Post Bone Marrow Transplantation Research Articles

Bone marrow architecture reconstitution after hematopoietic grafting: A cross-sectional study

Abstract Objective Changes in bone marrow (BM) architecture and its remodeling after hematopoietic stem cell transplantation (HSCT) following a reduced-intensity conditioning regimen for treating malignant hematologic diseases are poorly documented. We assessed these changes and their correlation with the clinical course to investigate the dynamics of this process. Material and methods Hematology patients who received a reduced intensity conditioning (RIC) were followed after successful allogeneic or autologous HSCT. Patients had at least 100 days after hematopoietic grafting. Post-transplant BM architecture was assessed by expert examination of a trephine biopsy using age-adjusted scores and histopathology results were contrasted with clinical post-transplant evolution to determine meaningful associations. Results Twenty-seven HSCT recipients, 19 allogeneic and 8 autologous, were studied at a median 155 (100–721) days post-transplant. Eleven (40.7%) had hypocellular biopsies with around 50% of the expected cell composition for a normal individual at that age. Patients with increased fibrosis had lower peripheral white blood cell counts compared to those with normal reticulin distribution (p = 0.015). A decrease in overall survival (OS) was documented in the group with more severe myelofibrosis at a median 813 days of follow-up. Infectious complications were more frequent in patients receiving an allogeneic (n = 6) compared with the recipients of an autologous transplant (n = 0). Conclusions Despite normal peripheral blood count and composition, significant post-transplant hypocellularity, revealing incomplete bone marrow reconstitution, was documented after HSCT in patients conditioned with a RIC regimen. Severe BM fibrosis was associated to decrease OS.

CXCR4 (CD184) expression on stem cell harvest and CD34+ cells post-transplant

Objectives/backgroundCXCR4 is a receptor for stromal-derived factor-1 (SDF-1), a molecule that has a chemotactic activity for lymphocytes and is important in homing of hematopoietic stem cells to their adult marrow. We evaluated the CXCR4 (CD184) expression in the harvest cells and in the post-transplant bone marrow (BM) and its relation to engraftment, as determined by the consensus criteria and chimerism. MethodsThis is a prospective study which included 30 patients undergoing hematopoietic stem cell transplantation; 15 patients received autograft and 15 patients received allograft on dates between January 2012 and May 2014. We assessed CD184 (CXCR4) using flow cytometry in the harvest cells together with post-transplant BM assessment on Day 28 and Day 90 for complete morphologic, molecular studies, and detection of CD184 expression on CD34+ cells with chimerism studies on total peripheral blood mononuclear cells. ResultsDiagnoses of the enrolled patients were as follows: seven (24.1%) with acute myeloid leukemia, eight (27.6%) with multiple myeloma, four (13.8%) with acute lymphoblastic leukemia, three (10.3%) with non-Hodgkin lymphoma, two (6.9%) with myelodysplastic syndromes, two (6.9%) with aplastic anemia, two (6.9%) with chronic myeloid leukemia, one (3.4%) with Hodgkin lymphoma, and one (3.4%) with plasmacytomas. One patient died and was excluded from the study because there were not enough data about engraftment. There was no statistical significance between the level of CD184 in stem cell harvest and the prediction of successful engraftment (p>0.05) as well as in Day 28 BM sample (p>0.05), whereas there was a statistical significance between the level of CD184 in Day 90 BM sample and the occurrence of successful engraftment (p=0.002). ConclusionSDF-1/CXCR4 axis plays a crucial role in engraftment; however, more studies are warranted to assess their expression post-transplant. Evaluating the ligand (chemokine, SDF-1) or its receptor (CXCR4) may serve as potential surrogate markers for assessment of engraftment.

Immune and Hematological Reconstitution after Allogenic Bone Marrow Transplantation in Tunisian Pediatric Recipients: Prospective Study and Tunisian Experience Report

AIM: A regular monitoring of the immune reconstitution mainly based on the quantitative determination of lymphocyte T subpopulation. This is prospective analysis for 1 year in Tunisian children treated with allogenic intrafamilial bone marrow transplantation. Methods: We conducted a prospective analysis for 1 year follow up enrolling 25 children treated with allogenic intrafamilial bone marrow transplantation among them two cases of Peripheral hematopoietic transplantation and placental cord blood transplantation including: aplastic anemia (6 cases), hemoglobinopathies (12 cases), myelodysplastic syndrome (1 case), 2 cases of Acute lymphocytic leukemia, a case of congenital amegacarycytosis and 3 cases of primary immunodeficiency with lack of expression of major MHC class II. All subjects received different conditioning regimens according to the indication. Our study consisted of a regular monitoring of the immune reconstitution mainly based on the quantitative determination of lymphocyte T subpopulation. So, these tests were routinely requested to 1 month, 2 months, 3 months, 6 months, 9 months and 12 months post- bone marrow transplantation. Results: The average time of engraftment was 18 days corresponding to neutrophil recovery (12-24). For the T cell recovery, a rate of CD4 + T lymphocytes > 200/ mm3 was provided within an average of 2.5 months (1-7). The average time to obtain CD8+ T lymphocytes >200 /mm3 was 2 months (1-5). The humoral immune reconstitution was made within an average of 2 months (1-4). A ratio of CD4+ / CD8+ T lymphocytes (>1) was obtained within 10 months and a half (1-24). Univaried analysis showed a significant correlation between the bone marrow sex matched and the faster reorganization of CD8 + T cells (p = 0.042). Moreover, a quantity of CD34 +> 6x 106/ kg was significantly associated with the recapture of a formula lymphocyte T CD4+ / CD8+ (> 1) (p=0.03). Conclusion: The immune recovery post bone marrow transplantation in children began with myeloid lineage then lymphoid B then lymphoid T. The inversion of the ratio CD4 +/CD8+ T lymphocytes, seemed to be influenced on the one hand by the high content of CD34 + cells in the graft as well as the type of conditioning on the other hand by the CMV infection since it accelerates significantly CD8+ T lymphocyte reconstitution.

VH1 Family Immunoglobulin Repertoire Sequencing after Allogeneic Hematopoietic Stem Cell Transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), recovery of humoral immunity is essential to protect from life-threatening infections. However, monitoring the humoral immune system after transplantation with standard techniques in the clinical routine is imprecise. Here, we performed sequencing of mononuclear bone marrow cells to characterize the VH1-repertoire of switched B cells of healthy volunteers and patients undergoing HSCT. Analysis of healthy bone marrow donors and patients showed virtually no clonally related sequences between individuals. Interestingly, clonally related sequences were present in pre- and post-transplantation bone marrow of patients undergoing HSCT for acute myeloid leukemia treatment. We consistently observed such related B cell clones, irrespective of conditioning regimen, donor source or time post transplantation. In general, repertoire diversity was lower in post-HSCT as compared to pre-HSCT samples. However, post-HSCT repertoires retained highly mutated sequences, despite immunosuppressive therapy and presence of T cell deficiency after HSCT. These observations identify key properties of the recovering B cell compartment and provide a conceptual framework for the surveillance of humoral immunity after allogeneic transplantation.

Diagnosis of cytomegalovirus pneumonia by quantitative polymerase chain reaction using bronchial washing fluid from patients with hematologic malignancies

BackgroundThe incidence of cytomegalovirus (CMV) pneumonia is increasing in patients diagnosed with hematologic malignancies. The utility of CMV-DNA viral load measurement has not been standardized, and viral cut-off values have not been established. This study was designed to investigate the utility of CMV quantitative real-time PCR (qRT-PCR) using bronchial washing fluid.MethodsWe retrospectively reviewed the microbiologic and pathologic results of bronchial washing fluid and biopsy specimens in addition to the patients' clinical characteristics.ResultsA total of 565 CMV qRT-PCR assays were performed using bronchial washing fluid from patients with hematologic malignancies. Among them, 101 were positive for CMV by qRT-PCR; of these, 24 were diagnosed with CMV pneumonia and 70 with CMV infection, and 7 were excluded due to a diagnosis of invasive pulmonary aspergillosis rather than viral pneumonia. The median CMV load determined by qPCR was 1.8 × 105 copies/mL (3.6 103-1.5 × 108) in CMV pneumonia patients and 3.0 × 103 copies/mL (5.0 × 102-1.1 × 105) in those diagnosed with CMV infection (P < 0.01). Using the ROC curve, the optimal inflection points were 18,900 copies/mL (137,970 IU/mL) in post-bone marrow transplantation (BMT) patients, 316,415 copies/mL (2,309,825 IU/mL) in no-BMT patients and 28,774 copies/mL (210,054 IU/mL) in all patients.ConclusionsThe CMV titers in bronchial washing fluid determined by qRT-PCR differed significantly between patients diagnosed with CMV pneumonia and those with CMV infection. The viral cut-off values in bronchial washing fluid were suggested for the diagnosis of CMV pneumonia, which were different depending on the BMT status.

Relationship between Autoimmune Diseases and Imbalances in Helper/Suppressor T-cell Populations after Rituximab-containing Chemotherapyfor Non-Hodgkin Lymphoma

Immunologic abnormalities including autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP) have been described in patients with non-Hodgkin lymphoma. In some cases, anti-red cell or platelet antibodies are produced by lymphoma cells. However, cases of the occurrence of autoimmune diseases without recurrence of lymphomas have been also reported. The relationship between autoimmune diseases with post-bone marrow transplantation and Hodgkin disease may be attributed to immune dysfunctions, particularly those involving T cells. This autoimmune phenomenon may be related to imbalances in helper/suppressor T-cell populations. Imbalances in helper and suppressor T-cell populations have been reported after R-CHOP chemotherapy, and the recovery of serum IgG and CD4+ counts was observed more than 2 years after R-CHOP therapy in patients with B-cell lymphoma. In this report, we present a case of an 87-year-old man who was treated with rituximab-containing chemotherapy and maintained complete remission. However, the immunophenotyping of peripheral blood and bone mallow mononuclear cells revealed a reversed CD4/CD8 ratio. Two years later, he developed ITP. He was treated with intravenous immunoglobulin and eltrobopag, and thrombocytopenia improved. Five years later, he developed pneumonia and sudden Coombs-positive hemolytic anemia caused by autoantibodies against D antigen and thrombocytopenia without the recurrence of lymphoma. He was treated with prednisolone and a pulse dose of methylprednisolone; however, his response to therapy was poor and he subsequently died. We herein report a case who have been showed reversed imbalances in the helper/suppressor T cell populations over 2 years after R-CHOP therapy, developed AIHA and ITP without recurrence of lymphoma. The long-term monitoring of T-cell counts after rituximab containing chemotherapies is important, and careful attention to infection signs.

Utility of the inspiratory phase in high-resolution computed tomography evaluations of pediatric patients with bronchiolitis obliterans after allogeneic bone marrow transplant: reducing patient radiation exposure.

ObjectiveTo evaluate the utility of the inspiratory phase in high-resolution computed tomography (HRCT) of the chest for the diagnosis of post-bone marrow transplantation bronchiolitis obliterans.Materials and MethodsThis was a retrospective, observational, cross-sectional study. We selected patients of either gender who underwent bone marrow transplantation and chest HRCT between March 1, 2002 and December 12, 2014. Ages ranged from 3 months to 20.7 years. We included all examinations in which the HRCT was performed appropriately. The examinations were read by two radiologists, one with extensive experience in pediatric radiology and another in the third year of residency, who determined the presence or absence of the following imaging features: air trapping, bronchiectasis, alveolar opacities, nodules, and atelectasis.ResultsA total of 222 examinations were evaluated (mean, 5.4 ± 4.5 examinations per patient). The expiratory phase findings were comparable to those obtained in the inspiratory phase, except in one patient, in whom a small uncharacteristic nodule was identified only in the inspiratory phase. Air trapping was identified in a larger number of scans in the expiratory phase than in the inspiratory phase, as was atelectasis, although the difference was statistically significant only for air trapping.ConclusionIn children being evaluated for post-bone marrow transplantation bronchiolitis obliterans, the inspiratory phase can be excluded from the chest HRCT protocol, thus reducing by half the radiation exposure in this population.

A Calcineurin Inhibitor Does Not Affect the Post-Transplantation Cyclophosphamide -Induced Regulatory T Cell Expansion after Bone Marrow Transplantation in a Murine Chronic Gvhd Model

Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Prolonged administration of a calcineurin inhibitor, such as cyclosporin A (CsA), does not decrease the risk of chronic GVHD. Indeed, we and others have reported that long-term use of CsA impairs reconstitution of bone marrow-derived regulatory T cells (Tregs) and increases susceptibility to chronic GVHD. Recently, the administration of post-transplantation cyclophosphamide (PTCY) has been used widely in GVHD prophylaxis and lower incidences of acute and chronic GVHD have been reported. Here, we evaluated the effects of PTCY and CsA on Tregs and cytokine production in effector T cells using the well-defined chronic GVHD mouse model of B10.D2 (H2d) cells into BALB/c (H2d) mice. Sublethally-irradiated recipient BALB/c mice were transplanted with 8.0 × 106 T cell-depleted bone marrow cells and 2.0 × 106 T cell-replete spleen cells from donor B10.D2 mice. PTCY was administered by intraperitoneal injection at days 3 and 4 post bone marrow transplantation (BMT). CsA was injected peritoneally once daily, starting on day 0 of BMT until the day of analysis. Administration of PTCY, with or without CsA, improved the clinical chronic GVHD score significantly at day 50 versus the controls and the CsA-alone group (controls: 2.8 ± 0.34, PTCY: 0.5 ± 0.0, p < 0.01; CsA alone: 1.8 ± 0.73, PTCY-CsA: 0.5 ± 0.0, p < 0.01). There was no significant difference in the GVHD score between the PTCY and PTCY-CsA groups. Histopathological examinations of the skin at day 50 after BMT also showed significantly reduced chronic GVHD damage in the PTCY group versus the controls and the CsA-alone group (controls vs. PTCY, p < 0.001; CsA alone vs. PTCY, p < 0.01). Flow cytometry analysis of the cells isolated from the peripheral lymph nodes 28 days after BMT revealed that the proportions of Tregs to CD4+ T cells in the PTCY (25.8 ± 2.1%) and PTCY-CsA (29.5 ± 2.2%) groups were remarkably higher than the controls (8.3 ± 2.7%) and CsA-alone (6.7 ± 1.6%) groups (p < 0.05). There was no significant difference in the Treg ratio between the PTCY and PTCY-CsA groups. Intracellular staining showed that IFN-γ single-positive and IFN-γ/IL-17 double-positive CD4 T cells were well suppressed in the PTCY and PTCY-CsA groups versus the controls and CsA-alone group (controls: 38.2 ± 3.5%, PTCY: 11.9 ± 3.1%, p < 0.01; CsA alone: 46.9 ± 2.2%, PTCY-CsA: 17.9%, p < 0.01). Our data suggest that PTCY treatment attenuated chronic GVHD via enhanced reconstitution of donor Tregs and suppression of IFN-γ/IL-17-producing effector T cells. Moreover, CsA does not appear to hinder this suppressive effect of PTCY. This result seems helpful for understanding how PTCY prevents chronic GVHD in clinical settings. DisclosuresMaeda:Mundipharma KK: Research Funding.

Neuropsychological Impairments in Hematological Malignancies Patients Expected to Allo-BMT

Introduction: Hematological malignancies treatment is associated with various side effects, some of them are inversive, other - not. The psychiatric disorders including deficits in cognition functions frequently occur (Vybornykh et al. Int J. of Hematol Res 2016; 2 (1) 103-114). Cognitive impairments (for example, in attention, memory domains) in pre and post bone marrow transplantation (BMT) period are common disabling disturbance, associated with further decrease in quality of life among hematological malignancy patients. Neuropsychological impairment understanding can facilitate to patients future rehabilitation. It is very important to study of existing cognitive impairments in the pre-allo-BMT period.Aim: To define the main neuropsychological impairments before BMT.Materials: 32 patients with various hematological malignancies in pre-allo-BMT period with multiple myeloma (MM) (n=16), acute lymphoblastic leukemia (ALL) (n=10), acute myeloid leukemia (AML) (n=6). Mean age of the 32 study participants (13 men and 19 women) was 31.6 (+-8.5 years). Neuropsychological assessment was made by using Luria neuropsychological battery, also self-reported questionnaire was used. Statistical analyzes were performed using the licensed package Statistica for Windows 5.5.Results and discussion: In 75% (n=24) patients observed decrement (15.7% population norms) in one or more cognitive domain (more 1-1.5 st.dev.). Results in attention, memory, motor skills and complex executive function tests were most likely to be decreased (р. = 0.01). 1-1.5 st.dev. was denoted as mild deficiency and 2-3 st.dev denoted as moderate to total deficiency; within this criteria: 75% (n=24) of participants had at least mild deficits in psychomotor skills, 50 %(n=16) moderate to total (including impairment in kinetic and kinesthetic praxis). 37,5% had deficits in executive function (n=7 - mild and n=5 - moderate to total impairment). 87% (n=28) had deficits in verbal memory (n=10 mild and n=18 moderate to total). 34% (n=11) in attention (n=4 - mild, n=7 - moderate to total). Also it was found the significant correlation (р=0.05) between patients cognitive impairments self-reports and results of neuropsychological assessment which confirmed deficits. In 25% (n=8) patients were no observations of discernible symptoms of neuropsychological impairments. Therefore on the basis of qualitative and quantitative analysis of neuropsychological approach and results of neuropsychological assessment can be supposed that patient’s cognitive impairments probably connected with deficits in basal structures and left hemisphere.Conclusion: Memory, attention, motor skills and complex executive functions are most damaged cognitive domains (probably depends on preceded chemotherapy and some others risk factors) in patients with hematological malignancies before BMT, but their intimate investigation can help in prospective neuropsychological rehabilitation and socialization of patients, so further research of cognitive impairments in dynamics needed to be done. DisclosuresNo relevant conflicts of interest to declare.

Genomic Discovery, Prognosis, and Target Therapy Development

For over three decades now, the t(14;18)(q32.3;q21.3) has been synonymous with the indolent B cell malignancy, follicular lymphoma (FL). Through the widespread uptake of next generation sequencing, we recognize that these tumors acquire many additional genetic aberrations, targeting several distinct signaling pathways (HVEM, mTORC1, NOTCH, NF-κB and JAK-STAT) all of which fall onto a backbone of epimutations that arise in nearly all FL tumors and preferentially target the histone methyl (KMT2D) and acetyl (CREBBP) transferase machinery. We are still in the early stages of understanding how these mutations impact on the biology of FL, their weight of contribution to FL initiation, progression and transformation and indeed whether we can harness this new knowledge to improve on existing treatment. At the very least, we now realize that the acquisition of these mutations and onset of FL may occur several years earlier from the study of rare examples of clonally-related tumors developing post bone marrow transplantation in both donor and recipient. This has helped formulate the notion of an uncharacterized common progenitor pool of B cells (CPC) that are capable of seeding each new genetically distinct episode of disease and providing an explanation for the relapsing remitting nature of FL. We can also now infer the nature of the apparent early driver mutations that seemingly propagate the disease in all FL clones, including the CPC, by using a gene’s mutation load or clonal composition as a surrogate marker. It is apparent that many epimutations represent early events and appear to alter normal B cell programming by favoring sets of genes that promote proliferation and survival while switching off anti-tumor mechanisms, with CREBBP-mutated FLs for example showing a decreased antigen presentation gene signature. The prospect of following a precision medicine approach informed by a patient’s mutation composition should be encouraged given the frequency of activating EZH2 mutations, an underappreciated role of the NF-κB pathway and mutations in the mTORC1 pathway, that are unique to FL, together with the availability of targeted therapeutic agents. There is of course a need to focus efforts on the 20% of patients that succumb to early disease relapse, and the emergence of a new clinical-genetic risk model (m7-FLIPI), as an outcome predictor offers a promising first step in developing a robust risk stratification approach. We do however need to embrace these new opportunities and embed biomarker discovery and validation studies as part of all FL trial programs. Indeed, a case can be made for utilizing material from specific retrospective studies, where updated mutation analysis may demonstrate a potential novel predictive and therapeutic biomarker (e.g. RRAGC mutations/mTOR inhibitors). Altogether, while the genetic portrait of FL is still far from complete, we are now much closer to delivering a final canvas and with it the prospect of changing the landscape of future FL treatment. DisclosuresFitzgibbon:Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

A marker chromosome in post-transplant bone marrow.

Detection of small supernumerary marker chromosomes in karyotype analysis represents a diagnostic challenge. While such markers are usually detected during cytogenetic studies of constitutional chromosome abnormalities, they have also been found in specimens submitted from patients with acquired malignancies. We report here the detection of a marker chromosome in a bone marrow specimen from a patient who received a bone marrow transplantation. We discuss the importance of proper characterization and interpretation of marker chromosomes in clinical practice.

Post-bone marrow transplant thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) is a systemic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and organ failure. Post-bone marrow transplant TMA (post-BMT TMA) is a life-threatening condition that has been reported to afflict between 0.5 and 63.6% of BMT patients. The incidence of post-BMT TMA is affected by evolving therapies such as conditioning regimens. The etiology of post-BMT TMA is thought to be multifactorial, including the effects of immunosuppressive agents, viral infections, TBI and GvHD. A growing body of evidence highlights the importance of complement system activation and endothelial damage in post-BMT TMA. Although plasmapheresis has commonly been used, its therapeutic rationale for the majority of post-BMT TMA cases is unclear in the absence of circulatory inhibitors. It has become possible to target complement activation with eculizumab, a drug that blocks the terminal complement pathway. Early studies have highlighted the importance of anti-complement therapies in treating post-BMT TMA. Moreover, finding complement gene mutations may identify patients at risk, but whether such patients benefit from prophylactic anti-complement therapies before BMT remains to be studied. This review focuses on diagnostic criteria, pathophysiology, treatment and renal outcomes of post-BMT TMA.

Safety and effectiveness of percutaneous cholecystostomy in critically ill children who are immune compromised.

Acalculous cholecystitis is known to develop in critically ill patients without cystic duct obstruction. In the past, treatment for acalculous cholecystitis has been cholecystectomy; however, many children who are critically ill are Percutaneous cholecystostomy is likely the procedure of choice in this subgroup of patients. To assess the safety and effectiveness of percutaneous cholecystostomy in critically ill and immune-compromised children with acalculous cholecystitis. Retrospective review of immune-compromised and critically ill children who underwent percutaneous cholecystostomy between 2006 and 2013. Diagnostic imaging performed included ultrasound, CT and hepatobiliary scintigraphy. Every percutaneous cholecystostomy was performed using imaging guidance. Ten critically ill and immune-compromised children with acalculous cholecystitis underwent percutaneous cholecystostomy. Seven boys and 3 girls, ranging in age from 10months to 15years 8months, were treated. Six of the immune-compromised children had received a bone marrow transplant for leukemia (5 children) or severe combined immunodeficiency (SCID) (1 child), and ranged from 18 to 307days post bone marrow transplant at the time of their percutaneous cholecystostomy. Of the remaining four immune-compromised children with acalculous cholecystitis who underwent percutaneous cholecystostomy, two had leukemia, one had SCID and lymphoma, and the fourth was undergoing treatment for undifferentiated germ cell tumor. The 10 percutaneous gallbladder drains were placed using a transhepatic approach, except one unintentional transperitoneal approach. There were no complications. Three gallbladder drains were removed in Interventional Radiology. Those three patients had a return to normal gallbladder function and didn't require cholecystectomy. Two drains were removed during cholecystectomy and another as an outpatient. Four patients died in the hospital due to multiorgan system failure, with indwelling gallbladder drains. Percutaneous cholecystostomy is a safe procedure in immune-compromised and critically ill children with acalculous cholecystitis. Percutaneous cholecystostomy may obviate the need for future cholecystectomy.

Abstract 914: Pioglitazone and metformin as potential chemopreventative treatments in Fanconi anemia related oral squamous carcinoma

Abstract Fanconi anemia (FA) is a genetic disorder which leads to bone marrow failure and cancer due to mutations in DNA repair mechanisms. FA is also a solid tumor-prone disease, affecting patients decades earlier and at a rate several hundred-fold higher than the general population necessitating regular cancer surveillance. To better understand the features of post-BMT oral SCC tumors and assist in development of preventative, survival-enhancing therapies for FA patients, we established a cell line and treated it with a panel of emerging chemopreventatives and natural compounds. An adjunct therapy with a minimal side effect profile will help this population in which chemotherapy and radiation are particularly damaging. We established a cell line from a post bone marrow transplant FA patient with a T2N2bM0 oral SCC grown as an adherent monolayer culture. The chemopreventative compound panel we used included Pioglitazone and Metformin. We also tested the synthetic retinoid Bexarotene, and commonly used chemotherapeutics Taxol and Cisplatin. MTT assays determined cell viability after 1, 2, and 3 days of treatment. Clonogenic assays were performed to determine survival and proliferation over 1-2 weeks. Reporter gene assays were performed to determine the effect of treatments on several target genes, to investigate pathway response and potential candidate biomarkers. Our results show Pioglitazone at all concentrations (5, 10, 20, 40μM) decreases cell proliferation compared to solvent controls (P&amp;lt;0.0001). Metformin (1 - 100μM) unexpectedly increased cell proliferation or was otherwise equal in growth with the controls, with the exception of the high dose (10mM) which decreased cell proliferation (P&amp;lt;0.0001). Taxol, Cisplatin, and Bexarotene were also found to decrease cell proliferation in a dose-dependent manner. Clonogenic assays demonstrated dose-dependent decreases in colony forming ability with Pioglitazone and Bexarotene, however no significant changes were shown with Metformin treatments. Reporter gene assays revealed that PPAR ligand binding activity was upregulated in FA cells treated with Pioglitazone (P&amp;lt;0.02 at 40μM, P = 0.0003 at 10μM) compared to controls. Conversely PPAR activity was decreased compared to controls in FA cells treated with Metformin (P&amp;lt;0.0001 for all concentrations of Metformin) and Bexarotene (P&amp;lt;0.0001 for all concentrations of Bexarotene 1, 2, 4μM). Pioglitazone seems to be a promising chemopreventative adjunct treatment for HNSCC in FA patients. Further studies are underway to evaluate the contribution of decreased cell proliferation versus apoptotic mechanisms. Metformin does not appear to decrease tumor cell proliferation at doses up to 100 uM in our FA cell line. Citation Format: Kim Miller, Beverly Wuertz, Frank G. Ondrey. Pioglitazone and metformin as potential chemopreventative treatments in Fanconi anemia related oral squamous carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 914. doi:10.1158/1538-7445.AM2015-914

Donor Derived Myelodysplastic Syndrome in a Pediatric Allogeneic Transplant Recipient

235 Donor Derived Myelodysplastic Syndrome in a Pediatric Allogeneic Transplant Recipient Amos Gaikwad2,3,4,5, Challice L. Bonifant2,3,4, Pulivarthi H Rao2,3,4, Dolores Lopez-Terrada1,4, Michael Cubbage2,3,5, Robert Krance2,4, and Andrea M. Sheehan1,2,4 Departments of Pathology1, Pediatric-Hematology-Oncology2, Texas Children’s Cancer & Hematology Centers3, Baylor College of Medicine4 and Texas Children’s Hospital5 Background: Emergence of leukemic transformation post-bone marrow transplantation (BMT) is reflective of disease recurrence in the vast majority of cases. However, de novo development of myelodysplastic syndrome (MDS) or frank leukemia can also occur in donor derived hematopoietic cells. Though rare, recognition of this entity is critically important given the implications for treatment design and disease monitoring. The phenomenon can also provide a unique platform to study leukemogenesis and the role of bone marrow environment. Utilization of current cytogenetic and flow cytometry techniques can easily and accurately identify dysplastic cell origin and this information should be included in routine disease monitoring post-transplantation. Objective: Presentation of a single case to highlight the diagnosis of donor-derived leukemia based on combined cytogenetic and flow cytometric techniques. Case presentation and results: A 17 year old male patient presented with pancytopenia and was subsequently found to contain 26% blasts in his bone marrow (BM) expressing CD117 and CD13 consistent with acute myeloid leukemia (AML). Cytogenetic analysis showed 32% of the BM cells contained a 13q14.3 deletion. He was treated on the Children’s Oncology Group (COG) protocol AAML0531 (standard arm) and achieved remission after two cyles of chemotherapy. Following completion of his next cycle, he proceeded to a mismatched related donor allogeneic stem cell transplant. His mother was a 5/6 HLA match. He received two CD34+ enriched donor lymphocyte infusions in the post-transplant period to strengthen engraftment. Four years post-transplant, the patient showed 15% blasts in BM expressing CD34 in addition to CD117 and CD13, similar to the initial clone. Cytogenetic analysis demonstrated 99.8% donor cells (XX) with monosomy 7 in 78% of the cells and no evidence of 13q14.3 deletion, indicating a new clone derived from the donor cells. Conclusions: Donor derived leukemia is a rare, but clinically important entity. When considering the presented case, flow cytometry was critical in initially detecting the abnormal blast population as well as the malignant cells post BMT that directed the cytogenetic analysis to examine the donor cells. Therefore, the synergistic review of cytogenetic, and flow cytometric data is necessary to accurately follow disease at diagnosis and at relapse.

Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation.

Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.

Polyomaviruses and disease: is there more to know than viremia and viruria?

Purpose of reviewPolyomavirus nephropathy (PVN) mainly caused by BK virus (BKV) remains the most common productive viral infection of the kidney. Over the past decade, clinical interest often focused on BK viremia and viruria as the diagnostic mainstays of patient management. The purpose of this review is to discuss viral nephropathy in the context of BK viremia and viruria and new strategies to optimize diagnostic accuracy and patient management. The emerging roles of polyomaviruses in oncogenesis, salivary gland disease, and post-bone marrow transplantation as well as novel Polyomavirus strains are highlighted.Recent findingsAreas of investigation include proposals by the Banff working group on the classification of PVN and studies on PVN progression and resolution, including the role cellular immune responses may play during reconstitution injury. New noninvasive strategies to optimize the diagnosis of PVN, that is, the urinary ‘polyomavirus-haufen’ test and mRNA expression levels for BKV in the urine, hold great promise to accurately identify patients with viral nephropathy. Tools are now available to separate ‘presumptive’ from ‘definitive’ disease in various patient cohorts including individuals post-bone marrow transplantation. Recent observations also point to a currently underrecognized role of polyomaviruses in oncogenesis post-transplantation and salivary gland disease in patients with HIV-AIDS.SummaryThis review summarizes recent studies on PVN and the significance of the BKV strain in disease. Current paradigms for patient management post-(renal) transplantation are discussed in the setting of new observations. Issues that still require clarification and further validation are highlighted.

21: Dysplastic Features in Posttransplant Bone Marrow Biopsies May Precede but Not Predict Relapse

21: Dysplastic Features in Posttransplant Bone Marrow Biopsies May Precede but Not Predict Relapse

G488(P) A novel use for eculuzimab: successful treatment of a child with transplant associated microangiopathic haemolytic anaemia

Microangiopathic haemolytic anaemia (MAHA), as part of thrombotic microangiopathy (TMA) post bone marrow transplant, is well documented yet its management continues to challenge clinicians. Outside the transplant setting, classical thrombotic thrombocytopenia is often successfully treated with plasma exchange therapy, but there is evidence to suggest that is not beneficial for post–transplant patients particularly when acute graft-versus-host disease (GVHD) is present. Post–transplant TMA is characterised by MAHA, thrombocytopenia, elevated LDH and abnormal renal function. Fever and neurological impairment may also feature. In children, management has mainly been supportive, with a focus on withdrawing risk factors. The aetiology is unconfirmed and is likely to be multifactorial. Infection, GVHD, irradiation and immunosuppressive drugs have been implicated as has the use of ATG in conditioning and CMV seronegativity. Outside of the transplant setting it is thought that inappropriate complement pathway is central to the development and treatment of TMAs and it has been hypothesised that complement dysregulation caused by ciclosporin may be responsible for some cases of post-transplant TMA. Eculizumab is a monoclonal antibody that acts on the complement pathway by preventing the cleavage of C5 into C5a and C5b, which forms part of a cytotoxic membrane attack complex. Eculizumab is licensed for use in adults and adolescents to treat atypical haemolytic uraemic syndrome and is the standard treatment for paroxysmal nocturnal haemoglobinuria in adults with emerging reports of its use to treat children with the same condition. There is also increasing evidence to support its use in children with atypical haemolytic uraemic syndromeand adult patients with TMA following renal transplant. We present the case of an 18-month-old boy with Mucopolysaccharidosis Type 1 who following a bone marrow transplant, developed anaemia, thrombocytopenia, renal impairment with difficult to manage hypertension and raised LDH. A diagnosis of MAHA was reached and eculizumab was given after minimal improvement was seen after discontinuing ciclosporin. This resulted in an improvement in blood pressure and renal function, reduction in LDH and urine protein–creatinine ratio and reduced need for transfusion.

No evidence of transfusion transmission of Adenovirus and Epstein-Barr virus infections in paediatric recipients post-bone marrow transplant.

Adenovirus and Epstein-Barr virus can cause significant morbidity and mortality in paediatric patients post-bone marrow transplant. The source of infection is thought to be either reactivation of latent viruses or primary infection. We have investigated whether transfusion of blood components from viraemic donors could provide a route of primary infection in these patients and sought the prevalence of viraemia in the blood donor population from England. In 32 linked donor/recipient samples and 300 unselected blood donors, we found no evidence to suggest that these infections in paediatric bone marrow transplant recipients had been acquired from transfused blood components.