Norihiro Murakami

Recurrent MYB rearrangement in blastic plasmacytoid dendritic cell neoplasm

Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Service d’Hématologie Biologique, GH Pitié-Salpêtrière, Paris, France; Service de Biostatistique et Informatique Médicale, Hôpital Saint Louis, Paris, France.; Département de génétique, GH Pitié-Salpêtrière, Paris, France; INSERM U1170, Institut Gustave Roussy, Villejuif, France; Département d’Hématologie, Hôpital Becquerel, Rouen, France; Pôle d’Hématologie, Hôpital Brabois, Vandoeuvre-les-Nancy, France; Service d’Hématologie Clinique, Hôpital d’Argenteuil, Argenteuil, France and Karyopharm Therapeutics, Newton, MA, USA E-mail: santos.susin@crc.jussieu.fr or florence.nguyen-khac@psl.aphp.fr These authors contributed equally to this work. Senior coauthorship.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow‐up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10‐year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6–20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

A novel IFIH1 mutation in the pincer domain underlies the clinical features of both Aicardi-Goutières and Singleton-Merten syndromes in a single patient

DEAR EDITOR, Gain-of-function mutations in IFIH1 encoding interferon-induced helicase C domain-containing protein 1 were identified in a spectrum of human disease phenotypes including the overlap between Aicardi–Gouti eres syndrome (AGS) and Singleton–Merten syndrome (SMS). Here we describe a case with a novel IFIH1 missense mutation in the pincer domain. Our patient, a Japanese girl, first seen at 7 years of age, is the younger of two siblings born to nonrelated parents with no family history of any similar disorder. At the age of 6 months, she was noticed as having dry skin with mild erythema. She demonstrated erythematous cheeks and ichthyosis on the extremities (Fig. 1a). Since then, she has shown the following clinical features: low height (SD –2 8) and low weight (SD –1 7), osteopenia (Fig. 1d), poor dentition (Fig. 1e), myopia of the right eye and amblyopia of the left eye, and bilateral calcification of the deep frontal lobes and the globus pallidi on computed tomography (Fig. 1f). She has moderate motor and mental retardation. She can walk with another person’s assistance or by using a handrail. At the age of 8 years, she was able to run 30 metres. She can feed herself by using dentures. She can almost use the toilet by herself. She is not always spastic, but she does have a febrile seizure every year. She showed low IgA and IgM, and high IgE and IgG levels. Her ichthyotic skin was improved by bathing with baking soda. Following ethical approval, informed written consent was obtained in compliance with the Declaration of Helsinki guidelines. Whole-exome capture was performed (peripheral blood genomic DNA from the patient and both parents) by in-solution hybridization using SureSelect All Exon 50 Mb Version 5 0 (Agilent, Santa Clara, CA, U.S.A.) followed by massively parallel sequencing (HiSeq2500; Illumina, San Diego, CA, U.S.A.) with 150-bp paired-end reads. In total, 24 717 single-nucleotide substitutions were identified in this patient: 10 031 homozygous and 14 686 heterozygous. After filtering, 14 previously unreported heterozygous variants were found de novo. Within these variants, a nonsynonymous heterozygous mutation was identified in IFIH1 (c.2561T>A; p.Met854Lys), which was confirmed by Sanger sequencing. The mutation had not been described in our in-house database (data for 777 Japanese exomes), nor in the gnomAD Database (http://gnomad.broadinstitute.org/), which includes data for 123 136 whole exomes and 15 496 whole genomes. We confirmed paternity/maternity by single-nucleotide polymorphisms on exome data. In silico analysis with PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and the SIFT (http://sift.jcvi.org/) predict the mutation to be ‘damaging’. We did not identify potentially pathogenic mutations in genes implicated in inherited ichthyoses. A skin biopsy specimen from the right lower leg showed compact hyperkeratosis, parakeratosis with hypogranulosis, a few dyskeratotic cells and mild vacuolar degeneration in the epidermis. A number of facts support the pathogenicity of the present mutation (p.Met854Lys). Firstly, the mutation was absent in both parents, suggesting a de novo condition. Secondly, the mutation affects a residue that is highly conserved among different species (Fig. 1b). The corresponding methionine is a highly conserved amino acid residue that shows homology not only within the IFIH1, but also to human RIG-I and LGP2, which also encode the other antiviral RNA helicase proteins. Thirdly, the mutation lies on the pincer domain, which connects helicase domain 2 (Hel2) and the C-terminal domain (CTD), and is composed of two a helices (aP1–aP2) that emerge from the last b-strand of Hel2. p.Met854Lys is located at the first helix, aP1. Although the pincer is neither an ATPase core domain nor a direct RNA-binding site, it transmits information between Hel2 and CTD, regulating the behaviour of IFIH1, which is essential for RNA sensing of IFIH1. Immunohistochemical stainings revealed that p-STAT3 was strongly expressed in the nuclei of keratinocytes in the patient (Fig. 1h), although there were no significant changes in the staining of IFIH1. The interferon (IFN)-a receptor, the receptor of type I IFN, activates Janus kinase 1 and tyrosine kinase 2. Phosphorylation of the receptor by these kinases results in the recruitment of STAT proteins, phosphorylation, dimerization and nuclear translocation. The three predominant STAT complexes (STAT1, STAT2 and STAT3) that form in response to type I IFN control distinct gene expression programmes. Our observations suggest the possibility that type I IFN signalling is activated in the patient’s epidermis. All 11 previously reported pathogenic mutations in IFIH1 were located in the Hel1 or Hel2 domains and were associated with upregulated type I interferon signalling (a summary of the data is available from the authors. The present proband shows characteristic clinical features of AGS (intracranial

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.

Integrated molecular profiling of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.