were required to be in remission for 3 months post HSCT and have available d+28 and d+90 peripheral blood T cell chimerism data. Seven HI patients with graft rejection, 5/232 with early (2%-all MYA) and 2/232 with late (0.1%-1 MYA and 1 RIC), were not included in the analysis. No MR patients developed graft rejection. Days to ANC > 500 cells x 103/ul, CD3/4 and CD3/8 counts at d+28 and d+90, peak fever after DLI infusion as a rough measure of alloreactivity, and number of patients requiring post HSCT donor lymphocytes for mixed chimerism were also examined. Independent samples T test was used for chimerism and peak fever comparisons.The final study group contained 201 patients treated on a 2 step MYA [12 Gy total body irradiation (TBI)] or RIC (fludarabine/TBI based) regimen. Patients had various hematological malignancies and 6 had aplastic anemia. Data is contained in the table.Mean T cell chimerism was higher in HI versus MR recipients at d+28 and d+90 (p= 0.001 both time points). In 3 MR recipients, donor T cell chimerism was < 70% through d+90. Four (9%) MR versus no HI recipients received post HSCT DLI for mixed chimerism in the absence of recurrent disease. Chimerism differences did not affect time to myeloid engraftment or lymphoid reconstitution. While higher median peak temperatures after DLI were observed in HI versus MR recipients, some patients in the latter population were also highly febrile. Those MR recipients with higher fevers had a higher mean donor engraftment percentage at d+28 than those with lower fevers, but the difference was not significant (96.61% vs 93.95%, p=0.193).The data presented here supports the concept that MHC mismatching in HI HSCT is associated with a higher frequency of alloreactive T cells in the DLI which more efficiently eliminate residual host lymphocytes prior to CY administration resulting in more complete donor lymphoid chimerism. Interestingly, further support for this notion was provided by the MR recipient data where higher fevers, a surrogate for more robust immunologic activation, translated into the more rapid achievement of full donor chimerism. This finding was potentially due to higher alloreactivity between some MR recipient/donor pairs from non-MHC polymorphisms. Persistence of small numbers of host lymphocytes, which have also been rendered tolerant by CY administration, does not, in and of itself, lead to graft failure/rejection. However, concerns that low degree of chimerism might contribute to higher risk of relapse led to administration of post-transplant DLIs to push donor lymphoid chimerism closer to 100%. The impact if any, of delayed complete chimerism on GVHD and relapse requires further evaluation and may dictate changes to the 2 step regimen for the MR group. [Display omitted] DisclosuresPorcu:Innate Pharma: Consultancy.

Reduced-Intensity Conditioning with Fludarabine, Melphalan, and Total Body Irradiation for Allogeneic Hematopoietic Cell Transplantation: The Effect of Increasing Melphalan Dose on Underlying Disease and Toxicity

Disease relapse and toxicity are the shortcomings of reduced-intensity conditioning (RIC) for allogeneic hematopoietic cell transplantation (alloHCT). We hypothesized that adding total body irradiation (TBI) to and decreasing melphalan (Mel) from a base RIC regimen of fludarabine (Flu) and Mel would increase cytoreduction and improve disease control while decreasing toxicity. We performed a phase II trial of Flu 160 mg/m2, Mel 50 mg/m2, and TBI 400 cGy (FluMelTBI-50, n = 61), followed by a second phase II trial of Flu 160 mg/m2, Mel 75 mg/m2, and TBI 400cGy (FluMelTBI-75, n = 94) as RIC for alloHCT. Outcomes were compared with a contemporaneous cohort of 162 patients who received Flu 125 mg/m2 and Mel 140 mg/m2. Eligibility criteria were equivalent for all 3 regimens. All patients were ineligible for myeloablative/intensive conditioning. The median (range) follow-up for all patients was 51 (15 to 103) months. Day 100 donor lymphoid chimerism and transplant-related mortality, neutrophil and platelet engraftment, acute and chronic graft versus host disease incidence, overall survival (OS), and progression-free survival (PFS) were equivalent between FluMel, FluMelTBI-50, and FluMelTBI-75. Stomatitis wasdecreased for FluMelTBI versus FluMel (P < .01). PFS for patients not in complete remission on alloHCT was improved for FluMelTBI-75 versus FluMel (P = .03). On multivariate analysis, OS (P = .05) and PFS (P = .05) were significantly improved for FluMelTBI-75 versus FluMel. FluMelTBI-75 is better tolerated than FluMel, with improved survival and disease control.

Reduced Non Relapse Mortality (NRM) and Survivable Acute Graft Versus Host Disease (GVHD) in the Current Era of Myeloablative Ex Vivo T-Cell Depleted (TCD) Transplantation

Introduction:Little is known about the lethality of acute GVHD (aGVHD) in T -cell depleted (TCD) allogeneic hematopoietic stem cell transplantation (HSCT). We examined the incidence of aGVHD and its relative contribution to non relapse mortality (NRM) in a cohort of consecutive TCD HSCT at a single institute.Methods:We report 132 consecutive subjects who had undergone TCD HLA-identical sibling HSCT between 2006 and 2016 for hematologic malignancies. All subjects received conditioning with Fludarabine 125mg/m2, Cytoxan 120 mg/kg and 1200 cGy total body irradiation (TBI) (<age 55 years) or 400-600 cGy (³age 55) followed by either a CD34+ selected (n=112) or CD3/19 depleted (n=20) peripheral blood stem cell graft with a T cell addback of 5-50x10e4CD3+/kg (Miltenyi CliniMACS, resulting in 3.5 to 4-log TCD). Low dose cyclosporine was used until day 21 for GVHD prophylaxis.Thirty-six subjects were over the age of 55 years, 66 were females and 66 were males. Transplant indications were acute leukemia (92), myelodysplastic / myeloproliferative syndromes (26), lymphoid malignancies (7) and chronic myelogenous or myelomonocytic leukemia (7). Fifty-three percent of subjects were at a high risk of relapse. The median follow-up post HSCT was 4.3 years.Results:The incidence of Grade 1-2 and 3-4 aGVHD were 51.6% and 19%, respectively. Of these, 15% of aGVHD was steroid refractory and was treated with infliximab/basiliximab or with mesenchymal stromal cells. Extensive or limited chronic GvHD was observed in 30% and 24% of subjects. 74% of those at risk developed cytomegalovirus (CMV) reactivation. The Kaplan-Meier (KM) estimate for overall survival (OS), NRM and cumulative incidence of relapse (CIR) for the entire cohort was 71%, 14.6% and 24% respectively at one year, and 52%, 32.5% and 39% at 5 years. We considered the impact of CMV reactivation, slow donor lymphoid chimerism and steroid refractory aGVHD on NRM. However there was no significant impact from CMV reactivation or slow (> 31 days) achievement of complete donor lymphoid chimerism. Significantly improved outcomes were noted for those transplanted beyond 2012: OS, NRM and CIR being 82%,6.2% and 20% at one year, and 68%, 6.2% and 41% at 3 years. Cox proportionate hazard modeling identified steroid refractory aGVHD (HR 4.0, P=0.007) and transplant prior to 2012 (HR 6.7, P=0.001) as significant factors impacting NRM.Conclusions:T cell addback after ex vivo TCD HSCT was associated with a significant burden of aGVHD. Steroid refractory aGVHD impacted NRM, but slow lymphoid engraftment, disease risk, CMV reactivation and age did not. Significant improvements in NRM in the current era suggest greatly improved salvage of steroid refractory aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High-Resolution (HR) HLATyping Can Confirm the Diagnosis of Graft Versus Host Disease (GVHD)After Orthotopic Liver-Transplantation (OLT)

Acute graft versus host disease (GVHD) following orthotopic liver transplantation (OLT) is a rare but severe disease. GVHD diagnosis can often be delayed, since the symptoms are non-specific. We present a case of a 62 year-old male with end-stage liver disease who underwent OLT. There were no major peri-transplant complications. Patient presented ~4 weeks post-OLT, with fever,cough, throat discomfort, and rash; and was treated for suspected infection with broad-spectrum antimicrobials. ~5 weeks post-OLT, patient became pancytopenic, and a skin biopsy was suspicious for GVHD. Bone marrow biopsy was performed and revealed extreme pancytopenia with necrosis. The individual pre-OLT patient and donor types were compared at the serologic equivalent and high-resolution (HR) HLA-type, without clear indication of risk for GVHD, and or graft rejection. HR HLA-typing was performed on bone marrow cells obtained post-OLT and compared with the pre-OLT specimen to demonstrate donor lymphoid chimerism. HR typing results from the patient’s bone marrow post-OLT indicated the presence of all four alleles, and along with the clinical features,confirmed the diagnosis of GVHD. This case demonstrates howHR HLA typing can aid in rapid diagnosis and early recognition of GVHD post-OLT.

Early T-Lymphocyte Chimerism Kinetics Is Influenced By Conditioning Regimen in Reduced Intensity Allogeneic Stem Cell Transplantation

Introduction: Disease relapse remains one of the major causes of treatment failure in hematopoietic stem cell transplant (HSCT) following reduced-intensity conditioning (RIC) regimens. Donor chimerism assessment early after HSCT may help identify patients at risk for relapse who may benefit from additional therapeutic interventions but its precise role in this setting is unclear. In addition, the differential impact of conditioning regimen is not well studied.Aim:1. Study the impact of early full T-lymphocyte chimerism (T-lymphocyte chimerism ≥ 90% on day 30 to 60) on relapse incidence (RI) and acute graft versus host disease (aGVHD) in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) following RIC-HSCT.2. Looking at the tempo of achievement of complete donor T-lymphocyte chimerism based on the two most common RIC regimens, Fludarabine/Melphalan (FM) and Fludarabine/Busulfan (FB).Method:After IRB approval, patients with AML or MDS who underwent RIC-HSCT with FB or FM between 2008 and 2014 at our institution were identified. Post-transplantation CD3 (lymphocyte) and CD33 (myeloid) donor chimerism were measured per protocol using PCR-based analysis of short tandem repeats. Categorical and continuous variables were analyzed using Pearson's chi-squared and Wilcoxon / Kruskal-Wallis, respectively. Survival estimates were calculated using the Kaplan-Meier and log-rank test. RI was estimated accounting NRM on a competing event analysis using Grey's model. Cox proportional regression was used to compute hazard ratios (HR).Results:Baseline characteristics: Out of 134 patients who received FM or FB, 75 patients had evaluable early sorted chimerism data. 42 and 33 patients received FB and FM, respectively. Patient, disease and transplant variables were balanced in both FM and FB (Table 1). The median follow up of the cohort was 37 months (2.4-43) and at last follow up 29.1% of patients relapsed and 59% died. All patients achieved early complete myeloid chimerism whereas T-lymphocyte chimerism was complete in 50 patients and mixed in 25 patients. Characteristics of early lymphocyte chimerism by status are shown in table 1. Notably, likelihood of achieving complete T-lymphocyte chimerism ≥ 90% at 60 days was 100% versus 47% in FM and FB respectively (p <0.0001).Acute graft versus host disease: There was no significant difference in incidence of grade II-IV aGVHD between patients with full early donor T-lymphocyte chimerism in comparison to those with early mixed donor chimerism (46% vs. 48%, p =0.87).Impact of full chimerism and conditioning regimen on RI and overall survival (OS): Among the entire cohort, patients achieving early full T-lymphocyte chimerism had a significantly lower RI compared to those with mixed chimerism with a HR of 0.38 (CI 0.14-0.97; p=0.044) but OS was not significantly different with HR 0.69 (CI 0.33-1.51; p=0.34). In patients conditioned with FB, there was no difference in 2-year RI between complete or mixed early T-lymphocyte chimerism at 38.2% and 40.7% respectively (p = 0.9). After FM conditioning, all patients achieved early T-lymphocyte chimerism and the 2-year RI was 6.9%. Achievement of early complete lymphocyte chimerism post FB in comparison to FM was associated with significantly higher risk of relapse (HR 8.6 CI 2.07-57, p=0.0025). Furthermore, there was a trend towards worse OS with FB vs. FM with a median of 23 vs. 44 months, respectively (p = 0.058).Conclusion:Similar to prior reports, we observed that full T-lymphocyte chimerism is associated with decreased relapse risk without an increase in aGVHD. Our findings also suggest that RIC with FM yields a higher incidence of early complete T-lymphocyte chimerism and is significantly associated with decreased risk of relapse compared to FB. There was a greater risk of relapse in the FB group, even among those achieving early complete donor lymphoid chimerism. This needs further validation given the small sample size. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The key determinants of donor lymphoid chimerism support the diagnosis of acute graft versus host disease after liver transplant and predict the clinical outcome

Management of acute Graft versus Host Disease (aGvHD) following liver transplant (LT) is critical for reducing mortality from this life-threatening disease. The aim of this study was to examine how Donor Lymphoid Chimerism (DLC) determinants associate with the diagnosis and prediction of clinical outcome of aGvHD after LT. We retrospectively analyzed 17 LT patients (2007-2015) who were clinically suspected and DLC supportive of aGvHD. The DLC was tested by short tandem repeat with an initial 5% as positive cut off. The DLC determinants were: initial positive DLC after 30 days post-LT (InPD>30D); initial positive DLCs in whole blood, T, NK, and CD8+ cells; peak DLCs of >5% in whole blood, >50% in each of T, NK, CD8+ cells respectively; positive DLC in both CD8+ and NK cells at initial phase, DLC 50% in both CD8+ and NK cells at peak phase. Clinical outcome was determined over the following 40-day duration of treatment and/or aGvHD related motility (>40D DuT/M). Logistic regression was used to analyze >40D DuT/M in response to all the variables of DLC determinants. Of 17 aGvHD patients, the prevalence of 100% (16/16) in CD8+ cell was markedly higher than the prevalence of 41% (7/17) in whole blood, 59% (10/17), in T cell, and 38% (6/16) in NK cell at the initial detection of positive DLC. Amongst all analyzed variables of DLC determinants, InPD>30D and DLC 50% in both CD8+ and NK cells at peak phase were all significantly associated with >40D DuT/M. There was a mildly significant association with the individual variables of initial phase positive DLC in T and NK cells, and peak phase >50% DLC in T and NK cells. Regardless of initial phase or peak phase, the individual variables of DLC in whole blood and CD8+ cell had no significant relationship with >40D DuT/M (Figure 1). Initial positive DLC of CD8+ cell is a sensitive and reliable indicator supporting the diagnosis of aGvHD. Both delayed diagnosis and/or peak phase DLC over 50% in both CD8+ and NK cells strongly predicts aGvHD related negative impact following LT.Download : Download full-size image

Full Donor Myeloid Engraftment with Minimal Toxicity in Dyskeratosis Congenita Patients Undergoing Allogeneic Bone Marrow Transplantation without Radiation or Alkylating Agents

Dyskeratosis congenita (DC) is an inherited bone marrow failure (BMF) syndrome caused by defects in telomere biology genes. BMF occurs in approximately 80% of patients by 30 years of age and, in addition to pulmonary failure and malignancies, remains a major contributor to mortality. Hematopoietic cell transplantation (HCT) cures BMF in DC patients but is associated with a high incidence of graft failure and transplant-related mortality. Radiation and/or alkylating agents (e.g. cyclophosphamide, busulfan, melphalan) used in conditioning may have a role in the poor outcomes in DC patients following HCT. Recently, reduced-intensity conditioning approaches have improved short-term outcomes, but concerns remain about the long-term effects of DNA damaging agents in these regimens, given the multisystem disease and lifelong predisposition to malignancy conferred by telomere dysfunction.Radiation and alkylating agents are mainstays of allogeneic HCT preparative regimens: their highly effective myeloablative and immunosuppressive properties provide a niche for donor hematopoietic progenitors and decrease graft rejection. Based on a rationale that presumes (1) niche availability in DC patients with BMF, and (2) an intrinsic replicative defect in DC hematopoietic cells, we hypothesized that engraftment would be feasible in DC patients using a regimen of immunosuppressive therapy alone.Here, we report that in a single-institution prospective study, 4 consecutive patients with DC have undergone successful HCT using a radiation- and alkylator-free preparative regimen. Patients received alemtuzumab and fludarabine, followed by bone marrow transplantation (BMT) from unrelated donors. Cyclosporine A and mycophenolate mofetil were used for graft versus host disease (GVHD) prophylaxis. The primary outcome measures were neutrophil engraftment at day +30 and overall survival at day +100. Secondary outcome measures included acute toxicities and rates of graft failure, infections, and GVHD.The first two patients were 18 month-old twins with TINF2 mutations and transfusion-dependent BMF who received a BMT from a fully HLA-matched unrelated donor. The third patient was an 18 year-old female with CTC1 mutations and transfusion-dependent BMF, who underwent a B-mismatched unrelated donor BMT. The fourth patient, a 22 year-old male with clinical DC and short telomeres, had significant pulmonary disease (diffusing capacity of lung for carbon monoxide 28% of predicted) and BMF, and underwent a fully HLA-matched unrelated donor BMT.All 4 patients engrafted neutrophils by day +30, and are alive and well with follow-up ranging from 6 months (1 patient) to 2 years (3 patients). Platelet transfusion-independence occured between day +4 to +18, and red cell transfusion-independence occured between day 0 to +90. No patients required transfusions thereafter. All patients showed full donor myeloid chimerism by day +60. 3 of 4 patients showed full donor lymphoid chimerism by year 2; the fourth patient has high and increasing mixed donor lymphoid chimerism (60%) at day +180. There were no significant, unexpected toxicities, bacterial or fungal bloodstream or tissue infections. All patients were monitored weekly by serum PCR for CMV, EBV, adenovirus and HHV6 until day +100; only one patient showed CMV reactivation which was controlled with pre-emptive ganciclovir therapy. There was no acute GVHD. Only one patient developed chronic GVHD with limited skin involvement controlled with topical steroids.These results provide promising early data for engraftment and a favorable short-term toxicity profile using a radiation- and alkylator-free conditioning regimen in HCT for BMF due to DC. This approach could spare DC patients the acceleration of non-hematologic complications and malignancies, which has been attributed to HCT, and improve long-term survival. The limited toxicity of the alemtuzumab/fludarabine regimen may also allow patients with severe disease-related co-morbidities such as pulmonary dysfunction to safely undergo HCT. To our knowledge, this is the first prospective HCT study to achieve full donor myeloid engraftment in a series of patients following conditioning without radiation or alkylating agents. DisclosuresOff Label Use: alemtuzumab - conditioning for bone marrow transplantation fludarabine - conditioning for bone marrow transplantation cyclosporine A - graft versus host disease prophylaxis in bone marrow transplantation mycophenolate mofetil - graft versus host disease prophylaxis in bone marrow transplantation.

Mixed Chimerism: Good News or Bad News?

Mixed Chimerism: Good News or Bad News?

49-P: MULTI-SUBSET DONOR LYMPHOID CHIMERISM PATTERNS DURING ACUTE GRAFT VERSUS HOST DISEASE AFTER LIVER TRANSPLANT

We monitored donor lymphoid chimerism (DLC) long term patterns by STR to investigate their clinical relevance to aGvHD. 81 out of 879 liver transplant patients (2005-2013) who clinically suspected as aGvHD were tested for DLC. Short tandem repeat (STR) assay for DLC was run on the peripheral blood, T, NK, and CD8 + T cells. Cases with positive DLC in any preparation (>5%) were monitored until resolution of DLC or patient expiration. Three characteristic patterns of subset DLC were observed. The first pattern (5 patients) is characterized by low level D% that is detected only in CD8 + T cell on initial positive test (Fig. 1). In the second pattern, four patients on the initial positive DLC, had relatively higher CD8 + T cell D% combined with positive T cell D% (Fig. 1). These two patterns were associated with effective aGvHD control with complete resolution of DLC within a mean of 21 days. In the third pattern, four patients with positive DLC in all CD8 + T cells, NK cells and T cell were associated with longer duration for DLC resolution (114 days) (Fig. 2) and failure of treatment (Fig. 2). Detection of DLC in both NK and CD8 + T cell preparations is associated with longer duration to DLC resolution and higher mortality rate. These results suggest that multi-subset DLC testing is a useful tool to support the diagnosis of aGvHD and predict the response to treatment.

Rapid complete donor lymphoid chimerism and graft-versus-leukemia effect are important in early control of chronic lymphocytic leukemia

Eradication of minimal residual disease (MRD) after allotransplantation in persons with chronic lymphocytic leukemia (CLL) is associated with lower rates of relapse. Rapid engraftment of donor lymphocyte elements can contribute to MRD control, but it remains unclear whether this strategy will benefit patients. In this study, we report the incidence of MRD eradication and graft-versus-host disease (GvHD) in persons with rapid versus later donor T lymphocyte engraftment after lymphodepleting chemotherapy and reduced intensity conditioning (RIC) allotransplantation. Twenty-seven subjects received lymphodepleting chemotherapy to facilitate donor engraftment followed by fludarabine and cyclophosphamide RIC and a blood cell allograft. MRD was monitored by multicolor flow cytometry after transplantation. Complete donor T lymphoid chimerism (TLC) and myeloid chimerism (MC) were achieved in 25 subjects at a median of 28 days (range, 14-60 days) and 21 days (range, 14-180 days), respectively. Achieving complete donor TLC by day 14 versus day 28 or later correlated with occurrence of grade 2 or higher acute GvHD (90% [95% confidence interval {CI}, 78-100%] versus 35% [95% CI, 16-54%]; p = 0.014) and better control of MRD in the bone marrow at day 100, median 0% (range, 0-0.1%) versus 8.5% (range, 0-92%; p = 0.016). Among 11 persons with early donor TLC, none had progressive disease, and seven died of treatment -related mortality (TRM). In persons with later development of TLC, 8 of 16 had progressive disease and 2 died of TRM. Time to donor myeloid chimerism had no effect on outcomes. Rapid establishment of donor TLC resulted in more complete eradication of early MRD, but greater incidence of acute GvHD and TRM in persons with CLL undergoing RIC allotransplantation.

Acquired Hemophilia A in an African-American Male After Stem Cell Transplant for Sickle Cell Disease: Successful Treatment with Recombinant Porcine Factor VIII (OBI-1) and Tolerance Induction with Rituximab and Prednisone

AbstractAbstract 4631A 44 year old African-American asplenic male with sickle cell disease (HbSS) underwent allogeneic HSCT in September 2009. Upon engraftment, the patient had 97% donor myeloid, and 30% donor lymphoid chimerism, and he was free of his sickle cell disease with HbA 97%, HbA2 2.9%,and HbF <1%. Post-transplant he was maintained on sirolimus for GVHD prevention, Bactrim for PCP prophylaxis, and PCN-VK due to his asplenic state.Twenty eight months later, he experienced onset of minor soft tissue hemorrhage associated with a long aPTT, and low FVIII activity (14% of normal), two weeks after an upper respiratory infection. vWF levels were elevated (antigen 263% & activity 246%), and the FIX level (113%), PT (13.2 seconds), and fibrinogen (395 mg/dL) were normal. He had stable FVIII levels of 10–14% for two months, but then had acute onset of pain, swelling and decreased grip strength in the right hand, consistent with a compartment syndrome. He was treated with Humate-P at a dose of 3000 U every 12 hours, which initially normalized the aPTT and corrected the FVIII to 121%, permitting fasciotomy of the palmar and volar surfaces of the forearm to treat his acute compartment syndrome. After 4 days’ treatment on the same 3000 U Q 12 hour dose of Humate P, the recovered FVIII levels declined to only 18%. He was switched to Kogenate with no change in FVIII recovery, then rhFVIIa at doses of 90 μg/kg Q 4 hrs was started eleven days after surgery. Despite inhibitor bypassing therapy, the patient had recurrent pain, swelling, decreased grip strength, decreased range of motion, and numbness in the right hand, consistent with recurrent bleeding. His human FVIII inhibitor titer was 12 BIAU, but < 1 BIAU for porcine FVIII. Recombinant porcine FVIII (OBI-1) was obtained for use under an expanded access provision of IND 16395, and 200 units per kg were given 16 days after surgery, and a second dose of 100 units per kg was given 19 days after surgery. A FVIII level of 540 U/dL was obtained 20 minutes after the first treatment, and the volume of distribution was 2652 mL and the half life was 16.3 hrs. Within 8 hours the pain was diminished (4/10 vs. 7/10), and grip strength measured by dynamometer went from 8 lbs to 55 lbs (compared to unaffected left hand grip strength of 110 lbs). After the 2nd dose of OBI-1 a FVIII level of 621 U/dL was obtained 20 minutes later, and the volume of distribution was 2287 ml, and the half life was 14.7 hrs. Within two weeks the patient had improved range of motion, pain free status, and right hand grip strength of 82 lbs. The patient’s grip strength was restored to 101 lbs, 76 days later with physiatry and occupational therapy hand program.Immunosuppression with rituximab and high dose corticosteroids was begun at the onset of OBI-1 porcine factor VIII treatment; cyclophosphamide was not used to minimize risk for loss of the stem cell graft that cured his sickle cell disease. The inhibitor titer to human FVIII declined from 12 BIAU to less than 0.5 BIAU within three weeks, and there was no loss of donor erythropoiesis (HbA remained >96%). Concomitantly, the patient’s endogenous FVIII rose to >100% of normal by day 22, and remained normal after completion of three doses of rituximab and a complete taper of prednisone over the next two months.The patient had an H2 FVIII haplotype sequence, whereas his donor was heterozygous for the FVIII H1 and H2 haplotypes. A study of stored samples showed that at the onset of mild soft tissue hemorrhage the Bethesda inhibitor titer against a recombinant FVIII with H2 haplotype sequence (Recombinate) was higher (4.2 BIAU) than that against H1 (Kogenate) or “mixed” haplotypes derived from pooled plasma (1.9 BIAU). However, by the time of his compartment syndrome hemorrhage the differential reactivity of neutralizing titers to the H2 haplotype FVIII was not as pronounced (5.2 BIAU for H2 haplotype vs. 4–4.5 BIAU for all other haplotypes/products), suggesting that any initial specificity for the H2 FVIII haplotype had been diminished. No inhibition of OBI-1 porcine factor VIII was seen prior to treatment, or in the 90 day period after treatment. Interestingly, FVIII binding antibodies could be detected prior to the viral infection preceding the overt clinical inhibitor and soft tissue hemorrhage.In summary we demonstrate use of recombinant porcine FVIII in a patient with a compartment syndrome due to acquired hemophilia, and elimination of the inhibitor, while preserving the transplant that corrected his sickle cell disease. Disclosures:Lee:Inspiration Biopharmaceuticals Inc: Employment.

Dose-Adjusted EPOCH-Rituximab Combined With Fludarabine Provides an Effective Bridge to Reduced-Intensity Allogeneic Hematopoietic Stem-Cell Transplantation in Patients With Lymphoid Malignancies

There is currently no standard chemotherapy regimen for patients with lymphoid malignancies being considered for reduced-intensity conditioning allogeneic hematopoietic stem-cell transplantation (RIC-alloHSCT). The ideal regimen would provide disease control and result in lymphocyte depletion to facilitate engraftment. To this end, we developed a novel regimen by adding fludarabine to dose-adjusted continuous-infusion etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus with or without rituximab (DA-EPOCH-F/R). One hundred forty-seven patients with lymphoid malignancy (median age, 50 years) who had heavily pretreated (median prior regimens, three) and chemo-refractory (47%) disease were treated with DA-EPOCH-F/R before RIC-alloHSCT. Patients received one to three consecutive cycles until achieving lymphocyte depletion (CD4(+) count < 200/μL) or progressive disease. Overall response rate was 41%; 39% of patients had stable disease. Toxicity included grade 4 neutropenia in 65% and thrombocytopenia in 25% of patients. DA-EPOCH-F/R resulted in lymphocyte depletion (P < .001), which was inversely associated with serum interleukin (IL) 7 and IL-15 levels. Of 147 patients, 143 patients proceeded to RIC-alloHSCT. Patients with lower CD3(+) (P < .001), CD4(+) (P < .001), and CD8(+) (P < .001) T-cell counts after DA-EPOCH-F/R were more likely to achieve full donor lymphoid chimerism by day +14 after transplant. Relative to nonresponders to DA-EPOCH-F/R, patients with complete and partial response had increased event-free survival (77.4 v 4.8 months; P < .001) and overall survival (98.5 v 16.2 months; P < .001). DA-EPOCH-F/R safely provides tumor cytoreduction and lymphocyte depletion, thereby offering a bridge to RIC-alloHSCT in patients with aggressive lymphoid malignancies.

Acute Gvhd Is a Strong Predictor of Full Donor cd3+ t Cells Chimerism (TCC) After Reduced Intensity Conditioning (RIC) for Allogeneic Stem Cell Transplantation (Allo-SCT)

Abstract 4545The monitoring of chimerism is a standard procedure for assessing hematopoietic engraftment and achievement of full donor lymphoid chimerism after RIC based Allo-SCT. Post graft donor lymphocyte infusions are often decided on this evaluation. These studies have however a cost issue, all the more no consensus presently exists on when and how often to perform them.We retrospectively analysed the impact of acute GvHD in the prediction of allograft chimerism in our RIC program where TCC was serially assessed at 30, 60 and 90 days after Allo-SCT. We selected patients with hematologic malignancies (with the exclusion of myelofibrosis) transplanted between 2001 and 2010 after Fludarabine-Busulfan-ATG RIC from a HLA identical donor. 115 patients fulfilled all criteria including at least one T cells chimerism (TCC) determination between day 30 and 120. Allo-SCT was performed from familial donor in 92 patients (80%) and from MUD in 23 patients (20%). The conditioning regimen consisted of fludarabine (90 to 180 mg / m2), Busulfan (8 mg / kg orally or 6.4 mg / kg IV) and rabbit anti-thymocyte globulin (ATG) (2.5 or 5 mg /kg). As for chimerism study, recipient peripheral blood T lymphocytes were positively sorted by a mix of anti-CD4 and CD8 immunomagnetic beads (Dynal, Compiègne, France). T-cell purity was controlled by flow cytometry and was always > or =95%. Genomic DNA was amplified using fluorescent PCR primers for polymorphic variable number tandem repeats (VNTR) or short tandem repeats (STR). Mixed T-cell chimerism was defined as between 5 and 94% recipient cells, and full chimerism was defined as the presence of more than 95% donor cells.Full TCC was achieved in 94 patients (82%) at a median of 77 (30–120) days post transplant.Fifty eight patients (50.4%) developed acute GvHD. The cumulative incidence of Grade 2–4 GvHD in our population is 32% (95% CI 23–41).Overall the results showed that each of the 37 patients developing grade ≥ 2 AGVHD had a Full TCC prior day 120. On the other hand, all mixed chimerism were documented in patients not presenting Grade≥2 AGVHD (21 of the 78 patients (27%) without grade ≥ 2 AGVHD) (p=.002). No other parameter (ATG dose, Donor type…) achieved this level of individual prediction.These results, in a very homogenous population, are in line with the concept that full TCC is more likely to occur when patient develops significant aGVHD. Although they deserve further and deeper confirmation in different populations, they address the value of systematic routine chimerism surveillance (outside clinical studies) in patients presenting acute GvHD following RIC Allo-SCT. The modulation of TCC determination might represent an interesting cost and resources saving. Disclosures:No relevant conflicts of interest to declare.

EPOCH-FR: A novel salvage regimen for patients with lymphoid malignancies being considered for reduced-intensity allogeneic hematopoietic stem cell transplantation.

6536 Background: There is no standard chemotherapy regimen for patients (pts) with lymphoid malignancies being considered for reduced intensity allogeneic hematopoietic stem cell transplantation (RI-alloHSCT). The ideal regimen would result in disease control and recipient lymphocyte depletion and have limited toxicity. We developed a novel regimen consisting of continuous infusion etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus fludarabine ± rituximab, depending on CD20+ expression (EPOCH-FR). Methods: 147 pts, median age 50 yrs (range 21-71), with high-risk lymphoid malignancies (47% with chemo-resistant disease to last regimen, median 3 prior regimens [range 1-13]) were given EPOCH-FR (74% dose-adjusted) prior to RI-alloHSCT. Pts received 1 (48%), 2 (21%), or 3 (31%) cycles of EPOCH- FR until they were lymphodepleted (CD4+ count < 100/μl) or had progressive disease. Results: Overall response rate (RR) was 41% (16% CR, 25% PR); 39% had SD. RR of FCC/CLL>DLBCL/HL>TCL (p = 0.047). More EPOCH-FR cycles were associated with higher RR (17% vs. 33% vs. 84%) (p < 0.0001). 17% of pts with chemo-resistant disease had CR/PR. EPOCH- FR resulted in significant lymphodepletion (pretreatment median CD3+ = 574/μl, CD4+ = 244/μl, and CD8+ = 249/μl vs. posttreatment CD3+ = 184/μl, CD4+ = 97/μl and CD8+ = 68/μl (p < 0.0001). Pts with greater lymphodepletion of CD3+ (p=0.038) and CD4+ (p=0.017) were more likely to achieve CR/PR. Toxicity in 263 cycles included Grade 4 thrombocytopenia in 25%, neutropenia in 65%, and non-heme toxicity in 6%. Of 147 pts, 143 proceeded to RI-alloHSCT. Pts with greater lymphodepletion of CD3+ (p = 0.0007), CD4+ (p = 0.0004), and CD8+ (p = 0.0019) were more likely to achieve full donor lymphoid chimerism by Day +14 post-transplant. Following transplant, pts with CR/PR vs SD/PD to EPOCH-FR had median EFS = 77.4 vs. 4.8 mos (p < 0.0001) and OS = 98.5 vs. 16.2 mos (p = 0.0006) respectively. Conclusions: EPOCH-FR safely provides disease response and lymphodepletion in pts with lymphoid malignancies being considered for RI-alloHSCT. No significant financial relationships to disclose.

46-W: Donor lymphoid chimerism kinetics in a successfully treated case of acute graft versus host disease after orthotopic liver transplantation

46-W: Donor lymphoid chimerism kinetics in a successfully treated case of acute graft versus host disease after orthotopic liver transplantation

Double Negative T Cells Influence TCR VB Variablity to Induce Allotolerance.

Immune alloresponsiveness following allogeneic HSCT is influenced by the dynamics of immune reconstitution and development of allotolerance. In general, tolerance is induced by thymic clonal deletion (central) and apoptosis or suppression of alloresponsive lymphocytes by regulatory T cells in the periphery. We have recently demonstrated that the size of the TCR repertoire within the CD4 and CD8 compartments can be assessed using VB spectrum by flow cytometry, and expansions/losses of individual TCR VB families can be used as a surrogate marker of TCR variability. (Exp. Hem. 32: 1010–1022; Br. J. Haematol. 129:411–419). Additionally, regulatory T cells can also impact the clonal contractions and expansions within the TCR VB repertoire. Various types of regulatory T cells have been described including CD4+CD25+, CD8+, NK T−cells, and CD3+CD4/CD8− double negative T cells (DN Tregs). In our current study we investigated the role of DN Tregs on the restoration of immune repertoire diversity. We hypothesized that alloresponsiveness clinically detected as a manifestation of GvHD may be associated with oligoclonal T−cell expansions, and in this context decreased numbers of regulatory T cells suggest deficient tolerizing function by regulatory T cells including DN Tregs. Here we studied a cohort of 60 HSCT recipients (AML, CML, CLL, NHL, AA, and PV), of which 25 patients received matched unrelated donor grafts and 35 received matched sibling donor grafts. Blood was sampled between 2003–2006 at monthly intervals after HSCT, and flow cytometry for TCR repertoire in CD4 and CD8 cells as well as the numbers of DN cells were recorded. Additionally, separate samples were collected for measurement of chimerism and were included in analysis when donor lymphoid chimerism was > 60%. A subset analysis was performed based on the presence/absence of GvHD. For the 27/60 (45%) patients with episodes of GvHD, results were obtained at the time of diagnosis of GvHD (grade > 2), while for patients in whom notable GvHD was not captured, the steady−state values at corresponding times were used for analysis. For all patients serial evaluations were available. For the purpose of this study, significant VB expansions/contractions were defined as +/− 2 standard deviation over the average VB family size. Using Cox proportional hazards analysis to identify univariate risk factors for GVHD, CD8 VB TCR contractions > 14 VB families (58.3% contraction of entire CD4 VB repertoire) constituted a strong indicator for increased risk (HR=7.61, p=0.011). This observation is consistent with the fact that oligoclonality of alloreactive T cell clones is frequently accompanied by a significant contraction/loss of remaining VB families and may herald heightened alloresponsiveness as a manifestation of GvHD. Estimation for correlation by Pearson's correlation coefficient also demonstrated that percentage of DN cells strongly correlated with a normalization of CD4 VB TCR repertoire (lower number of expansions; N=57, R= −0.51, p=0.027), supporting our hypothesis that DN cells participate in peripheral tolerance and suppress proliferative, alloresponsive CD4 clones. In summary, our results further characterize TCR variability post HSCT and define the role of DN cells in the induction of allotolerance.

Early Recovery of Thymus-Derived Naïve T Cells in Pediatric Patients (pts) Treated with Non-Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation (NMSCT) for Cancer.

Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration.Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis.Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p<0.0001).Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers. [Display omitted] [Display omitted]

Definition of Cytochrome P450 1A2 as a Target Autoantigen in the Hepatitic Variant of Hepatic Chronic Graft-Versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) of the liver mimicking autoimmune hepatitis is an increasingly well recognized complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The target antigens and pathogenesis of this complication are poorly understood. We describe a case of hepatitic cGVHD in which cytochrome P450 1A2 (CYP1A2) was identified as a target antigen. A 47 year old male received an HLA matched sibling transplant for acute myeloid leukemia with normal cytogenetics following 3 cycles of idarubicin, cytarabine and etoposide remission induction therapy. Conditioning was with fludarabine 50mg/m2 and cyclophosphamide 120 mg/m2. Both donor and recipient were CMV IgG positive. Standard cyclosporin and short course methotrexate GVHD prophylaxis was used. Due to mixed lymphoid chimerism, cyclosporin was tapered from D94, and a donor leukocyte infusion (DLI) given at D124. One month following DLI, concomitant with attainment of 100% donor lymphoid chimerism, the patient developed florid oral changes of cGVHD and striking elevation of hepatic transaminases (ALT and AST peaked at 3604 (R 0–55 IU/L) amd 2241 (R 0–45 IU/L) respectively) with relatively minor elevation in hepatic ductal markers (GGT 327, R 0–60 IU/L). Liver biopsy was consistent with autoimmune hepatitis. Multiple recipient serum samples at this time demonstrated the presence of a high titre anti-liver microsomal (LM) antibody (titre 1:1280 by indirect immunofluorescence) reminiscent of hydralazine-induced hepatitis and immunofixed an antigen of approximately 51 kDa by immunoblotting using human liver homogenate. Neither pre-transplant recipient nor donor sera were reactive. Immunoblotting for a panel of 13 CYP450 antigens demonstrated that the antibody was specific for CYP1A2. This specificity was confirmed with blocking and inhibition experiments. Sequencing of the entire coding region of the CYP1A2 gene did not identify any coding variants present in the recipient but not the donor. Oral prednisolone resulted in brisk improvement in hepatitis and oral cGVHD, and was ceased at 12 months post transplant. The patient remains well with no further GVHD over 2 years post transplant. This is the first description of a target antigen in hepatitic cGVHD. The mechanism is unclear, but does not appear to be a CYP1A2 polymorphism acting as a minor histocompatibility antigen. Induction of hepatic autommune-like responses have been described in liver transplant recipients receiving calcineurin inhibitors, and it is possible that cyclosporin facilitated an autoimmune-type response in this case. Characterisation of autoantibody responses in additional cases of hepatitic cGVHD will be of great interest. [Display omitted]