Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

Who's your data? Primary immune deficiency differential diagnosis prediction via machine learning and data mining of the USIDNET registry.

PURPOSE: There are currently more than 480 primary immune deficiency (PID) diseases and about 7000 rare diseases that together afflict around 1 in every 17 humans. Computational aids based on data mining and machine learning might facilitate the diagnostic task by extracting rules from large datasets and making predictions when faced with new problem cases. In a proof-of-concept data mining study, we aimed to predict PID diagnoses using a supervised machine learning algorithm based on classification tree boosting. METHODS: Through a data query at the USIDNET registry we obtained a database of 2396 patients with common diagnoses of PID, including their clinical and laboratory features. We kept 286 features and all 12 diagnoses to include in the model. We used the XGBoost package with parallel tree boosting for the supervised classification model, and SHAP for variable importance interpretation, on Python v3.7. The patient database was split into training and testing subsets, and after boosting through gradient descent, the predictive model provides measures of diagnostic prediction accuracy and individual feature importance. After a baseline performance test, we used the Class Weighting Hyperparameter, or scale_pos_weight to correct for imbalanced classification. RESULTS: The twelve PID diagnoses were CVID (1098 patients), DiGeorge syndrome, Chronic granulomatous disease, Congenital agammaglobulinemia, PID not otherwise classified, Specific antibody deficiency, Complement deficiency, Hyper-IgM, Leukocyte adhesion deficiency, ectodermal dysplasia with immune deficiency, Severe combined immune deficiency, and Wiskott-Aldrich syndrome. For CVID, the model found an accuracy on the train sample of 0.80, with an area under the ROC curve (AUC) of 0.80, and a Gini coefficient of 0.60. In the test subset, accuracy was 0.76, AUC 0.75, and Gini 0.51. The positive feature value to predict CVID was highest for upper respiratory infections, asthma, autoimmunity and hypogammaglobulinemia. Features with the highest negative predictive value were high IgE, growth delay, abscess, lymphopenia, and congenital heart disease. For the rest of the diagnoses, accuracy stayed between 0.75 and 0.99, AUC 0.46-0.87, Gini 0.07-0.75, and LogLoss 0.09-8.55. DISCUSSION: Clinicians should remember to consider the negative predictive features together with the positives. We are calling this a proof-of-concept study to continue with our explorations. A good performance is encouraging, and feature importance might aid feature selection for future endeavors. In the meantime, we can learn from the rules derived by the model and build a user-friendly decision tree to generate differential diagnoses.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Severe combined immunodeficiency: improved survival leading to detection of underlying liver disease.

BACKGROUND: Adenosine deaminase deficiency (ADA) is an autosomal recessive disorder leading to severe combined immunodeficiency (SCID). It is characterized patho-physiologically by intracellular accumulation of toxic products affecting lymphocytes. Other organ systems are known to be affected causing non-immune abnormalities. We aimed to conduct a cross sectional study to describe liver disease in autosomal recessive ADA-SCID. METHODS: Single center retrospective analysis of genetically confirmed autosomal recessive ADA-SCID was performed. Liver disease was defined as ≥1.5x the gender specific upper limit of normal (ULN; 33 IU/L for males and 25 IU/L for females) alanine aminotransferase (ALT) or moderate and severe increase in liver echogenicity on ultrasound. RESULTS: The cohort included 18 patients with 11 males. The median age was 11.5 (3.5-30.0 years) and median BMI percentile was 75.5 [36.75, 89.5]. All patients received enzyme replacement therapy at the time of evaluation. Seven (38%) and five (27%) patients had gene therapy (GT) and hematopoietic stem cell transplant (HSCT) in the past. Five patients had 1.5x ALT level more than 1.5x the U. Liver echogenicity was mild in 6 (33%), moderate in 2 (11%) and severe in 2 (11%) patients. All patients had normal Fibrosis-4 Index and Non-alcoholic fatty liver disease fibrosis biomarker scores indicating absence of advanced fibrosis in our cohort. Of 5 patients who had liver biopsies, steatohepatitis was noted in 3 patients (NAS score of 3,3,4). DISCUSSION: Non-immunologic manifestations of ADA-SCID have become more apparent in recent years as survival improved. We concluded that steatosis is the most common finding noted in our ADA-SCID cohort.

Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome.

BACKGROUND: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). OBJECTIVE: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. METHODS: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. RESULTS: We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by Th1 effector T cells. CONCLUSIONS: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.

Busulfan Pharmacokinetics in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Gene Therapy.

The pharmacokinetics of low-dose busulfan (BU) were investigated as a nonmyeloablative conditioning regimen for autologous gene therapy (GT) in pediatric subjects with adenosine deaminase-deficient severe combined immunodeficiency disease (ADA SCID). In 3 successive clinical trials, which included either γ-retroviral (γ-RV) or lentiviral (LV) vectors, subjects were conditioned with BU using different dosing nomograms. The first cohort received BU doses based on body surface area (BSA), the second cohort received doses based on actual body weight (ABW), and in the third cohort, therapeutic drug monitoring (TDM) was used to target a specific area under the concentration-time curve (AUC). Neither BSA-based nor ABW-based dosing achieved a consistent cumulative BU AUC; in contrast, TDM-based dosing led to more consistent AUC. BU clearance increased as subject age increased from birth to 18 months. However, weight and age alone were insufficient to accurately predict the dose that would consistently achieve a target AUC. Furthermore, various clinical, laboratory, and genetic factors (eg, genotypes for glutathione-S-transferase isozymes known to participate in BU metabolism) were analyzed, but no single finding predicted subjects with rapid versus slow clearance. Analysis of BU AUC and the postengraftment vector copy number (VCN) in granulocytes, a surrogate marker of the level of engrafted gene-modified hematopoietic stem and progenitor cells (HSPCs), demonstrated gene marking at levels sufficient for therapeutic benefit in the subjects who had achieved the target BU AUC. Although many factors determine the ultimate engraftment following GT, this work demonstrates that the BU AUC correlated with the eventual level of engrafted gene-modified HSPCs within a vector group (γ-RV versus LV), with significantly higher levels of granulocyte VCN in the recipients of LV-modified grafts compared to recipients of γ-RV-transduced grafts. Taken together, these findings provide insight into low-dose BU pharmacokinetics in the unique setting of autologous GT for ADA SCID, and these dosing principles may be applied to future GT trials using low-dose BU to open the bone marrow niche.

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.

Pulmonary Disease Burden in Primary Immune Deficiency Disorders: Data from USIDNET Registry.

PURPOSE: Pulmonary manifestations are common in patients with primary immunodeficiency disorders (PIDs) but the prevalence, specific diseases, and their patterns are not well characterized. METHODS: We conducted a retrospective analysis of pulmonary diseases reported in the database of the United States Immunodeficiency Network (USIDNET), a program of the Immune Deficiency Foundation. PIDs were categorized into 10 groups and their demographics, pulmonary diagnoses and procedures, infections, prophylaxis regimens, and laboratory findings were analyzed. RESULTS: A total of 1937 patients with various PIDs (39.3% of total patients, 49.6% male, average age 37.9 years (SD = 22.4 years)) were noted to have a pulmonary disease comorbidity. Pulmonary diseases were categorized into broad categories: airway (86.8%), parenchymal (18.5%), pleural (4.6%), vascular (4.3%), and other (13.9%) disorders. Common variable immune deficiency (CVID) accounted for almost half of PIDs associated with airway, parenchymal, and other pulmonary disorders. Pulmonary procedures performed in 392 patients were mostly diagnostic (77.3%) or therapeutic (16.3%). These patients were receiving a wide variety of treatments, which included immunoglobulin replacement (82.1%), immunosuppressive (32.2%), anti-inflammatory (12.7%), biologic (9.3%), and cytokine (7.6%)-based therapies. Prophylactic therapy was being given with antibiotics (18.1%), antifungal (3.3%), and antiviral (2.2%) medications, and 7.1% of patients were on long-term oxygen therapy due to advanced lung disease. CONCLUSIONS: Pulmonary manifestations are common in individuals with PID, but long-term pulmonary outcomes are not well known in this group of patients. Further longitudinal follow-up will help to define long-term prognosis of respiratory comorbidities and optimal treatment modalities.

Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID) in the US Immunodeficiency Network (USIDNet) Registry.

Clinical data from ADA-SCID patients registered in the U.S. Immunodeficiency Network (USIDNet) Repository were analyzed. Sixty-four ADA-SCID patients born between 1981 and 2017 had clinical data entered by their local (or home) enrolling institution. Median age at diagnosis was 1 month for those with a positive family history and 3 months for those without a prior family history, with some diagnosed at birth and one as late as 9 years of age. Overall survival was 79.7%, which increased to 94.1% since 2010. These patients had multiple infections and pulmonary, gastrointestinal, and neurological complications. The majority received enzyme replacement therapy (ERT) at some time, including 88% of those born since 2010. Twenty-six patients underwent allogeneic hematopoietic stem cell transplant (HSCT). HSCT successfully supported survival (17/26, 65%) using a variety of cell sources (bone marrow, mobilized peripheral blood, and cord blood) from sibling, family and unrelated donors. Nineteen patients underwent autologous HSCT with gene therapy (GT) using retroviral and lentiviral vectors and all are surviving. The prognosis for patients with ADA-SCID has continued to improve but these patients do have multiple early and potentially long-term conditions that require medical monitoring and management.

Neutropenia Is an Underrecognized Finding in Pediatric Primary Immunodeficiency Diseases: An Analysis of the United States Immunodeficiency Network Registry.

BACKGROUND: The frequency of neutropenia in pediatric primary immunodeficiency disorders (PIDDs) is unknown and potentially underappreciated. Our study aimed to determine the overall frequency and severity of neutropenia in children diagnosed with a PIDD entered in the United States Immunodeficiency Network (USIDNET) patient registry. PROCEDURE: Neutropenia data and demographic/clinical information from 1145 patients younger than 21 years of age was obtained from the USIDNET registry. RESULTS: Neutropenia is more common in PIDD patients entered within the USIDNET registry than previously appreciated. There was a >10% occurrence rate of neutropenia in all broad primary immunodeficiency categories as well as in nearly all individual PIDDs. Neutropenia frequency was greater in African American pediatric PIDD patients than in white or Asian patients. The degree of neutropenia did not associate with mortality in pediatric patients with a PIDD. CONCLUSION: Although our study did not assess the frequency of PIDD in patients presenting with neutropenia, the possibility of a primary immune disorder should be considered in patients with idiopathic neutropenia.

Role of Allogeneic Hematopoietic Stem Cell Transplant for Chronic Granulomatous Disease (CGD): a Report of the United States Immunodeficiency Network.

PURPOSE: Chronic granulomatous disease (CGD) is a primary immunodeficiency for which allogeneic hematopoietic stem cell transplant (HSCT) offers potential cure. Direct comparison of HSCT to non-HSCT management in the North American population was performed to identify clinical factors associated with overall survival (OS) and transplant-related survival (TRS). METHODS: Retrospective review of CGD subjects enrolled in the United States Immunodeficiency Network. Survival was estimated by the Kaplan-Meier method and modeled by proportional hazards regression. RESULTS: We identified 507 patients (66% CYBB mutants) diagnosed in 1953-2016. Fifty underwent allogeneic HSCT. Median follow-up was 9.1 years after diagnosis (0-45.8 years). OS was negatively associated with CYBB mutation (HR = 6.25; p = 0.034) and not associated with HSCT (88% v. 85% ± HCT) (HR = 1.26; p = 0.65). Transplant at ≤ 14 years old was associated with improved TRS (93% v. 82% at T + 60 months) (HR = - 4.51; p = 0.035). Patients transplanted before 15 years old had fewer severe infections pre-HSCT (mean 0.95 v. 2.13; p = 0.047). No mortality was reported in patients receiving stem cells from matched siblings. Infection incidence declined post-HSCT in subjects with greater than or equal to four infections pre-HSCT (p = 0.0010). Compared to non-HSCT patients ≥ 15 years old, post-transplant survivors had higher mean performance score (93.2 v. 85.9; p = 0.0039) and lower frequency of disability (11% v. 52%; p = 0.014). CONCLUSION: Allogeneic HSCT was associated with reduced infection incidence and improved functional performance, but not with a change in overall survival. Transplant-related survival was elevated in patients undergoing HSCT before 15 years old. Consider HSCT prior to late adolescence in patients with severely diminished reactive oxygen intermediate synthesis, particularly if a matched sibling is available.

Cancer in primary immunodeficiency diseases: Cancer incidence in the United States Immune Deficiency Network Registry.

BACKGROUND: We evaluated the overall and site-specific incidence of cancer in subjects with primary immunodeficiency diseases (PIDD) enrolled in the United States Immune Deficiency Network (USIDNET) registry compared with age-adjusted cancer incidence in the Surveillance, Epidemiology and End Results Program (SEER) database. OBJECTIVE: We hypothesized that subjects with PIDD would have an increased incidence of cancer due to impaired immune function. METHODS: Overall and site-specific cancer incidence rates were evaluated in subjects with PIDD (n = 3658) enrolled in the USIDNET registry from 2003 to 2015 and compared with age-adjusted incidence rates in the SEER database. RESULTS: We observed a 1.42-fold excess relative risk of cancer in subjects with PIDD compared with the age-adjusted SEER population (P < .001). Men with PIDD had a 1.91-fold excess relative risk of cancer compared with the age-adjusted male population (P < .001), while women with PIDD had similar overall cancer rates compared with the age-adjusted female population. Of the 4 most common malignancies in men and women in SEER (lung, colon, breast, and prostate cancers), we found no significant increase in these diagnoses in subjects with PIDD. Significant increases in lymphoma in both men (10-fold increase, P < .001) and women (8.34-fold increase, P < .001) with PIDD were observed. CONCLUSIONS: Excess incidence of cancer occurred in subjects with PIDD. An excess of lymphoma in specific PIDD populations principally drove this increased incidence, while no increased risk of the most common solid tumor malignancies was observed. These data point to a restricted role of the immune system in protecting from specific cancers.

Actionable diagnosis of neuroleptospirosis by next-generation sequencing.

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases.

The ability of T-cells to respond to foreign antigens and to appropriately regulate this response is crucial for maintaining immune homeostasis. Using combinatorial peptide libraries, we functionally measured broad T-cell reactivity and observed impaired reactivity in established models of T-cell receptor repertoire restriction and in previously unrecognized disease contexts. By concurrently analyzing T-regulatory and T-effector cells, we show strong functional correlation between these subsets in healthy individuals and, strikingly, that alterations of this balance are associated with T helper type 2 (Th2)-mediated disease in a lymphopenic setting. Finally, we demonstrate that peptide-based priming of polyclonal naive cells with relatively low concentrations skews toward Th2 differentiation. These findings provide unique insight into the pathophysiology and functional consequences of abnormal T-cell repertoires and into differentiation of human naive T-cells.

Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Myeloid dysplasia and bone marrow hypocellularity in adenosine deaminase-deficient severe combined immune deficiency.

Genetic deficiency of adenosine deaminase (ADA) can cause profound lymphopenia and result in the clinical presentation of severe combined immune deficiency (SCID). However, because of the ubiquitous expression of ADA, ADA-deficient patients often present also with nonimmunologic clinical problems, affecting the skeletal, central nervous, endocrine, and gastrointestinal systems. We now report that myeloid dysplasia features and bone marrow hypocellularity are often found in patients with ADA-SCID. As a clinical correlate to this finding, we have observed vulnerability to antibiotic-induced myelotoxicity and prolonged neutropenia after nonmyeloablative chemotherapy. We have also noted that, in the absence of enzyme replacement therapy, absolute neutrophil counts of patients with ADA deficiency vary inversely with the accumulation of deoxynucleotides. These data have significant implications for the application of standard and investigational therapies to patients with ADA-SCID and support further studies to investigate the possibility that ADA deficiency is associated with a stem cell defect. These trials were registered at www.clinicaltrials.gov as #NCT00018018 and #NCT00006319.

Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis.

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.

Multicentric dermatofibrosarcoma protuberans in patients with adenosine deaminase-deficient severe combined immune deficiency.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t[17;22][q22;q13]) resulting in the COL1A1-platelet-derived growth factor ß(PDGFB) fusion gene. This malignancy is rarely diagnosed in childhood. OBJECTIVE: We observed an unexpected high incidence of this DFSP in children affected with adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) and set out to evaluate the association of these 2 clinical entities. METHODS: Twelve patients with ADA-SCID were evaluated with a complete dermatologic examination and skin biopsy when indicated. Conventional cytogenetic and molecular analyses (fluorescence in situ hybridization, RT-PCR, or both) were performed when possible. RESULTS: Eight patients were found to have DFSP. Six patients had multicentric involvement (4-15 lesions), primarily of the trunk and extremities. Most lesions presented as 2- to 15-mm, round atrophic plaques. Nodular lesions were present in 3 patients. In all cases CD34 expression was diffusely positive, and diagnosis was confirmed either by means of cytogenetic analysis, molecular testing, or both. The characteristic DFSP-associated translocation, t(17;22)(q22;q13), was identified in 6 patients; results of fluorescence in situ hybridization were positive for fusion of the COL1A1 and PDGFB loci in 7 patients; and RT-PCR showed the COL1A1-PDGFB fusion transcript in 6 patients. CONCLUSIONS: We describe a previously unrecognized association between ADA-SCID and DFSP with unique features, such as multicentricity and occurrence in early age. We hypothesize that the t(17;22)(q22;q13) translocation that results in dermal overexpression of PDGFB and favors the development of fibrotic tumors might arise because of the known DNA repair defect in patients with ADA-SCID. Although the natural course of DFSP in the setting of ADA-SCID is unknown, this observation should prompt regular screening for DFSP in patients with ADA-SCID.