Sandhya Kharbanda

Stable Long-Term Donor Engraftment following Reduced-Intensity Hematopoietic Cell Transplantation for Sickle Cell Disease

Reduced-intensity conditioning (RIC) regimens have the potential to decrease toxicities related to hematopoietic stem cell transplantation (HCT) in patients with sickle cell disease (SCD) and thus make HCT a more acceptable therapeutic option for this group of patients. We report the results of 7 patients enrolled on a study to evaluate safety and efficacy of HCT using bone marrow from an HLA matched sibling donor following an RIC regimen for patients with high-risk SCD. The conditioning regimen consisted of busulfan, fludarabine, equine antithymocyte globulin, and total lymphoid irradiation with shielding of the liver, lungs, heart, and gonads on day 1. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mycophenolate mofetil. The regimen was well tolerated, and all patients had hematopoietic recovery. Six of 7 patients are stably engrafted off immunosuppression and without sickle cell-related symptoms at 2 to 8.5 years after HCT. Consistent with the complete resolution of SCD related symptoms observed in the 6 engrafted patients, erythropoiesis of complete or predominantly donor origin was detected by red blood cell-specific chimerism assays, despite their having persistent mixed chimerism in the mononuclear and lymphoid compartments. These findings demonstrate the curative potential of allogeneic HCT after an RIC regimen in patients with SCD.

Unrelated donor allogeneic hematopoietic stem cell transplantation for patients with hemoglobinopathies using a reduced-intensity conditioning regimen and third-party mesenchymal stromal cells

Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m(2)), melphalan (140 mg/m(2)), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward.

Gene Expression Analysis in Radiotherapy Patients and C57BL/6 Mice as a Measure of Exposure to Ionizing Radiation

Abstract Dose assessment after radiological disasters is imperative to decrease mortality through rationally directed medical intervention. Our goal was to identify biomarkers capable of qualitative (nonirradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure. Using real-time quantitative PCR, biodosimetry genes were identified in blood samples from cancer patients undergoing total-body irradiation. Time- (5, 12, 23, 48 h) and dose- (0–8 Gy) dependent changes in gene expression were examined in C57BL/6 mice. A training set was used to derive weighted voting classification algorithms (nonirradiated/irradiated) and continuous regression (dose assessment) models that were tested in a separate validation set of mice. Of eight biodosimetry genes identified in cancer patients (ACTA2, BBC3, CCNG1, CDKN1A, GADD45A, MDK, SERPINE1, Tnfrsf10b), expression of BBC3, CCNG1, CDKN1A, SERPINE1 and Tnfrsf10b was significantly (P < 0.05) increased in irradiated mice. CCNG1 and CDKN1A expression segregated irradiated mice from controls with an accuracy, specificity and sensitivity of 96.3, 100.0 and 94.4%, respectively, at 48 h. Multiple linear regression analysis predicted doses for the 0-, 1-, 2-, 4-, 6- and 8-Gy treatment groups as 0.0 ± 0.2, 1.6 ± 1.0, 2.9 ± 1.4, 5.1 ± 2.0, 5.3 ± 0.7 and 10.5 ± 5.6 Gy, respectively. These results suggest that gene expression analysis could be incorporated into biodosimetry protocols for qualitative and quantitative assessment of radiation exposure.

Improved Outcomes after Autologous Bone Marrow Transplantation for Children with Relapsed or Refractory Hodgkin Lymphoma: Twenty Years Experience at a Single Institution

The purpose of this study is to evaluate the survival of pediatric patients undergoing autologous bone marrow transplantation (auBMT) for relapsed or refractory Hodgkin lymphoma (rrHL) and to identify factors that might contribute to their outcome. We reviewed the records and clinical course of 89 consecutive rrHL patients ≤ 21 years old who underwent auBMT at Stanford Hospitals and Clinics and the Lucile Packard Childrens Hospital, Stanford between 1989 and 2012. We investigated, by multiple analyses, patient, disease, and treatment characteristics associated with outcome. Endpoints were 5-year overall and event-free survival. Our findings include that cyclophosphamide, carmustine, and etoposide (CBV) as a conditioning regimen for auBMT is effective for most patients ≤ 21 years old with rrHL (5-year overall survival, 71%). Transplantation after the year 2001 was associated with significantly improved overall survival compared with our earlier experience (80% compared with 65%). Patients with multiply relapsed disease or with disease not responsive to initial therapy fared less well compared with those with response to initial therapy or after first relapse. Administration of post-auBMT consolidative radiotherapy (cRT) also appears to contribute to improved survival. We are able to conclude that high-dose chemotherapy with CBV followed by auBMT is effective for the treatment of rrHL in children and adolescents. Survival for patients who undergo auBMT for rrHL has improved significantly. This improvement may be because of patient selection and improvements in utilization of radiotherapy rather than improvements in chemotherapy. Further investigation is needed to describe the role of auBMT across the entire spectrum of patients with rrHL and to identify the most appropriate preparative regimen with or without cRT therapy in the treatment of rrHL in young patients.

Humans are colonized by many uncharacterized and highly divergent microbes

Blood circulates throughout the entire body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analysing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype

Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

A pediatric case of T-cell prolymphocytic leukemia.

T‐cell Prolymphocytic Leukemia (T‐PLL) is a rare entity, and to date has never been reported in children. Here, we describe the first pediatric case of T‐PLL in a 16‐year old male and review his clinical course through treatment. He underwent therapy with alemtuzumab and pentostatin, which was successful in inducing initial remission. He then underwent an allogeneic matched sibling stem cell transplant following a myeloablative conditioning regimen and remains disease‐free 1.5 years after diagnosis. Pediatr Blood Cancer 2015;62:1061–1062.

Pericardial Effusion Following Hematopoietic Cell Transplantation in Children and Young Adults Is Associated with Increased Risk of Mortality

Hematopoietic cell transplantation (HCT) is curative for many pediatric malignant and nonmalignant disorders but is associated with significant morbidity and mortality, including the development of pericardial effusion (PEF). We report the results of a retrospective chart review performed to assess the incidence, risk factors, and prognostic significance of PEF in pediatric HCT recipient at Lucile Packard Childrens Hospital of Stanford University. A total of 119 patients undergoing HCT between January 2010 and December 2013 were selected through the hospitals Pediatric Stem Cell Transplant Program database. A retrospective chart review, including review of documentation, correspondence, imaging reports, laboratory values, and death records, was performed to collect data. The overall incidence of PEF in our population was 21%. Risk factors for the development of PEF included unrelated donor transplants and cord blood as the stem cell source (P = .005), whereas HLA mismatch approached significance (P = .05). The risk for development of PEF was found to not be significantly associated with acute or chronic graft-versus-host disease (GVHD), age at transplantation, sex, conditioning regimen, or viral reactivation status. Of interest, 6 of the 119 patients were found to have transplant-associated thrombotic microangiopathy (TA-TMA). Four of those 6 patients developed PEF, suggesting TA-TMA as a risk factor for PEF. Eight of the 25 patients who developed PEF (32%) required pericardiocentesis. Five out of the 8 patients requiring pericardiocentesis died owing to causes unrelated to the procedure or to PEF itself. Pericardial fluid testing in 4 of these patients (50%) was positive for human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, and/or adenovirus. Finally, of significant interest, patients with PEF had a statistically significant higher likelihood of mortality compared with those without PEF (44% versus 17%; P = .007).

Invasive Fungal Disease in Pediatric Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant

Invasive fungal disease (IFD) remains a major cause of morbidity and mortality in pediatric patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed the outcome of 152 consecutive pediatric patients who underwent allogeneic HSCT from 2005 to 2012: 126 of these without a history of IFD and 26 with IFD before HSCT. Antifungal prophylaxis agent was determined by the primary transplant attending. The rate of IFD after HSCT among patients with or without prior IFD was similar (7.7% with and 7.1% without a history of fungal disease before transplant). Mortality in these 2 populations did not differ (35% vs. 28%, P=0.48, &khgr;2). Patients deemed at higher risk for IFD were generally placed on voriconazole prophylaxis; however, this did not affect rates of posttransplant IFD. All-cause mortality in patients with posttransplant IFD was significantly higher than those without posttransplant IFD (67% vs. 21%, P<0.0001,&khgr;2). Identifying risk factors for posttransplant IFD remains a high priority to improve outcome of HSCT.

BDCM: a novel B‐cell line with genetic and functional similarity to dendritic cells

Summary. We describe a B‐cell line, BDCM, which arose spontaneously from culture of a pheresis product from a patient with M5a myeloid leukaemia. Cell growth was associated with autocrine activation of the signal transducer and activator of transcription 1 (Stat‐1) transcription factor. Although the cells expressed several B‐cell surface markers and had a rearranged immunoglobin J region, they also exhibited several characteristics associated with dendritic cells. These included extensive surface projections, cross‐priming ability and strong T cell‐stimulating capability. In addition, the cytokine production profile of BDCM cells was nearly identical to that of mature monocyte‐derived dendritic cells.