Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Hematopoietic cell transplant in pediatric acute myeloid leukemia after similar upfront therapy; a comparison of conditioning regimens.

The impact of conditioning regimen prior to hematopoietic cell transplant (HCT) in pediatric AML-patients is not well studied. We retrospectively analyzed the impact of Busulfan-Cyclophosphamide (BuCy), Busulfan-Cyclophosphamide-Melphalan (BuCyMel) and Clofarabine-Fludarabine-Busulfan (CloFluBu) in pediatric AML-patients, with similar upfront leukemia treatment (NOPHO-DBHconsortium), receiving an HCT between 2010 and 2015. Outcomes of interest were LFS, relapse, TRM and GvHD. 103 patients were included; 30 received BuCy, 37 BuCyMel, and 36 CloFluBu. The 5-years LFS was 43.3% (SE ± 9.0) in the BuCy group, 59.2 % (SE ± 8.1) after BuCyMel, and 66.7 % (SE ± 7.9) after CloFluBu. Multivariable Cox regression analysis showed a trend to lower LFS after BuCy compared to CloFluBu (p = 0.07). BuCy was associated with a higher relapse incidence compared to the other regimens (p = 0.06). Younger age was a predictor for relapse (p = 0.02). A strong correlation between Busulfan Therapeutic Drug Monitoring (TDM) and lower incidence of aGvHD (p < 0.001) was found. In conclusion, LFS after BuCyMel and CloFluBu was comparable, lower LFS was found after BuCy, due to higher relapse incidence. CloFluBu was associated with lower incidence of aGvHD, suggesting lower toxicity with this type of conditioning. This finding is also explained by the impact of Busulfan monitoring.

Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma.

BACKGROUND: MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation. METHODS: A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line. RESULTS: The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells. CONCLUSION: The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.