Partially CD3+-Depleted Unrelated and Haploidentical Donor Peripheral Stem Cell Transplantation Has Favorable Graft-versus-Host Disease and Survival Rates in Pediatric Hematologic Malignancy.

Most children who may benefit from stem cell transplantation lack a matched related donor. Alternative donor transplantations with an unrelated donor (URD) or a partially matched related donor (PMRD) carry an increased risk of graft-versus-host-disease (GVHD) and mortality compared with matched related donor transplantations. We hypothesized that a strategy of partial CD3+/CD19+ depletion for URD or PMRD peripheral stem cell transplantation (PSCT) would attenuate the risks of GVHD and mortality. We enrolled 84 pediatric patients with hematologic malignancies at the Children's Hospital of Philadelphia and the Children's Hospital of Wisconsin between April 2005 and February 2015. Two patients (2.4%) experienced primary graft failure. Relapse occurred in 23 patients (27.4%; cumulative incidence 26.3%), and 17 patients (20.2%) experienced nonrelapse mortality (NRM). Grade III-IV acute GVHD was observed in 18 patients (21.4%), and chronic GVHD was observed and graded as limited in 24 patients (35.3%) and extensive in 8 (11.7%). Three-year overall survival (OS) was 61.8% (95% confidence interval [CI], 50.2% to 71.4%) and event-free survival (EFS) was 52.0% (95% CI, 40.3% to 62.4%). Age ≥15 years was associated with decreased OS (P= .05) and EFS (P= .05). Relapse was more common in children in second complete remission (P = .03). Partially CD3+-depleted alternative donor PSCT NRM, OS, and EFS compare favorably with previously published studies of T cell-replete PSCT. Historically, T cell-replete PSCT has been associated with a higher incidence of extensive chronic GVHD compared with limited chronic GVHD, which may explain the comparatively low relapse and NRM rates in our study cohort despite similar overall rates of chronic GVHD. Partial T cell depletion may expand donor options for children with malignant transplantation indications lacking a matched related donor by mitigating, but not eliminating, chronic GVHD.

Sirolimus is effective in relapsed/refractory autoimmune cytopenias: results of a prospective multi-institutional trial.

Patients with autoimmune multilineage cytopenias are often refractory to standard therapies requiring chronic immunosuppression with medications with limited efficacy and high toxicity. We present data on 30 patients treated on a multicenter prospective clinical trial using sirolimus as monotherapy. All children (N = 12) with autoimmune lymphoproliferative syndrome (ALPS) achieved a durable complete response (CR), including rapid improvement in autoimmune disease, lymphadenopathy, and splenomegaly within 1 to 3 months of starting sirolimus. Double-negative T cells were no longer detectable in most, yet other lymphocyte populations were spared, suggesting a targeted effect of sirolimus. We also treated 12 patients with multilineage cytopenias secondary to common variable immunodeficiency (CVID), Evans syndrome (ES), or systemic lupus erythematosus (SLE), and most achieved a CR (N = 8), although the time to CR was often slower than was seen in ALPS. Six children with single-lineage autoimmune cytopenias were treated and only 2 responded. Sirolimus was well tolerated with very few side effects. All of the responding patients have remained on therapy for over 1 year (median, 2 years; range, 1 to 4.5 years). In summary, sirolimus led to CR and durable responses in a majority of children with refractory multilineage autoimmune cytopenias. The responses seen in ALPS patients were profound, suggesting that sirolimus should be considered as a first-line, steroid-sparing treatment of patients needing chronic therapy. The results in other multilineage autoimmune cytopenia cohorts were encouraging, and sirolimus should be considered in children with SLE, ES, and CVID. This trial was registered at www.clinicaltrials.gov as #NCT00392951.

Hematopoietic Stem Cell Transplantation for X-Linked Thrombocytopenia With Mutations in the WAS gene.

X-linked thrombocytopenia (XLT) is a mild form of the Wiskott-Aldrich syndrome (WAS) caused by mutations in the WAS gene. A recent retrospective study of the clinical outcome and molecular basis of a large cohort of XLT patients demonstrated that although overall survival is excellent, event free survival is severely affected with conservative treatment. To answer the question whether hematopoietic stem cell transplantation (HSCT) offers a viable alternative therapeutic option in XLT, we retrospectively investigated the outcome of HSCT in a cohort of 24 XLT patients who received HSCT between 1990 and 2011 at 14 transplant centers in the United States, Italy, Germany, Canada, and Japan. The engraftment rate was 100% and the overall survival rate was 83.3%. Of the four non-survivors, 2 underwent splenectomy prior to HSCT and died of sepsis, and two of aspergillus infections associated with severe GVHD. In all but one patient, pretransplant complications were resolved by HSCT. Our data indicate that HSCT following myeloablative conditioning is curative and associated with acceptable risks as a treatment option for XLT.

Allogeneic hematopoietic cell transplantation for XIAP deficiency: an international survey reveals poor outcomes.

There have been no studies on patient outcome after allogeneic hematopoietic cell transplantation (HCT) in patients with X-linked inhibitor of apoptosis (XIAP) deficiency. To estimate the success of HCT, we conducted an international survey of transplantation outcomes. Data were reported for 19 patients. Seven patients received busulfan-containing myeloablative conditioning (MAC) regimens. Eleven patients underwent reduced intensity conditioning (RIC) regimens predominantly consisting of alemtuzumab, fludarabine, and melphalan. One patient received an intermediate-intensity regimen. Survival was poor in the MAC group, with only 1 patient surviving (14%). Most deaths were from transplantation-related toxicities, including venoocclusive disease and pulmonary hemorrhage. Of the 11 patients who received RIC, 6 are currently surviving at a median of 570 days after HCT (55%). Preparative regimen and HLH activity affected outcomes, and of RIC patients reported to be in remission from HLH, survival is 86% (P = .03). We conclude that MAC regimens should not be used for patients with XIAP deficiency. It is possible that the loss of XIAP and its antiapoptotic functions contributes to the high incidence of toxicities observed with MAC regimens. RIC regimens should be pursued with caution and, if possible, efforts should be made to ensure HLH remission before HCT in these patients.

Use of combination chemotherapy for treatment of granulomatous and lymphocytic interstitial lung disease (GLILD) in patients with common variable immunodeficiency (CVID).

PURPOSE: A subset of patients with common variable immunodeficiency (CVID) develops granulomatous and lymphocytic interstitial lung disease (GLILD), a restrictive lung disease associated with early mortality. The optimal therapy for GLILD is unknown. This study was undertaken to see if rituximab and azathioprine (combination chemotherapy) would improve pulmonary function and/or radiographic abnormalities in patients with CVID and GLILD. METHODS: A retrospective chart review of patients with CVID and GLILD who were treated with combination chemotherapy was performed. Complete pulmonary function tests (PFTs) and high-resolution computed tomography (HRCT) scans of the chest were done prior to therapy and >6 months later. HRCT scans of the chest were blinded, randomized, and scored independently (in pairs) by two radiologists. The differences between pre- and post-treatment HRCT scores and PFT parameters were analyzed. RESULTS: Seven patients with CVID and GLILD met inclusion criteria. Post-treatment increases were noted in both FEV1 (p=0.034) and FVC (p=0.043). HRCT scans of the chest demonstrated improvement in total score (p=0.018), pulmonary consolidations (p=0.041), ground-glass opacities (p=0.020) nodular opacities (p=0.024), and both the presence and extent of bronchial wall thickening (p=0.014, 0.026 respectively). No significant chemotherapy-related complications occurred. CONCLUSIONS: Combination chemotherapy improved pulmonary function and decreased radiographic abnormalities in patients with CVID and GLILD.

Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited.

C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient's serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient's serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient's C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, review the literature on C4-NeF and comment on its detection in the clinical laboratory.

Newborn screening for severe combined immunodeficiency; the Wisconsin experience (2008-2011).

Severe combined immunodeficiency is a life-threatening primary immune deficiency characterized by low numbers of naïve T cells. Early diagnosis and treatment of this disease decreases mortality. In 2008, Wisconsin began newborn screening of infants for severe combined immunodeficiency and other forms of T-cell lymphopenia by the T-cell receptor excision circle assay. In total, 207,696 infants were screened. Seventy-two infants had an abnormal assay. T-cell numbers were normal in 38 infants, abnormal in 33 infants, and not performed in one infant, giving a positive predictive value for T-cell lymphopenia of any cause of 45.83% and a specificity of 99.98%. Five infants with severe combined immunodeficiency/severe T-cell lymphopenia requiring hematopoietic stem cell transplantation or other therapy were detected. In summary, the T-cell receptor excision circle assay is a sensitive and specific test to identify infants with severe combined immunodeficiency and severe T-cell lymphopenia that leads to life-saving therapies such as hematopoietic stem cell transplantation prior to the acquisition of severe infections.

Malignancies after hematopoietic cell transplantation for primary immune deficiencies: a report from the Center for International Blood and Marrow Transplant Research.

We describe the incidence of malignancy in patients with primary immunodeficiency disorders (PIDD) following hematopoietic cell transplantation (HCT). From the Center for International Blood and Marrow Transplant Research, 2266 PIDD patients who had undergone allogeneic HCT between 1968 and 2003 were identified. Patient, disease, and transplant factors for development of malignancy were examined and pathology reports for reported malignancies reviewed independently by a pathologist for confirmation. The incidence of malignancy was highest for Wiskott-Aldrich syndrome (3.3%), with an overall incidence of 2.3% for PIDD. Post-HCT malignancy was confirmed for 52 of 63 reported cases. Forty-five of 52 patients developed posttransplant lymphoproliferative disorders (PTLD) at a median of 3 months post-HCT. Of these, 26 had received T cell-depleted (TCD) bone marrow. Three patients who developed myelodysplastic syndrome had received TCD marrow and total body irradiation. Three patients developed a solid tumor. Patients with PIDD are at a relatively low risk of developing malignancies post-HCT compared with their historic risk of cancer. The most frequent malignancy or lymphoproliferative disorder was early-onset PTLD. As in other HCT recipients, TCD appears to correlate with PTLD development. Our results lend support to the hypothesis that immune reconstitution in PIDD following HCT leads to a decrease in cancer risk.

Making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease.

PURPOSE: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management. METHODS: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management. RESULTS: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling. CONCLUSIONS: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting.

Recent decrease in acute graft-versus-host disease in children with leukemia receiving unrelated donor bone marrow transplants.

Unrelated donor (URD) bone marrow transplantation (BMT) is an effective treatment for leukemia in children, but its success is threatened by graft-versus-host disease (GVHD) and relapse. In this report, we describe the incidence of and risk factors for GVHD over time in children receiving URD BMT. We analyzed outcomes of 638 myeloablative URD BMTs performed between 1990 and 2003 to treat acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia, or myelodysplastic syndrome MDS, using the Center for International Blood and Marrow Transplant Research (CIBMTR) database. All recipients were under age 18 years and had available high-resolution HLA typing for HLA-A, -B, -C, and -DRB1. Overall, 27% of the recipients developed acute GVHD (aGVHD) grade III-IV; the risk was significantly higher in children receiving T cell-replete grafts compared with those receiving T cell-depleted grafts (odds ratio [OR] = 3.12; 95% confidence interval [CI] = 2.02 to 4.83; P < .0001). Acute GVHD significantly reduced the risk of relapse in children with ALL (OR = 0.34; 95% CI = 0.13 to 0.86; P = .0052), but not in those with AML (OR = 0.58; 95% CI = 0.22 to 2.98; P = .26). The risk of aGVHD was higher in children undergoing transplantation in 1990-1998 (n = 365) compared with those doing so in 1999-2003 (OR = 1.93; 95% CI = 1.27 to 2.91; P = .002). We conclude that outcomes have changed significantly over time, with a reduced risk of aGVHD associated with the more recent transplantations.

A scheme for defining cause of death and its application in the T cell depletion trial.

The primary cause of death (COD) provides important information in many studies of hematopoietic stem cell transplantation (HSCT). A panel of experts critically assessed the CODs submitted by 15 transplantation centers for 281 patients who died in a randomized multicenter trial of unrelated HSCT. The panel reviewed the CODs reported by the transplantation centers, which used the Center for International Blood and Marrow Transplant Research and National Marrow Donor Program COD reporting form. The panel determined that the existing criteria for primary and contributing CODs lacked sufficient stringency for uniform interpretation. A hierarchy was developed and applied to the T cell depletion project. Using its scheme, the panel reclassified 157 CODs (56%) reported by the transplantation centers. The changes resulted in increased recognition of graft-versus-host disease as the primary COD and a concomitant decrease in attribution of the primary COD to infection. This algorithm promotes consistent assignment of primary and contributing CODs for patients with leukemia or lymphoma who expire after myeloablative allogeneic HSCT.

Influence of T-cell depletion on chronic graft-versus-host disease: results of a multicenter randomized trial in unrelated marrow donor transplantation.

Donor-derived T cells have been proposed to play a role in pathogenesis of chronic graft-versus-host disease (cGVHD). The impact of ex vivo T-cell depletion (TCD) on cGVHD was analyzed in a randomized multicenter trial involving unrelated donor marrow transplants. A total of 404 patients diagnosed with hematologic malignancies received a total body irradiation-based myeloablative conditioning regimen. GVHD prophylaxis included TCD plus cyclosporine (CSA) or unmodified grafts with CSA plus methotrexate (M/C). Median recipient age was 31.2 years (range, 0.5-55.6 years); median follow-up time since randomization was 4.2 years. The mean number of T cells infused was 1 log lower on the TCD arm. The incidence of cGVHD at 2 years was similar between the TCD and M/C arms, 29% versus 34% (P = .27), respectively. Survival at 3 years from diagnosis of cGVHD was also similar, (TCD 51% versus M/C 58%; P = .29). The proportion of patients with cGVHD who discontinued immunosuppression at 5 years was not different (TCD 72% versus M/C 63%; P = .27), and incidence of serious infections and leukemia relapse were similar on both treatment arms. In spite of a significant reduction of acute GVHD, TCD did not reduce the incidence of cGVHD or improve survival in patients who developed cGVHD.

Unrelated marrow transplantation for children with acute lymphoblastic leukemia in second remission.

Allogeneic bone marrow transplantation (BMT) may be curative for more patients than chemotherapy for the child with relapsed acute lymphoblastic leukemia. This study reviewed the outcomes of 363 children with acute lymphoblastic leukemia in second remission who received unrelated donor BMT from 1988 to 2000 in order to define prognostic factors that affect leukemia-free survival (LFS). Median patient age was 9 years (range, 0-19 years), and median follow-up 29 was months (range, 0-125 months). The median duration of first remission was 24 months (range, 0-109 months). Prognostic factors, including age, duration of first remission, HLA matching, and graft-versus-host (GVH) disease, were analyzed using both univariate and multivariate analyses. Overall survival was 38%, and LFS was 36% at 5 years. LFS was significantly worse for patients 15 years or older (log-rank, P =.009). HLA matching was associated with improved LFS. Acute GVH disease developed in 71%, with 29% having grades III-IV. The incidence of chronic GVH disease was 39% for patients who survived more than 80 days and was significantly higher for female patients receiving marrow from female donors (P =.0009). Transplantation-related mortality was 42% and was associated with HLA mismatches, age 15 years and older, and first remission less than 12 months. The 5-year estimate for relapse was 22%, with first remission at least 6 months associated with a lower risk. Results of unrelated donor BMT appear similar to multi-institutional studies of matched related donor BMT, and this approach appears to be curative for many patients. However, innovative approaches are needed for patients with initial remissions of less than 6 months and for older teenagers.

Production of macrophage migration inhibitory factor by human and murine neuroblastoma.

Tumor cells avoid immune recognition by subverting the ability of the immune system to mount an inflammatory response that generates cytotoxic effector cells. This can be achieved through cytokine production by the tumor itself. Our objective was to determine the cytokine profile of neuroblastoma (NB) lesions in tumor vaccine models. We found that the murine NB cell line, Neuro2a, secretes macrophage migration inhibitory factor, MIF, a multifunctional cytokine with the potential to block effective immune responses to a tumor. Patient-derived NB cell lines were also found to produce MIF. MIF production by NB was documented at the level of RNA by RNAse protection, soluble cytokine production by ELISA, and in a macrophage migration assay. Our studies also confirmed reports of IL-6 production by human NB cell lines. NB culture-derived MIF was also shown to activate tumor cell migration. This supports the hypothesis that MIF is a tumor-derived cytokine that may play a role in NB aggressiveness and evasion of immune recognition.

Monitoring and modulation of Epstein-Barr virus loads in pediatric transplant patients.

A major risk faced by bone-marrow and solid organ transplant patients is the development of post-transplant lymphoproliferative disease or post-transplant lymphoma (PTLD). In pediatric transplantation, PTLD onset is often associated with a rapid rise in Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells (PBMC). We have analyzed EBV viral loads in PBMC over time using real-time quantitative PCR in 56 patients, 19 of which have been followed for more than 1 year. In nine patients; eight bone marrow (BMT) and one kidney transplant, PTLD was associated with a rapid rise in viral load, exceeding 1 x 10(5) EBV genomes/microg of PBMC-derived DNA. Four of these patients exceeded 1 x 10(6) EBV genomes/microg PBMC DNA. All patients with viral loads exceeding 1 x 10(5) EBV genomes/microg PBMC DNA were clearly at high risk for transplant-associated mortality, with only six of nine surviving. Importantly, only one of these deaths was directly attributable to EBV. A second elevated state of EBV load, defined as exceeding 2 x 10(4) EBV genomes/microg PBMC, was seen in a total of 12 BMT, kidney, heart, and liver transplant patients. These patients did not appear to be at immediate lethal risk for PTLD and one EBV-attributable death was found in this group as well. Thirty-four transplant patients whose EBV viral load oscillated from undetectable to 10 000 EBV genomes/microg PBMC DNA are reported as well. The threshold for normal EBV viral load based on our combined experience with viral load analysis is defined as 1 x 10(4) EBV genomes/microg PBMC DNA. The ability to rapidly analyze EBV load allows rapid changes in viral load, such as those that occur with PTLD onset, and the impact of anti-CD20 antibody therapy to be rapidly detected.

Transfusion support with RBCs from an Mk homozygote in a case of autoimmune hemolytic anemia following diphtheria-pertussis-tetanus vaccination.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) in children, although unusual, is often associated with recent infection. Several reports have identified the diphtheria-pertussis-tetanus (DPT) vaccination as a possible trigger for AIHA. STUDY DESIGN AND METHODS: Life-threatening AIHA was diagnosed in a 6-week-old infant 5 days after receiving a DPT vaccination. The patient required daily transfusion and/or exchange transfusion for 3 weeks. RBCs from an Mk homozygote were found compatible with the patient's autoantibody. Transfusion of RBCs from an Mk homozygote and later RBCs from an individual (K.T.) with a variant glycophorin, Mi.VII, were required to sustain the patient's Hb level until autoantibody production ceased, as evidenced by a fall in antibody titer and the patient's Hct returning to normal. RESULTS: The DAT was positive (3+) with only anti-C3 on presentation. An IgM cold reactive autoantibody with probable anti-Pr specificity and high thermal amplitude (37 degrees C) was identified in the serum. The DAT was no longer positive after transfusion with compatible blood. CONCLUSION: This case represents life-threatening AIHA in an infant, temporally related to a DPT injection and responsive to a combination of immunosuppression and transfusion of rare compatible blood.