Parinda A. Mehta

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia

CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.

Reduced-intensity conditioning significantly improves survival of patients with hemophagocytic lymphohistiocytosis undergoing allogeneic hematopoietic cell transplantation

Recent experience suggests that reduced-intensity conditioning (RIC) regimens can improve the outcomes of patients with hemophagocytic lymphohistiocytosis (HLH) undergoing allogeneic hematopoietic cell transplantation (HCT). However, studies directly comparing RIC to myeloablative conditioning (MAC) regimens are lacking. Forty patients with HLH underwent allogeneic HCT between 2003-2009 at Cincinnati Childrens Hospital. Fourteen patients received MAC consisting of busulfan, cyclophosphamide, and antithymocyte globulin plus or minus etoposide. Twenty-six patients received RIC consisting of fludarabine, melphalan, and alemtuzumab. All patients engrafted. Acute graft-versus-host disease grades II to III occurred in 14% of MAC patients and 8% of RIC patients (P = .3171). Posttransplantation mixed donor/recipient chimerism developed in 18% of MAC patients and 65% of RIC patients (P = .0110). The majority of patients with mixed chimerism received intervention with reduction of immune suppression plus or minus donor lymphocyte infusion or stem cell boost, which stabilized or increased donor contribution to hematopoiesis and prevented relapse of HLH in all but 1 patient. Grade II to III graft-versus-host disease occurred in 5 of 14 RIC patients after donor lymphocyte infusion. The overall estimated 3-year survival after HCT was 43% (confidence interval = ± 26%) for MAC patients and 92% (confidence interval = ± 11%) for RIC patients (P = .0001). We conclude that RIC significantly improves the outcome of patients with HLH undergoing allogeneic HCT.

Reduced-intensity conditioning is effective and safe for transplantation of patients with Shwachman–Diamond syndrome

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment for the BM dysfunction seen in patients with Shwachman–Diamond syndrome (SDS). Historically, these patients have fared poorly with intensive conditioning regimens with increased regimen-related toxicity especially involving the heart and lungs. We report our institutional experience with a reduced-intensity-conditioning protocol in seven patients with SDS and BM aplasia or myelodysplastic syndrome/AML. The preparative regimen consisted of Campath-1H, fludarabine and melphalan. Four patients received matched related marrow and three received unrelated stem cells (two PBSCs and one marrow). All but one was 8 of 8 allele HLA matched. All patients established 100% donor-derived hematopoiesis. No patient in this cohort developed grades III–IV GVHD. One patient had grade II skin GVHD that responded to systemic corticosteroids and one had grade I skin GVHD, treated with topical corticosteroids. Two out of seven patients developed bacterial infections in the early post transplant period. Viral infections were seen in four out of seven patients and were successfully treated with appropriate antiviral therapy. All patients are currently alive. These data indicate that HSCT with reduced-intensity conditioning is feasible in patients with SDS and associated with excellent donor cell engraftment and modest morbidity.

Blood, and Not Urine, BK Viral Load Predicts Renal Outcome in Children with Hemorrhagic Cystitis following Hematopoietic Stem Cell Transplantation

BK virus is a significant cause of hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT). However, its role in nephropathy post-HSCT is less studied. We retrospectively evaluated clinical outcomes in pediatric HSCT patients with hemorrhagic cystitis. Although most of these patients had very high urine BK viral loads (viruria), patients with higher BK plasma loads (viremia) had significant renal dysfunction, a worse clinical course, and decreased survival. Patients with a peak plasma BK viral load of >10,000 copies/mL (high viremia) were more likely to need dialysis and aggressive treatment for hemorrhagic cystitis compared to patients with ≤ 10,000 copies/mL (low viremia). Conversely, most patients with low viremia had only transient elevations in creatinine, and less severe hemorrhagic cystitis that resolved with supportive therapy. Overall survival (OS) at 1 year post-HSCT was 89% in the low viremia group and 48% in the high viremia group. We conclude that the degree of BK viremia, and not viruria, may predict renal, urologic, and overall outcome in the post-HSCT population.

Perforin polymorphism A91V and susceptibility to B-precursor childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

Perforin plays a key role in the cytotoxicity of natural killer and cytotoxic T cells. Genetic mutations in the perforin gene (PRF1) give rise to approximately 30% cases of familial hemophagocytic lymphohistiocytosis. A frequent polymorphism, A91V (C to T transition at position 272), may impair processing of perforin protein to the active form, and has been suggested to increase susceptibility to childhood acute lymphoblastic leukemia (ALL). To investigate the role of A91V in ALL, we genotyped 2272 children with de novo ALL registered on the Pediatric Oncology Group ALL Classification study P9900 and 655 normal controls. Allele frequencies in the controls showed a very low frequency of the variant allele in blacks, 0.7% compared to 4% in white controls. In light of this, analysis was restricted to a comparison of white cases and controls only. Overall genotype frequencies were similar in white ALL cases and normal white controls (P=0.58), indicating that in contrast to the previous report, A91V polymorphism is not associated with increased risk of childhood ALL. PRF1 A91V frequency was significantly increased in children with BCR-ABL positive ALL (24 vs 8.5%; P=0.0048); however, this observation includes a relatively small number of cases and needs further exploration.

Early clinical indicators of transplant-associated thrombotic microangiopathy in pediatric neuroblastoma patients undergoing auto-SCT.

Patients undergoing auto-SCT for neuroblastoma present a unique population to study transplant-associated thrombotic microangiopathy (TA-TMA), due to standardized chemotherapy and later exposure to radiation and cis-retinoic acid (cis-RA). We retrospectively analyzed 20 patients after auto-SCT to evaluate early clinical indicators of TA-TMA. A total of 6 patients developing TA-TMA (30% prevalence) were compared with 14 controls. Four of six patients were diagnosed with TA-TMA by 25 days after auto-SCT. Compared with controls, TA-TMA patients had higher average systolic and diastolic blood pressure levels during high-dose chemotherapy and developed hypertension by day 13 after auto-SCT. Proteinuria was a significant marker for TA-TMA, whereas blood and platelet transfusion requirements were not. Serum creatinine did not differ between groups post transplant. However, patients with TA-TMA had a 60% decrease in renal function from baseline by nuclear glomerular filtration rate, compared with a 25% decrease in those without TA-TMA (P=0.001). There was no TA-TMA-related mortality. Significant complications included end-stage renal disease (n=1) and polyserositis (n=3). Patients with TA-TMA were unable to complete cis-RA therapy after auto-SCT. We suggest that careful attention to blood pressure and urinalysis will assist in the early diagnosis of TA-TMA, whereas serum creatinine seems to be an insensitive marker for this condition.

Cytidine Deaminase Genotype and Toxicity of Cytosine Arabinoside Therapy in Children with Acute Myeloid Leukemia

Cytosine arabinoside (ara‐C) is irreversibly deaminated to a non‐toxic metabolite by cytidine deaminase (CDA). A common polymorphism, A79C, in the gene encoding cytidine deaminase (CDA) changes a lysine residue to glutamine resulting in decreased enzyme activity. CDA A79C genotypes were determined in 457 children with acute myeloid leukaemia (AML) treated on the Children’s Cancer Group (CCG) 2941 and 2961 protocols and analyzed the impact of CDA genotype on therapy outcomes. Postinduction treatment‐related mortality (TRM) was significantly elevated in children with the CC genotype (5‐year TRM 17 ± 13% CC vs. 7 ± 4% AA, 5 ± 4% AC, P = 0·05). This was more notable in children who received idarubicin, fludarabine, ara‐C, and granulocyte colony‐stimulating factor (IDA‐FLAG; ara‐C = 7590 mg/m2) (5‐year TRM 24 ± 21% CC vs. 6 ± 6% AA, 6 ± 7% AC, P = 0·07) as consolidation therapy compared to idarubicin, dexamethasone, cytarabine, thioguanine, etoposide and daunomycin (IDA‐DCTER; ara‐C = 800 mg/m2) (5‐year TRM 15 ± 20% CC vs. 8 ± 6% AA, 4 ± 6% AC; P = 0·29). Relapse‐free survival was non‐significantly increased in children with the CC genotype treated with IDA‐FLAG (76 ± 20% CC vs. 59 ± 12% AA and 55 ± 14% AC; P = 0·40). These data indicate that children with a low activity CDA genotype are at increased risk of TRM with ara‐C based therapy for AML.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Chemotherapy for Myeloid Malignancy in Children With Fanconi Anemia

Children with Fanconi anemia (FA) have a markedly increased risk of developing myeloid malignancies. Historically, patients with FA and myeloid malignancy have extremely poor outcomes. There are currently no clinical trials or case series addressing the use of chemotherapy for children with FA, except in the context of preparative regimens for stem cell transplantation (SCT). In this report we describe the toxicity of a chemotherapy approach for patients with FA and myeloid malignancy to achieve cytoreduction prior to SCT.

Outcomes following hematopoietic cell transplantation for Wiskott–Aldrich syndrome

HLA-identical sibling donor transplantation remains the treatment of choice for Wiskott–Aldrich Syndrome (WAS). Since 1990, utilization of alternative donor sources has increased significantly. We report the hematopoietic cell transplantation (HCT) outcomes of 47 patients with WAS treated at a single center since 1990. Improved outcomes were observed after 2000 despite the increased number of alternative donors. Five-year OS improved from 62.5% (95% CI: 34.9% to 81.1%) to 90.8% (95% CI: 67.7% to 97.6%) for patients transplanted during 1990–2000 and 2001–2009, respectively. In multivariate analysis, transplant era significantly impacted OS (P=0.04), whereas age was only marginally significant (P=0.06, Cox proportional hazard analysis). No TRM occurred within the first 100 days among patients transplanted during 2001–2009 compared with 3/16 during 1990–2000, (P=0.03, Fishers exact test). The extent of HLA mismatch did not significantly affect the incidence of acute GVHD, chronic GVHD or survival. Post-HCT autoimmune cytopenias were frequently diagnosed after 2001: 17/31 (55%) patients. Their occurrence was not associated with transplant donor type (P=0.53), acute GVHD (P=0.74), chronic GVHD (P=0.12), or post-transplant mixed chimerism (P=0.50).