Sharat Chandra

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation

Background: X‐linked hyper‐IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients. Objectives: We sought to collect data on the clinical presentation, treatment, and follow‐up of a large sample of patients with XHIGM to (1) compare long‐term overall survival and general well‐being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT. Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan‐Meier method compared with log‐rank tests and modeled by using proportional hazards regression. Results: Twenty‐eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow‐up and vital status information. Sixty‐seven (38%) patients received HCT. The average follow‐up time was 8.5 ± 7.2 years (range, 0.1‐36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987‐1995; the hazard ratio was significantly less than 1 for diagnosis years 1995‐1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2‐10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft‐versus‐host disease, and most deaths occurred within 1 year of transplantation. Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964‐2013). However, survivors treated with HCT experienced somewhat greater well‐being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates.

Experience with Alemtuzumab, Fludarabine, and Melphalan Reduced-Intensity Conditioning Hematopoietic Cell Transplantation in Patients with Nonmalignant Diseases Reveals Good Outcomes and That the Risk of Mixed Chimerism Depends on Underlying Disease, Stem Cell Source, and Alemtuzumab Regimen

Alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens are increasingly used for the hematopoietic cell transplantation (HCT) of pediatric and young adult patients with nonmalignant diseases. Early experience suggests that these regimens are associated with good survival but a high incidence of mixed chimerism, which we have previously shown to be influenced by the alemtuzumab schedule. We hypothesized that the underlying diagnosis and donor graft source would also affect the development of mixed chimerism and that the majority of patients would survive RIC HCT without graft loss. To examine this, we conducted a retrospective study of 206 patients with metabolic diseases, non-Fanconi anemia marrow failure disorders, and primary immune deficiencies who underwent 210 consecutive RIC HCT procedures at Cincinnati Childrens Hospital. Ninety-seven percent of the patients engrafted. Mixed donor and recipient chimerism developed in 46% of patients. Patients with marrow failure had a low risk of mixed chimerism (hazard ratio [HR], .208; 95% confidence interval [CI], .061 to .709; P = .012). The risk of mixed chimerism was high in patients who received a cord blood graft (HR, 3.122; 95% CI, 1.236 to 7.888; P = .016). As expected, patients who received a proximal or higher dose per kilogram of alemtuzumab schedule also experienced higher rates of mixed chimerism (all HR > 2, all P < .05). At the time of last follow-up (median, 654 days; range, 13 to 3337), over 75% of patients had greater than 90% whole blood donor chimerism. A second transplantation was performed in 5% of patients. Three-year survival without retransplantation was 84% (95% CI, 71% to 98%) for patients who underwent transplantation with an HLA-matched sibling donor. Survival without retransplantation was negatively affected by lack of a matched related donor, increasing age, and development of grades III and IV acute graft-versus-host disease. We conclude that alemtuzumab, fludarabine, and melphalan RIC HCT offers good results for many patients and that the risk of developing mixed chimerism is influenced by underlying diagnosis, graft source, and alemtuzumab dosing.

Immune reconstitution and survival of 100 SCID patients post-hematopoietic cell transplant: A PIDTC natural history study

The Primary Immune Deficiency Treatment Consortium (PIDTC) is enrolling children with severe combined immunodeficiency (SCID) to a prospective natural history study. We analyzed patients treated with allogeneic hematopoietic cell transplantation (HCT) from 2010 to 2014, including 68 patients with typical SCID and 32 with leaky SCID, Omenn syndrome, or reticular dysgenesis. Most (59%) patients were diagnosed by newborn screening or family history. The 2-year overall survival was 90%, but was 95% for those who were infection-free at HCT vs 81% for those with active infection (P = .009). Other factors, including the diagnosis of typical vs leaky SCID/Omenn syndrome, diagnosis via family history or newborn screening, use of preparative chemotherapy, or the type of donor used, did not impact survival. Although 1-year post-HCT median CD4 counts and freedom from IV immunoglobulin were improved after the use of preparative chemotherapy, other immunologic reconstitution parameters were not affected, and the potential for late sequelae in extremely young infants requires additional evaluation. After a T-cell-replete graft, landmark analysis at day +100 post-HCT revealed that CD3 < 300 cells/μL, CD8 < 50 cells/μL, CD45RA < 10%, or a restricted Vβ T-cell receptor repertoire (<13 of 24 families) were associated with the need for a second HCT or death. In the modern era, active infection continues to pose the greatest threat to survival for SCID patients. Although newborn screening has been effective in diagnosing SCID patients early in life, there is an urgent need to identify validated approaches through prospective trials to ensure that patients proceed to HCT infection free. The trial was registered at www.clinicaltrials.gov as #NCT01186913.

K-Cl Cotransporter Gene Expression during Human and Murine Erythroid Differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1–7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

A Prospective Study of Alemtuzumab as a Second-Line Agent for Steroid-Refractory Acute Graft-versus-Host Disease in Pediatric and Young Adult Allogeneic Hematopoietic Stem Cell Transplantation

We describe a single-center prospective study of alemtuzumab as a second-line agent for steroid-refractory (SR) acute graft-versus-host disease (aGVHD) in pediatric and young adult allogeneic hematopoietic stem cell transplant recipients. Alemtuzumab was administered for grades II to IV aGVHD if patients did not improve within 5 days or worsened within 48 hours after corticosteroids. Interim analyses of alemtuzumab levels and response were performed after every 5 patients enrolled, resulting in 3 dosing cohorts, as follows: (1) .2 mg/kg alemtuzumab subcutaneously on days 1 to 5 (maximum of 31 mg over 5 days) and .2 mg/kg/dose (not exceeding 10 mg/dose) on days 15, 22, and 29; (2) .2 mg/kg alemtuzumab subcutaneously on days 1 to 5 (maximum of 43 mg over 5 days) and .2 mg/kg/dose on day 7, 10, 15, 22, and 29; and (3) .2 mg/kg subcutaneously on days 1 to 5 and .2 mg/kg/dose on day 7, 10, 15, and 22. Alemtuzumab levels were assessed before starting alemtuzumab and at days 1, 3, 6, 10, and 14 and weekly until day 99, where day 1 was the day of first alemtuzumab dose. Fifteen patients (median age, 10 years; range, 1.4 to 27) received alemtuzumab for grades II (6%), III (74%), and IV (20%) SR-aGVHD. The overall response rate was 67%, with complete response (CR) in 40%, partial response (PR) in 27%, and no response in 33%. The median day 6 alemtuzumab level was 2.79 µg/mL (interquartile range, 1.34 to 4.89) in patients with CR compared with .62 µg/mL (interquartile range, .25 to 1.45) in patients with PR + no response (P < .05). Ninety percent (n = 9) of patients with a CR or PR reduced corticosteroid doses within 8 weeks from first alemtuzumab dose. Side effects included fever (26%) and transient thrombocytopenia (53%). Asymptomatic viremias occurred in all patients but invasive viral disease occurred in 2 patients. One patient developed Epstein-Barr virus-post-transplantation lymphoproliferative disorder. Eighty percent (n = 12) of patients were alive at 6 months, of whom 53% (n = 8) were free of GVHD whereas 13% (n = 2) developed chronic GVHD. Alemtuzumab is an effective second-line agent for children and young adults with SR-aGVHD. Higher alemtuzumab levels are associated with CR. A real-time dose adjusted alemtuzumab study is needed to further optimize the dose of alemtuzumab in aGVHD.

Angiogenic growth factors augment K–Cl cotransporter expression in erythroid cells via hypoxia‐inducible factor‐1α

The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF‐ and PlGF‐mediated cellular signaling involved VEGF‐R1 and downstream effectors, specifically, PI‐3 kinase, p38 MAP kinase, mTOR, NADPH‐oxidase, JNK kinase, and HIF‐1α. VEGF and PlGF‐mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral‐mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF‐1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease. Am. J. Hematol. 89:273–281, 2014.

SCID genotype and 6-month posttransplant CD4 count predict survival and immune recovery

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4+ and CD4+CD45RA+ cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4+ cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

The genetic landscape of severe combined immunodeficiency in the United States and Canada in the current era (2010-2018)

In a 250 patient cohort from the US and Canada in the current era (2010-2018), we show that over 90% of patients with severe combined immunodeficiency (SCID) can be genetically-characterized.

Micafungin antifungal prophylaxis in children undergoing HSCT: can we give higher doses, less frequently? A pharmacokinetic study

Background Micafungin has a distinct advantage for antifungal prophylaxis in HSCT owing to its better safety profile, specifically in terms of hepatic and renal toxicity. In children, prophylactic micafungin is given as either 1 mg/kg every day or 3 mg/kg every other day. Objectives We performed a prospective single-centre observational study that investigated the pharmacokinetics (PK) of a single 5 mg/kg dose of micafungin in young children undergoing HSCT, to ascertain the eventual feasibility of twice-weekly prophylactic administration. Methods Nine children, ≤10 years of age undergoing HSCT, were enrolled and received a single intravenous dose of 5 mg/kg micafungin. Blood samples were obtained for PK analysis. Micafungin plasma concentration of >0.2 mg/L was chosen for target attainment (i.e. considered adequate prophylactic concentration). In addition, a population PK model was developed based on current and our previous PK study data. We also evaluated PK model-based simulation of PK profiles and target attainment using Monte Carlo simulation, for several dosing scenarios. Results Mean clearance was 15.3 mL/h/kg (range 11.0-21.4 mL/h/kg) and the mean elimination half-life was 11.6 h (range 7.8-16.6 h). The mean concentration at 96 h was 0.11 mg/L (range 0.03-0.26 mg/L). Eleven percent (n = 1) of patients achieved target attainment at the end of 96 h. Simulation data showed that 1 mg/kg daily dosing and 3 mg/kg alternate-day dosing strategies achieved at least 99% and 81% target attainment, respectively, whereas a 5 mg/kg with 3 day-interval dosing strategy resulted in 64%, 72% and 84% target attainments in patients with body weights of 10, 20 and 30 kg, respectively. Conclusions Micafungin at 5 mg/kg dosing did not achieve target attainment at the end of 96 h for antifungal prophylaxis in children undergoing HSCT. Simulation data suggest that a dosing strategy of micafungin at 5 mg/kg every 72 h is more likely to achieve target attainment in children with a higher body weight in comparison with children with a lower body weight. A cautious approach is advisable when using a high, but less frequent, dosing strategy in very young children.

Azacitidine and Sorafenib Therapy in a Pediatric Patient With Refractory Acute Myeloid Leukemia With Monosomy 7 and Somatic PTPN11 Mutation

Monosomy 7 is a well‐documented cytogenetic aberration in pediatric acute myeloid leukemia (AML) and may occur in combinations with molecular abnormalities including PTPN11 mutation. PTPN11 mutations contribute to leukemogenesis through upregulation of Ras pathway signaling. We present the case of a 3‐year‐old female with AML with monosomy 7 and somatic PTPN11 mutation who was refractory to conventional AML chemotherapy but responded to a novel regimen of azacitidine and sorafenib followed by stem cell transplantation. Combination therapy with azacitidine and sorafenib may be an effective therapeutic strategy for patients with AML with Ras pathway abnormalities.