Allogeneic Bone Marrow Recipients Research Articles

Acrobat: A Multicenter Prospective Study to Monitor Chimerism in Post Allogeneic Hemopoietic Cell Transplant Patients with AML, ALL, or MDS for Prediction of Engraftment and Relapse

BACKGROUND: Allogeneic hematopoietic cell transplantation (HCT) remains the treatment of choice for leukemia patients, yet its success is limited at least in part by disease relapse, which can be as high as 50% or more at two years post-HCT. While engraftment post-HCT is routinely measured by semi-quantitative fluorescent PCR of short tandem repeats (STRs), its low sensitivity (1-5%) limits its usefulness as a routine relapse monitoring tool. We have developed an ultra-sensitive, accurate, and precise microchimerism monitoring solution, AlloHeme that can potentially improve allogeneic HCT patient surveillance for engraftment and relapse. NGS-based AlloHeme can accurately measure microchimerism (mC, defined as <1% recipient DNA) with a limit of detection down to 0.035%. We hypothesized that detection of microchimerism and precise measurement of increasing mixed chimerism (iMC) using a highly sensitive test can predict disease relapse in patients post allo-HCT. OBJECTIVES: To study the role of mC and iMC measured by AlloHeme on relapse detection in patients with acute leukemias and myelodysplastic syndrome, we designed a prospective multicenter clinical trial - ACROBAT (Assessment of Chimerism in Recipients of Allogeneic Bone Marrow / Hematopoietic Cell Transplantation using AlloHeme Test). METHODS: The ACROBAT study will enroll approximately 260 allo-HCT recipients with AML, ALL and MDS across about 10 centers in the US. All patients will undergo serial chimerism monitoring in the first two years post-HCT. All AlloHeme testing will be conducted at CareDx's CLIA-certified/CAP-accredited laboratory. Chimerism will be monitored in the whole blood as well as for its constituent CD3+, CD15+ and CD33+ cellular subtypes. Bone marrow (whole bone marrow and CD34+ sub-type) chimerism will also be evaluated at specific timepoints. The study will determine the predictive value of mC and iMC for relapse detection and the lead time to relapse. Univariable and multivariable sub-distributional hazard regression models and joint models of longitudinal and time-to-event data will be used to determine the impact of mC and iMC measured at different time points on relapse post-HCT. A time-dependent ROC curve will be used to investigate the predictive ability of AlloHeme, MRD, and chimerism by STR for relapse detection. CONCLUSIONS: The ACROBAT study is a multicenter prospective clinical study designed to evaluate the role of increasing mixed chimerism and microchimerism for early relapse detection in patients who have undergone allo-HCT for AML, ALL or MDS.

Reduced GvHD in Recipients of BM Derived Versus G-CSF Mobilized Plasmacytoid Dendritic Cells: Role of Inducible IL-12

Introduction: BMT CTN 0201, a randomized clinical trial, demonstrated that overall survival is equivalent between recipients of allogeneic bone marrow (BM) vs. granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood grafts. Increased graft failure was observed in recipients of BM grafts, while recipients of G-CSF mobilized peripheral blood (G-PB) grafts had more chronic graft-versus-host disease (GvHD) (Anasetti NEJM 2012). Donor plasmacytoid dendritic cell (pDC) content is associated with increased overall survival and decreased acute GvHD related mortality among recipients of BM grafts from unrelated donors, but not G-PB grafts (Waller JCO 2014). We have previously shown that in murine bone marrow transplant (BMT), donor pDC from the bone marrow reduce GvHD and improve survival while maintaining graft-versus-leukemia effect when added to grafts containing purified hematopoietic stem cells (HSC) and T cells (Lu BLOOD 2012). We hypothesized that the difference in chronic GvHD observed in BMT CTN 0201 is due to donor pDC content and function that vary according to graft source differences: BM vs. G-PB.Methods and Results: To assess the effects of pDC source on overall survival and GvHD, we used an established MHC mismatched model of murine BMT (C57BL/6 to B10.BR). To test whether the graft source of donor pDC was relevant to GvHD, we used flow cytometry to sort populations of HSC and T cells from untreated donor mice and pDC from either untreated BM or G-CSF mobilized splenocytes. We performed a BMT with 5,000 HSC, 1 million T cells, and either 50,000 BM or G-PB pDC from C57BL/6 mice into B10.BR recipients. Overall survival was significantly reduced among recipients of G-PB donor pDC compared with recipients of BM pDC, while late stage GvHD was higher in recipients of G-PB pDC (Fig. 1).To determine possible differences in function of pDC from each graft source: BM vs. G-PB, we assessed cytokine and enzymatic profiles by flow cytometry. Intracellular levels of IL-12 was significantly higher in CpG stimulate purified BM pDC compared to G-PB pDC co-cultured with purified T cells stimulated by anti-CD3/CD28 beads (Fig. 2). Additionally, Th1 polarization was higher in T cells sitmulated with anti-CD3/CD38 and cocultured with BM pDC, resulting in higher TNFa and IFNy production from these T cells. Levels of intracellular cytokines IFNa and IL-10 and levels of IDO showed no significant differences between pDC from Bm vs. G-PB.The transplant detailed above was repeated with BM pDC from wild-type (WT) mice or IL-12 KO mice. Overall survival of recipients of IL-12 KO BM pDC was significantly reduced when compared with recipients of WT BM pDC (Fig. 3).Conclusion: These data confirm that donor BM pDC are protective against lethal GvHD and indicate a loss of GvHD protection in donor pDC from G-PB grafts due to decreased inducible IL-12 synthesis. Because both graft sources are widely used clinically, mechanisms to recapitulate the immune-modulating properties of BM pDC in pDC of G-PB grafts are of great interest to the BMT field. [Display omitted] DisclosuresWaller:National Institutes of Health: Research Funding; Celldex: Consultancy; Cerus: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Katz Foundation: Research Funding; AMGEN: Consultancy; Chimerix: Equity Ownership; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; PRA: Consultancy; Coulter Foundation: Research Funding.

IMMUNOGLOBULIN THERAPY IN TRANSPLANTATION OF ORGANS AND TISSUES. REVIEW

Relevance. The expediency of prescribing immunotherapy for the transplantation of solid organs is still the subject of discussions among transplantologists.&#x0D; Objective. An analysis of key clinical trials devoted to the study of various aspects of the efficacy and safety of intravenous immunoglobulin therapy in allogeneic solid organ recipients with consideration of possible indications for the use of immunotherapy in transplant practice according to the current evidence base.&#x0D; Materials and methods. Retrospective analysis of the literature in depth in 20 years (1996-2016) in science-based databases PubMed, Scholar. Different approaches, different immunomodulating strategies (monotherapy, combination), different fields of application (transplantology, immunodeficiency states) were considered.&#x0D; Results. Currently accumulated evidence is determined the efficacy and safety i/v immunoglobulin therapy in monotherapy or in combination with other immunomodulatory strategies such as plasmapheresis and rituximab for the prevention and treatment of rejection reactions of allogenic kidney, where present donor-specific antibodies. It also shows that i/v immunotherapy compensates secondary humoral immunodeficiency in recipients of allogeneic heart, thereby sharply reducing the risk associated with immunosuppression severe infections and increases the survival of patients. Of interest are studies on the efficacy of specific immunoglobulins in a transplantology.&#x0D; Conclusions. Although in the case of transplanttion of solid organs, there is less evidence base of immunotherapy efficacy than in recipients of allogeneic bone marrow, through the now accumulated evidence that normal i/v human immunoglobulin can be used advantageously for the treatment and prevention of various infectious and immune-dependent complications in certain subgroups of patients with internal organs transplants or in nonstandard clinical cases as part of personalized medicine.

Phase I Clinical Trial Evaluating Abatacept in Patient with Steroid-Refractory Chronic Graft Versus Host Disease

Introduction: Chronic graft versus host disease (cGVHD) remains a major source of morbidity and mortality following allogeneic transplantation. While corticosteroids remain first line therapy for cGVHD, they are associated with significant toxicity, and a substantial proportion of patients fail to completely respond. Treatments for steroid-refractory cGVHD are limited. While the pathophysiology of chronic GVHD is complex, activated T cells play a critical role, driven by allo-antigen stimulation. As such, inhibition of T cell activation via blockade of co-stimulation has potential as a therapeutic target in cGVHD. Abatacept is a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 and a fragment of the Fc domain of human IgG1 that has been modified to prevent complement fixation and antibody-dependent cellular cytotoxicity. Abatacept is the first drug in a class of agents termed "selective co-stimulation modulators." The CTLA-4 moiety of Abatacept binds specifically to CD80 and CD86 and down-modulates the CD28-mediated co-stimulation of T cells. We conducted a phase I clinical trial was conducted to evaluate the safety, clinical and immune effects of Abatacept in patients with steroid-refractory cGVHD.Methods: The study followed a 3+3 design with two escalating doses of Abatacept to determine the maximum tolerated dose (MTD): 3 mg/kg and 10 mg/kg. Dose-limiting toxicities (DLTs) were defined as Grade 3 or 4 toxicities judged to be probably or definitely related to Abatacept. Infection was not considered a DLT. Abatacept was administered for a total of 6 doses. Doses 1-3 were administered at two-week intervals. One month following Dose 3, Abatacept was given at four-week intervals for three doses (Doses 4-6). Inclusion criteria included recipients of allogeneic bone marrow or stem cell transplantation with myeloablative or reduced intensity conditioning, with cGVHD defined by NIH consensus criteria. Patients must have had treatment with ≥ 0.5 mg/kg/day of prednisone for at least 4 weeks. Patients with active malignant disease relapse or other active malignancy and patients with uncontrolled infection were excluded. Peripheral blood was drawn prior to each dose of Abatacept and following completion of therapy to assess the effect of treatment on circulating T cells. PD-1 expression on circulating T cells, and T cell expression of interferon gamma versus IL-10 was assessed by multichannel FACS analysis.Results: 17 subjects were treated. Three patients were treated at a dose of 3 mg/kg without DLT. Three evaluable patients completed treatment on cohort 2, at a dose of 10mg/kg without DLT. A forth participant withdrew consent following one dose of treatment and therefore is not evaluable. Ten patients were treated on an expansion cohort at a dose of 10mg/kg. We observed one grade 4 pulmonary infection, and three grade 3 pulmonary infections which resolved. Other Abatacept related adverse events included grade 2 gastritis (n=1), grade 2 pain (n=1), and grade 1 diarrhea (n=2), fatigue (n=2), rash (n=1), and skin pain (n=1). Of the 16 evaluable patients, 7 (44%) achieved a clinical partial response as defined by improvement of two disease systems based on the 2011 NIH consensus criteria. Abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders with a mean baseline dose of 27mg compared to a mean dose of 14mg 1 month following the 6th dose of Abatacept (p = 0.01). PD-1 expression on circulating CD4+ and CD8+ T cells increased from a mean of 3.4% and 2.7% respectively at baseline to a mean of 8.9% and 7.6% respectively at one month following the 6thdose of Abatacept in clinical responders (n=3; p<0.05). In contrast, no change in T cell expression of PD-1 was observed in non-responders. A shift from Th1 to Th2 cytokine secretion was observed in clinical responders, with a mean 2.8 fold decrease in interferon gamma and a mean 2.5 fold increase in IL-10 secretion by circulating T cell populations (n=4).Conclusion: Abatacept is well-tolerated in the treatment of steroid-refractory cGVHD. Abatacept resulted in the improvement in NIH cGVHD scores in 44% of patients with steroid-refractory GVHD with a significant decrease in prednisone dose. An increase in PD-1 expression and a skewing toward Th2 cytokines was observed in clinical responders. Based on this promising data, a phase II trial is being initiated. DisclosuresSoiffer:GentiumSpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Arnason:Gilead: Consultancy. Avigan:Astex: Research Funding; DCPrime: Research Funding. Rosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding.

Pre-treatment of allogeneic bone marrow recipients with the CXCR4 antagonist AMD3100 transiently enhances hematopoietic chimerism without promoting donor-specific skin allograft tolerance

Hematopoietic chimerism established by allogeneic bone marrow transplantation is known to promote donor-specific organ allograft tolerance; however, clinical application is limited by the need for toxic host conditioning and “megadoses” of donor bone marrow cells. A potential solution to this problem has been suggested by the observation that recipient bone marrow mobilization by the CXCR4 antagonist AMD3100 promotes chimerism in congenic bone marrow transplantation experiments in mice. Here we report that a single subcutaneous dose of 10mg/kg AMD3100 in recipient C57BL/6 mice was able to enhance hematopoietic chimerism when complete MHC-mismatched BALB/c donor bone marrow cells were transplanted 1h after drug dosing. However, levels of chimerism measured 30days post-transplantation were not sustained when mice were reexamined on day 90 post-transplantation. Moreover, transient chimerism induced by this protocol did not support robust donor-specific skin allograft tolerance. Using the same transient immunosuppression protocol, we confirmed that “megadoses” of donor bone marrow cells could induce durable chimerism associated with donor-specific skin allograft tolerance without AMD3100 pre-treatment. We conclude that in this protocol AMD3100 pretreatment may empty bone marrow niches that become reoccupied by allogeneic donor hematopoietic progenitor cells but not by true long-lived donor hematopoietic stem cells, resulting in short-lived chimerism and failure to support durable donor-specific allograft tolerance.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Treatment of Metastatic Renal Cell Carcinoma With CAIX CAR-engineered T cells: Clinical Evaluation and Management of On-target Toxicity

Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 10(9) CAR T cells. CTC grade 2-4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 10(9) T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed "on-target" toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Moving Successful Virus-specific T-cell Therapy for Hematopoietic Stem Cell Recipients to Late Phase Clinical Trials

There are now multiple effective strategies for the activation and expansion, or direct selection of virus-specific T cells that may be able to eliminate a range of virus infections in the immunocompromised host. In this mini-review, we explain how the stage is set for their rigorous evaluation in large-scale clinical trials. Virus infections are the cause of 30% or more of transplant-related deaths in recipients of T-cells depleted, allogeneic hematopoietic stem cell transplants (HSCT).1 Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviruses (AdV) are the most common culprits, but other common viruses like parainfluenza virus, respiratory syncytial virus, influenza viruses, polyomaviruses, and human herpesvirus 6 together contribute significant morbidity and mortality (Figure 1).2,3,4 Small molecule therapies are often ineffectual, always costly and frequently produce significant adverse effects. Figure 1 Relative frequency of viral infections after HSCT. Adv, adenovirus; BK, JC, KI, and WU (polyomaviruses); CMV, cytomegalovirus; H1N1, influenza strain Hemagglutinnin 1 Neuraminidase 1); HHV, human herpes virus; HSCT, hematopoietic stem cell transplant; ... Virus-specific T-cells derived from stem cell donors can prevent and treat post-transplant viral infections, in the recipients for whom they were intended, and also in partially human leukocyte antigen (HLA)-matched, third party recipients.5,6,7 The low toxicity and long-term protection provided by virus-specific T-cells compares favorably with the significant toxicities and short-term effects of most antivirals.5,6,8,9,10 Is it time, therefore, to begin the transfer of T-cell manufacturing from academic phase I/II clinical trials into hospital or industry-supported facilities so that virus-specific T-cells can be made available to all high-risk HSCT recipients? Several barriers prevent the broader use of virus-specific T-cell therapies after stem cell transplantation. While T-cell therapies for EBV, CMV, and AdV have clearly demonstrated their safety and efficacy both as prophylaxis and as therapy, for many other viruses, the antigens that induce protective T-cells have yet to be identified. Moreover, these infections may occur in <5% of patients making it difficult to perform the rigorous comparative effectiveness studies that will be required to show lower overall cost, fewer adverse effects, and equivalent or superior efficacy. Before any of these barriers can be breached effectively by the academic institutions who are the major developers of these T-cells therapeutics, we must select and optimize manufacturing strategies that are robust and scalable and have the lowest possible cost. Finally, the leap to late phase trials cannot be accomplished by academic institutions alone, but requires partnership with industry. This article will largely deal with selection and optimization of virus-specific T-cell manufacturing strategies.

An absence of donor TH17 differentiation ameliorates dermal tissue damage

To explore the functions of TH17 cell in cutaneous graft-versus-host disease (GVHD). A model of acute GVHD (aGVHD) was established with a major histocompatibility complex class I/II-disparate allogeneic bone marrow transplantation (BMT). Bone marrow monocytes and splenic T cells from donor C57/BL6 were enriched. The recipient BABL mice were irradiated ((60)Co source) with 7.5 Gy total body irradiation (TBI) and injected with 5 × 10(6) marrow monocytes and 5 × 10(5) T cells. The experimental mice were divided into 3 groups: lethal total body irradiation (TBI); allogeneic bone marrow transplantation (BMT) and recipients of halofuginone (HF). The symptoms of aGVHD were observed daily and detailed histopathologic analyses of recipient skin were performed at Day 6 post-transplantation. And Tri-color flow cytometry (FCM) was performed at Day 6 post-transplantation to measure the levels of interleukin (IL)-17, interferon (IFN)-γ and TH1/TH17. Clinical GVHD symptoms were observed in recipient mice. The administration of HF to lethally irradiated recipients led to very modest GVHD-induced cutaneous changes manifested predominantly by fur loss. However, the experimental animals receiving only allogeneic BMT showed significant fur loss and pathologic skin conditions. Consistent with the clinical evaluations, the histopathologic results demonstrated significantly increased pathologic cutaneous lesions in recipients undergoing only BMT. The median ratios of TH1/TH17 cells were 17.57 and 5.31 in the HF and BMT groups respectively. The difference had statistical significance (P < 0.05). The serum levels of IL-17 were(1.47 ± 0.18) and (2.81 ± 0.19) pg/ml in the TBI and BMT groups respectively (P < 0.05). But IL-17 could not be detected in the HF group. The serum levels of IFN-γ were (3.86 ± 0.32), (42.97 ± 0.42) and (9.89 ± 0.51) pg/ml in the TBI, BMT and HF groups respectively. The inter-group differences had statistical significance (P < 0.05). An absence of TH17 cell may alleviate the cutaneous GVHD but exacerbate the systemic GVHD.

Effects of Sublethal Irradiation on Patterns of Engraftment after Murine Bone Marrow Transplantation

Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of various immunosuppressive, yet nonmyeloablative preparative regimens that often include low-dose irradiation. To determine the effects of low-dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard endpoint flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10-90) post-BMT periods. To exclude the contribution of irradiation on reducing immunologic rejection, severely immune-deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sublethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrowcompared with nonirradiated recipients (P < .05). Bioluminescence imaging demonstrated that theeffect of TBI (P < .001) on chimerism occurred only after the first 7 days post-BMT. Flow cytometric analysison day 3 showed no increase in the number of donor cells in irradiated bone marrow, confirming that sublethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intrafemoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with i.v. administration (P< .05). Overall, the combination of TBI and i.v. administration of donor cellsprovided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sublethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.

Bronchiolitis Obliterans After Allogeneic Stem Cell Transplantation: a Single Center Retrospective Study of Risk Factors.

AbstractAbstract 4507Bronchiolitis obliterans (BO) is a late-onset pulmonary complication occurring after stem cell transplantation as a manifestation of chronic graft versus host disease (cGVHD). It is characterized by an insidious airflow obstruction leading to a high mortality.To identify risk factors of BO, we retrospectively analyzed 151 allogeneic bone marrow recipients allografted between 2006 and 2008 in our hematology center. The median age is 47 years (4 – 66 years). The ratio male/female is 2. 52 % of patients were allografted for AL, 13% for myelodysplastic syndrome, 10.5% for NHL, 3 % for CML, 4.6% for CLL, 4% for HL.Sixty four patients (42%) had a myeloablative conditioning regimen and 87 patients (58%) had reduced intensity conditioning regimen. Busulfan was used as a part of the conditioning in 45% of the transplantations and total body irradiation in 47%. The source of the graft is bone marrow in 32% of cases, peripheral stem cells in 57% of cases and umbilical cord blood in 11% of cases. In 50 % of the cases the donor is a sibling. GVHD prophylaxis is cyclosporine associated with MTX in 41% of the cases, cyclosporine alone in 25% of cases and cyclosporine with MMF in 30% of the cases.Patients had pulmonary function test pre transplant and at 3, 6, 12 months after transplantation and then every 6 months. BO was defined according to national institute of health (NIH) consensus for diagnosis and staging of cGVHD. NIH definition requires: absence of active infection, decreased FEV1 (<75% of predicted normal), evidence of airway obstruction with a ratio of FEV1 to forced vital capacity <70%, elevated residual volume of air (>120% of predicted normal) or an expiratory chest CT or lung biopsy that reveals air trapping or bronchiectasis.According to this definition, we found 11 cases of BO with a cumulative incidence at 3 years of 13%. BO appeared after a median time of 16 months (6m-25m). In univariate analysis, recipient age 50 y.o. and above (p=0.003), donor age 50 y.o and above (p=0.05), cGVHD (p=0.05), aGVHD (p=0.04), female donor to female recipient (p=0.001) and reduced intensity conditioning regimen (p=0.08) are associated with BO.In multivariate analysis, aGVHD (p=0.029) (RR 6.5, 95% confidence interval, 1.2%- 34.7%) and female donor to female recipient (p=0.028) (RR 5.45, 95%, confidence interval 1.19%-24.8%) are risk factors of BO. All the patients who developed BO where treated for GVHD at the time of onset of pulmonary symptoms. Among the 11 patients, 6 died and BO is the cause of death in 3 patients. The remaining 5 patients are alive with a BO controlled with immunosuppressive treatment. Conclusion:BO is a rare complication after allogeneic stem cell transplantation according to the consensus criteria. It is tightly linked to GVHD. Female donor to female recipient is strongly associated with the occurrence of this complication. This should be verified on a larger number of patients. A reinforced immunosuppressive treatment may control the progression of the disease and prolong the survival of those patients. Disclosures:No relevant conflicts of interest to declare.

Peripheral blood vs. bone marrow for molecular monitoring of BCR‐ABL1 levels in chronic myelogenous leukemia, a retrospective analysis in allogeneic bone marrow recipients

Molecular monitoring of the BCR-ABL1 transcript in chronic myelogenous leukemia (CML) using quantitative real-time PCR (RQ-PCR) can be performed using either bone marrow (BM) or peripheral blood (PB). However, a recent report by Stock et al. [International Journal of Oncology 28 (2006) 1099] questioned the reliability of PB samples for BCR-ABL1 detection as performed by RQ-PCR. We report a study on 114 CML patients who received allogeneic stem cell transplantation (ASCT), and who were monitored by RQ-PCR using paired samples of BM and PB: the total number of determinations was 428, with a median follow-up after transplant of 8 years. BCR-ABL1 transcript was undetectable or <0.1%, in 106 (49.57%) and 62 (29%) paired determinations, respectively. BCR-ABL1 was >0.1% in 36 (16.8%) paired determinations and was discordant in 10 (4.7%). Agreement between PB and BM results was quantified by the kappa test (k = 0.85; 95% CI 0.76-0.94). This study shows that BCR-ABL1 RQ-PCR monitoring of CML patients after ASCT with PB is concordant with BM in 95.3% of cases, and thus may be used to monitor the disease. This may be relevant when discussing both quality of life issues and the need for post-transplant monitoring with the patient.

What's New in Childhood Organ Transplantation

1. Debra Sudan, MD* 2. Emile A. Bacha, MD† 3. Eunice John, MD‡ 4. Amelia Bartholomew, MD§ 1. *Professor of Surgery; Director, Living Donor and Intestinal Rehabilitation Programs, University of Nebraska Medical Center, Omaha, Neb 2. †Associate Professor of Surgery; Senior Associate, Department of Cardiac Surgery, Harvard Medical School, Children's Hospital Boston, Boston, Mass 3. ‡Professor and Director, Pediatric Nephrology, University of Illinois at Chicago Medical Center, Chicago, Ill 4. §Associate Professor, Transplantation and Molecular Genetics, University of Illinois at Chicago Medical Center, Chicago, Ill 1. Describe the indications, immunosuppression therapy, outcomes, potential complications, and long-term issues of kidney transplantation. 2. Describe the indications, surgical techniques, immunosuppressive therapy, and outcomes of liver transplantation. 3. Describe the indications, surgical techniques, immunosuppressive therapy, potential complications, and prognosis of intestinal transplantation. 4. Describe the indications, surgical techniques, required mechanical support, immunosuppressive therapy, and outcomes of heart transplantation. 5. Describe the indications, immunosuppressive therapy, and outcomes of lung transplantation. 6. Delineate the role of the primary care physician in organ transplantation. Transplantation remains the most effective treatment for children who have end-organ failure. In this review, we summarize the current issues facing children and their treating physicians following transplantation of whole organs. Although recipients of allogeneic bone marrow can follow a similar clinical course as organ recipients, bone marrow transplantation as a therapy has additional unique posttransplant features that are not covered in this article. Regardless of transplant type, common themes emerge. Children differ from adults in their immune responses, in the way they metabolize many drugs, and in their susceptibility to many of the adverse effects of transplantation and immunosuppression. Drug regimens based on induction immunotherapy with interleukin-2 receptor antagonists or anti-lymphocyte antibody coupled with tacrolimus-based long-term immunosuppression have increased in prevalence. Long-term exposure to immunosuppressive medications, however, has led to increased drug-related morbidities. Hyperlipidemia, hypertension, cardiovascular disease, malignancy, and diabetes mellitus have emerged as significant concerns. Nonadherence to immunosuppressive regimens, particularly among adolescents, contributes to increased graft failure. Despite these obstacles, posttransplant pediatric patient and graft survival have been improving steadily. Nationwide data indicate that patient and graft survival rates are equivalent to or better than adult survival rates. Successful delivery of care for the long-term well-being of these patients demands communication between the patient and a complex of many physicians, from the earliest encounters with general pediatricians to more specialized care with …

Loss of IFNγR1 Signaling on Donor Bone Marrow Abrogates GVHD but Maintains Immunocompetence.

BACKGROUND: Acute graft versus host disease (GVHD) remains as the major complication after allogeneic bone marrow transplant (BMT) resulting in organ toxicity and immune dysfunction. Indeed, we have previously demonstrated that GVHD impairs responses to dendritic cell vaccines. The pathophysiology of GVHD involves preparative regimen-induced inflammation of target organs, release of inflammatory mediators such as gamma interferon (IFNg), and subsequent activation of alloreactive T cells. Given that IFNg can both contribute to GVHD and provide beneficial immune responses, we explored the potential role of IFNg on GVHD and post-transplant immunocompetence.METHODS: We utilized a minor histocompatibility antigen mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) as a means of controlling the induction of acute GVHD and to provide a source of immunocompetence in a thymectomized mouse. To study the role of IFNg on GVHD, we chose either IFNg receptor 1 (IFNgR1) −/− marrow or DLI to permit the normal production of IFNg in GVHD while influencing which cells that can respond to the cytokine. Normal C57BL/6 (B6) or IFNgR1 −/− B6 mice were used as bone marrow donors on day 0 into lethally irradiated, thymectomized B6 × C3H.SW (F1) mice. Normal B6 or IFNgR1 −/− DLIs given with or without a dendritic cell vaccine were introduced at days 14 and 28 post-BMT both to control the induction of GVHD and to provide a population of vaccine-responding cells. F1 recipients were observed for signs of GVHD. ELISPOT of the number of antigen-reactive IFNg-producing splenocytes were also performed to measure functional response to vaccine.RESULTS: The absence of IFNgR1 in the DLI abrogates GVHD as shown by % change in weight (B6 DLI = −6.3 +/− 4.7 vs. IFNgR1−/− DLI = 6.6 +/− 6.1, p=0.001) and allows for greater doses of DLI to be tolerated by the host, however, there is also decreased vaccine responses by ELISPOT (B6 DLI = 1631 vs. IFNgR1−/− DLI = 72, p=0.03). Surprisingly, using IFNgR1−/− bone marrow also abrogates GVHD as shown by % change in weight (B6 marrow = −6.3 +/− 4.7 vs. IFNgR1−/− marrow = 4.4 +/− 4.2, p less than 0.05) and splenocyte count (B6 marrow = 31.48 +/− 12.54 vs. IFNgR1−/− marrow = 63.54 +/− 15.92, p=0.008), but vaccine responses by ELISPOT can be restored to levels that are equivalent of syngeneic control mice, even in the presence of a normal B6 DLI (B6 marrow = 1631 vs. IFNgR1−/− marrow = 9283, p=0.0002). The abrogation of GVHD by IFNgR1−/− marrow does not appear to be a dominant effect since mixtures of IFNgR1 −/− and normal B6 bone marrow still cause GVHD.CONCLUSIONS: Recipients of allogeneic bone marrow and T cells developed GVHD and had decreased vaccine responses by ELISPOT. Loss of IFNgR1 on allogeneic donor lymphocytes abrogates their ability to cause GVHD, but also diminished their ability to respond to vaccine. Surprisingly, loss of IFNgR1 on a donor bone marrow-derived, non T cell results in equivalent abrogation of GVHD, while restoring immunocompetence through favorable responses to a vaccine. Further studies will attempt to identify the phenotype of the responsible bone marrow-derived cell. These results demonstrate a strategy of providing higher doses of DLIs to enhance anti-tumor activity without exacerbating GVHD, and thus, have implications for immune modulation post-allogeneic BMT.

Results of a Phase I/II-Study on PCR-Based Pre-Emptive Therapy with Valganciclovir or Ganciclovir for Active CMV Infection Following Reduced Intensity Allogeneic Stem Cell Transplantation.

The advent of reduced intensity conditioning(RIC) protocols for allogeneic haematopoietic stem cell transplantation(HSCT) has reduced the incidence of early transplantation related morbidity largely due to the attenuation of the conditioning intensity. Nevertheless, the incidence of CMV reactivation in these patients remains high. Herein we report the results of a multi-centre randomised prospective study to assess the bioavailability(auc0–12) of ganciclovir in the population of patients undergoing alemtuzumab-based RIC HSCT after oral administration of valganciclovir and secondly, the efficacy and safety of valganciclovir in the treatment of a CMV infection following allogeneic SCT. Recipients of allogeneic bone marrow or peripheral blood stem cell transplants with related or unrelated donors following reduced intensity conditioning, without proven graft versus host disease were eligible for this study. RIC protocols approved for the purposes of this study were FBC(fludarabine, busulphan, alemtuzumab) and FMC(fludarabine, melphalan,alemtuzumab). For inclusion into the study, patients had to have a detectable CMV DNA load in two consecutive blood specimens up to 120 days after transplant. Eligible patients were randomized to 1 of 2 treatment groups: group A received 900 mg oral valganciclovir(2 x 900 mg/d) for 14 days and group B received intravenous ganciclovir 5mg/kg/d twice daily for 14 days. All patients were monitored for 84 days(safety follow-up). pK profiles of ganciclovir were obtained after administration of the study drug in each study arm. pK assessments were scheduled at days 4 and 11. 27 patients were recruited into the study over a 24-month period from Jan 2005 to Dec 2006. The median age was 51 years(range:34–68). The median time to CMV reactivation was 43 days post-HSCT(range:20–114). 18 patients(67%) completed the allocated treatment resulting in CMV DNA load <10 copies/ml at a median of 14 days post-HSCT(range:7–28). In 9 cases, there were changes to the primary treatment regimen. In group A, treatment was modified in 5 cases; 3 due to rising CMV levels, 1 due to drug rash, 1 due to neutropenia. In group B, 4 patients had treatment modification; 3 patients due to rising CMV DNA levels, 1 had neutropenia. None of the patients in the study developed CMV invasive disease. The median value of the systemic clearance of ganciclovir was 11.8 l/h(95%CI: 8–15.6 l/h). The bioavailability of ganciclovir from valganciclovir(expressed as equivalents of ganciclovir) was 73%(95%CI, 34%–112%). The average exposure in the valganciclovir group(36.9 ± 14.9μg.h/ml) was significantly higher than in the ganciclovir cohort(27.9 ±7.5μg.h/ml). As a result, ganciclovir bioavailability in the subjects who received valganciclovir was 79%. In summary, when compared with intravenous ganciclovir, oral valganciclovir had high bioavailability with equivalent efficacy and safety in patients undergoing RIC HSCT. Use of valganciclovir can facilitate the out-patient treatment of patients with CMV reactivation post-HSCT with a potential reduction in hospitalization costs.

Post Transplant Lymphoproliferative Disorders (PTLD): Prevalence, Clinical Features and Outcome.

PTLD is a life threatening complication developing in 1–5% of transplant (Tx) recipients. Epstein Barr (EBV), Cytomegalovirus (CMV) and immunosuppression (IS) were identified as risk factors. Aggressive evolution and therapy related toxicity generate poor prognosis. We retrospectively analyzed data from 18 PTLD patients (pts) diagnosed at our hospital between March 1994- June 2007. All of them were at least one graft recipient and were under IS.RESULTS: Out of 1206 adult Tx recipients, 18 (1,5%) developed PTLD. Ninety-four percent were men, mean age 42,9 years (range: 18–72). Our study comprises 10 heart, 2 kidney, 2 lung, 1 heart-lung, 1 liver and 2 non related allogeneic bone marrow (BM) recipients. IS protocols included Cyclosporin A (16 pts), Azathioprine (8), Mycophenolate mofetil (7), Tacrolimus (4) and antitymocyte globulin (2). Pretx recipients serology for EBV and CMV were positive in 87 and 94% respectively. Sixty-six percent (12/18) disclosed late onset PTLD (median 49 months), all of them were solid organ (SO) recipients. BM recipients developed the disease within the first 6 months after Tx. Fifty percent presented extranodal involvement, 3/18 with BM infiltration and 3/18 showed lymphoma in the graft. Histologycal findings: 15/18 B cell lymphoma (8/15 large cell lymphoma), 1 peripheral T cell lymphoma, 1 early lesion infectious mononucleosis-like PTLD, 1 polymorphic hyperplasia PTLD. Immunophenotype: 89% were CD20+. Fifty percent of the group achieved response, 7/9 with complete response(CR). Three/9 pts obtained CR with reduction of IS alone.Treatment included Rituximab (63% pts), CHOP (56%) and radiotherapy (25%). Eleven/18 pts died, 4 due to disease progression, 6 by sepsis and 1 because of underlying hematological disease relapse.CONCLUSIONS: In our experience, PTLD was an uncommon complication. The late onset of the disease was the most frequent form of presentation among SO recipients. B cell lymphoma was the main diagnosis (83%). Fifty percent (9/18) achieved response and 78% of them showed CR. Disease progression and sepsis were the most important causes of death.

Detection of Bone Marrow-Derived Cells Expressing a Neural Phenotype in the Human Brain

Animal studies suggest that adult bone marrow cells have the potential to migrate into the brain and generate new neural cells. Because data on this physiologic repair mechanism in humans are lacking, we investigated bone marrow engraftment into the brain of bone marrow recipients after sex-mismatched transplantation. Brain sections of seven allogeneic female bone marrow recipients were examined. The Y-chromosome, which served as a natural marker of donor bone marrow-derived cells after male-to-female transplantation, was identified by in situ hybridization. The neural phenotype of Y-chromosome-positive cells was determined using neural nuclear protein (NeuN) immunohistochemistry. Y-chromosome-positive cells expressing NeuN were found within the first 3 months after transplantation in both the cerebrum and the cerebellum at a frequency of 0.003% to 0.013% of all neurons. These cells were observed only in patients with cerebral lymphocytic infiltration and graft-versus-host disease. Our data suggest that adult bone marrow cells are capable of generating cells that express the neural marker NeuN early after transplantation. Cells with this specific phenotype may contribute to tissue repair in brain regions remote from neurogenic zones.

FTY720 Inhibits Graft-Versus-Host Disease (GVHD) and Allogeneic Bone Marrow (BM) Graft Rejection Independent of Effects on T Cell Egress from Lymphoid Tissue.

FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not well understood. We studied FTY in murine GVHD and engraftment models as a single agent and in combination with known beneficial strategies. FTY (3 mg/kg orally d0-28) inhibited GVHD, increasing the median survival time (MST) of lethally irradiated C57BL/6 (B6) mice given BALB/c BM and a moderate T cell dose from 30d to 71d (p<.001) or a higher T cell dose from 7d to 32d (p<.006). FTY was additive with ex vivo activated and expanded CD4+CD25+ regulatory T cells (Tregs) for GVHD inhibition. In contrast to control mice which all died of GVHD by d70 (MST = 37d), mice receiving FTY or Tregs or both had 100 day survival rates of 50%, 71% and 100%, respectively. The additive effects of FTY and Tregs were reproduced in a second strain combination. Although we hypothesized that FTY trapped Tregs in the lymph nodes allowing for increased contact time for Tregs to inhibit GVHD effector T cells, imaging studies of green fluorescent protein (GFP)+ Tregs did not support this hypothesis. Moreover, imaging studies of GFP+ effectors did not indicate that FTY inhibited donor T cell egress from lymphoid tissues. To study alloantigen-specific responses, B6 TCR Tg CD8+ and CD4+ T cells that are reactive against BALB/c alloantigen were adoptively transferred into B6 rag mice prior to sublethal irradiation and infusion with BALB/c BM. FTY reduced the expansion of Tg CD8+ and CD4+ T cells in the spleen 10d after BMT by 80% and 78%, respectively. In addition to GVHD inhibition, FTY also promoted donor BALB/c BM engraftment in sublethally (5.0 Gy) irradiated B6 mice although donor chimerism was not stable in all mice. In contrast to 4 of 26 water-treated controls, all 29 FTY-treated mice (3 mg/kg d0-13) were engrafted at 1 month as assessed by PBL phenotyping (ave of 9% vs 68% donor. p<.001). However, by 3 months, the engraftment rate in FTY-treated mice had decreased to 12 of 28 mice. All 9 mice receiving FTY for an additional 2 weeks (d0-28) had high donor chimerism levels (>96%) at 1 month but chimerism was lost in 3 mice by 6 months after BMT. In a different strain combination, even a 2 wk FTY course increased donor chimerism that remained stable for 6 months. Imaging studies revealed that FTY did not prevent the egress of adoptively transferred host-type GFP T cells in allogeneic BM recipients. FTY was also tested in combination with anti-CD40L mAb, a potent engraftment-promoting agent in mice. In contrast to anti-CD40L mAb, FTY did not promote engraftment in mice conditioned with very low levels of irradiation (0.5–2.0 Gy) although the combination of anti-CD40L mAb and FTY resulted in superior stable engraftment rates and levels compared to anti-CD40L mAb as a single agent. Interestingly, FTY increased donor chimerism in syngeneic, as well as, allogeneic recipients. These data indicate that FTY is efficacious as a single or adjunct agent for the prevention of GVHD and graft rejection and further indicate that inhibition of lymphocyte egress was not essential for the beneficial effects of FTY in BMT recipients.

Graves’ disease following unrelated umbilical cord blood transplantation in pediatric patients

Graves’ disease (GD) is the most prevalent autoimmune disease in the United States with a peak incidence in the fifth to sixth decades of life and a female to male predominance of ∼10:1. In recipients of allogeneic bone marrow and peripheral blood stem cell transplants, GD recurs in previously affected recipients, occurs in unaffected recipients by adoptive transfer of autoimmune thyroiditis from an affected donor, and occurs less frequently in unaffected recipients transplanted with unaffected donors.