Allogeneic stem cell transplantation with reduced intensity conditioning for patients with adrenoleukodystrophy

Objective: The prognosis of adrenoleukodystrophy (ALD)with neurological involvement is generally dismal; however, allogeneic stem cell transplantation (SCT) is recognized as effective to stabilize or improve the clinical symptoms of ALD. Herein, we report the clinical outcomes of patients with ALD who consecutively underwent allogeneic stem cell transplantation with reduced intensity conditioning at our institution. Patients: Sixteen patients with ALD, who were symptomatic (n = 14) or presymptomatic (n = 2), received SCT from 2010 to 2016. The stem cell source was cord blood (n = 14), or bone marrow from a human leukocyte antigen identical sibling (n = 2). The conditioning regimen prior to transplantation was reduced intensity and consisted of fludarabine (125 mg/m2), melphalan (140 mg/m2) and low dose total body irradiation (TBI) of 4Gy (n = 15) or 3Gy (n = 1). Results: Primary engraftment was obtained in 11 patients, and 4 of the 5 patients who lost the primary graft received a second cord blood transplantation and were engrafted. Five years overall and event-free survival were 90.9% and 61.1% respectively, with a median of 45 months (range 16–91). Loes score stabilized or improved by 18 months after transplantation except for patients with internal capsule involvement. Conclusion: Allogeneic SCT with reduced intensity conditioning for patients with ALD was safely performed without major transplant-related complications even in symptomatic patients and neurological symptoms were stabilized after SCT in patients without internal capsule involvement. Keywords: Adrenoleukodystrophy, Allogeneic stem cell transplantation, Loes score, Very long chain fatty aci

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis. Keywords: CD19 CAR-T cell, piggyBac transposon, Integration site mapping, Tag-PC

Simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following 3 groups: high (HM), intermediate (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML were analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing-based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n = 99) using DREAM data identified HM (HM_DREAM; n = 35) and LM subgroups (LM_DREAM; n = 64). Of the 98 cases that could be compared with the international consensus classification, 90 HM (n = 30) and LM (n = 60) cases had 100% concordance with DREAM clustering results. Of the remaining 8 cases comprising the IM group, 4 were classified as belonging to the HM_DREAM group and 4 to the LM_DREAM group. A machine-learning classifier was successfully constructed using a support vector machine (SVM), which divided the validation cohort (n = 38) into HM (HM_SVM, n = 18) and LM (LM_SVM; n = 20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, we developed a robust methylation test using DREAM for patients with JMML. This simple and straightforward test can be easily incorporated into diagnosis to generate a methylation classification for patients so they can receive risk-adapted treatment in the context of future clinical trials