Kunihiko Moriya

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

IDH2 and TP53 mutations are correlated with gliomagenesis in a patient with Maffucci syndrome

We report on a 24‐year‐old woman who was diagnosed as having Maffucci syndrome with anaplastic astrocytoma. We analyzed the IDH1 and IDH2 mutations of enchondroma, hemangioma and anaplastic astrocytoma tissues and the same somatic mosaic mutation in IDH2 gene was identified in all these tissues. In addition, we identified additional mutation of the TP53 gene in anaplastic astrocytoma tissue but not in other benign tumors. This is the first report of the detection of an identical IDH2 mutation in multiple tissues and TP53 mutation in anaplastic astrocytoma in a patient with Maffucci syndrome. This case is unique and supports the IDH2‐dependent genetic pathway and second‐hit model for gliomagenesis.

Somatic BRAF c.1799T>A p.V600E Mosaicism syndrome characterized by a linear syringocystadenoma papilliferum, anaplastic astrocytoma, and ocular abnormalities

Genetic mosaicism for somatic mutations of oncogenes is common in genodermatoses, which can be complicated with extra‐cutaneous abnormalities. Here we describe an infant with a congenital anaplastic astrocytoma, a linear syringocystadenoma papilliferum, and ocular abnormalities. The BRAF c.1799T>A p.V600E mutation was detected in both the brain and skin tumor cells but not in the blood or normal skin cells, suggesting somatic mosaicsism for the mutation. Clinically, the brain tumor gradually became life threatening without any response to conventional chemotherapies including carboplatin, etoposide, and temozolomide. Vemurafenib, a BRAF p.V600E inhibitor, was administered daily after the detection of the BRAF mutation. This single‐agent therapy was dramatically effective against the anaplastic astrocytoma; the tumor regressed, the cerebrospinal fluid cell count and protein levels decreased to normal levels, and hydrocephalus resolved. Moreover, other lesions including a corneal cyst also responded to vemurafenib. The brain tumor continued shrinking after 6 months of treatment. We present a genodermatosis syndrome associated with BRAF c.1799T>A p.V600E mosaicism. This syndrome may represent a new entity in the mosaic RASopathies, partly overlapping with Schimmelpenning‐Feuerstein‐Mims syndrome, which is driven by mosaicism of HRAS and/or KRAS activating mutations. Screening for BRAF c.1799T>A p.V600E is especially useful for those with malignant tumors, because it is one of the most‐druggable targets.

Mesenchymal chondrosarcoma diagnosed on FISH for HEY1‐NCOA2 fusion gene

Mesenchymal chondrosarcoma (MC) is an extremely rare subtype of chondrosarcoma that has a small round‐cell sarcoma with focal cartilaginous differentiation, often with a pericytomatous vascular pattern. The non‐cartilaginous components are usually dominant, and such lesions might be confused with other small round‐cell tumors. Recently, a tumor‐specific HEY1‐NCOA2 fusion gene was identified in MC. Here we report the case of a 9‐year‐old boy who was diagnosed with MC by detection of HEY1‐NCOA2 fusion signals in almost 50% of tumor cells in tissue sections on fluorescence in situ hybridization (FISH). In this way, the tumor was definitively diagnosed as MC. This case suggests that the detection of the HEY1‐NCOA2 fusion gene on FISH is of diagnostic value for MC.

A case series of CAEBV of children and young adults treated with reduced‐intensity conditioning and allogeneic bone marrow transplantation: a single‐center study

Epstein–Barr virus (EBV)‐infected T or NK cells cause chronic active EBV infection (CAEBV). Allogeneic hematopoietic stem cell transplantation (HSCT) is curative treatment for CAEBV patients. However, chemotherapy prior to HSCT and optimal conditioning regimen for allogeneic HSCT are still controversial.

Novel Compound Heterozygous RTEL1 Gene Mutations in a Patient With Hoyeraal‐Hreidarsson Syndrome

To the Editor: Hoyeraal-Hreidarsson syndrome (HHS) is a clinically severe variant of dyskeratosis congenita (DC), which is characterized by a variety of features, including cerebellar hypoplasia, enteropathy, severe immunodeficiency, bone marrow failure (BMF), developmental delay, and typically short telomeres.[1,2] Although germline mutations in DKC1, TERT, TINF2, and PARN genes have been reported, the genetic basis of HHS is largely unclear. Several recent studies have identified a complex array of RTEL1 mutations responsible for the onset of DC/HHS.[3–5] Here, we describe a case of a 2-year-old female who had characteristic clinical features of HHS associated with novel compound heterozygous mutations of the RTEL1 gene. She was admitted at the age of 2 months because she had frequent bloody stool. Lower gastrointestinal series and colon fiberscopy showed rectal stenosis. She had thrombocytopenia and developed severe pancytopenia after varicella zoster infection at the age of 2 years. Bone marrow biopsy demonstrated a severely hypocellular marrow with clusters of hemophagocytosis. In addition to severe BMF, she had intrauterine growth retardation, developmental delay, andmicrocephaly (−5.0 SD). A brain MRI demonstrated cerebellar hypoplasia. The telomere lengths of the patient and her parents determined by Southern blot analysis were shorter than healthy controls, and the patient’s telomere length was shorter compared to her parents’ telomere length. We performed a mutational analysis of genes responsible for DC with the informed consent of the parents and approval of the Tohoku University Ethics Committee. No disease-causing mutation was found in the DKC1, TERT, TINF2, or PARN genes. All protein-coding exons of the RTEL1 gene were sequenced, demonstrating two different heterozygous mutations in the patient: (i) c.2956C>T,which resulted in a substitution of arginine to a stop codon (p.R986X) and (ii) c.3126A > C, which resulted in a substitution of glutamine to histidine (p.Q1042H). We successfully treated her with allogeneic cord blood stem cell transplantation (CBSCT) following reduced intensity conditioning (RIC). The patient has been alive for 2 years after CBSCT, and bone marrow was completely the donortype. However, gastrointestinal tract symptoms due to rectal stenosis remained, and she has been provided with total parenteral nutrition at home. Allogeneic hematopoietic stem cell transplantation (HSCT) is recommended for the management of BMF in patients with DC; however, transplant-related morbidity/mortality is common in myeloablative conditioning regimens. RIC regimens have recently been recommended as the standard for patients withBMFassociatedwithDC for reducing regimen-related toxicity.[6] Long-term follow-up after HSCT is required to conclude optimal conditioning regimens. Recently, genome-wide studies established an association between single-nucleotide polymorphisms in RTEL1 and increased susceptibility to brain tumors.[7] In addition, heterozygous RTEL1 mutations have been associated with familial pulmonary fibrosis.[8] HHS patients rarely develop brain tumor and pulmonary fibrosis, probably because of early death due to immunodeficiency. However, one can envisage that patients who carry a heterozygous RTEL1mutation may be at high risk of cancer and pulmonary disease. Future studies using patientderived RTEL1-deficient cells should help to clarify the multiple roles of RTEL1 as a genomic caretaker in humans.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs(flox/flox);mb1(cre/)(+):Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency

Background: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA‐ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N‐terminal truncations in I&kgr;B&agr; that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor &kgr; light chain enhancer of activated B cells (NF‐&kgr;B) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA‐ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in I&kgr;B&agr; have impaired noncanonical NF‐&kgr;B activity and defective lymphorganogenesis. Objective: We sought to establish genotype‐phenotype correlation in patients with AD EDA‐ID. Methods: A disease severity scoring system was devised. Stability of I&kgr;B&agr; mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF‐&kgr;B signaling in skin‐derived fibroblasts. Results: Disease severity was greater in patients with I&kgr;B&agr; point mutations than in those with truncation mutations. I&kgr;B&agr; point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF‐&kgr;B–dependent IL‐6 secretion and upregulation of the NF‐&kgr;B subunit 2/p100 and RELB proto‐oncogene, NF‐&kgr;B subunit (RelB) components of the noncanonical NF‐&kgr;B pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF‐&kgr;B–driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus &agr;‐lymphotoxin &bgr; receptor–stimulated fibroblasts from patients with point mutations compared with those with truncations. Conclusions: I&kgr;B&agr; point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF‐&kgr;B activity in patients with AD EDA‐ID.

Unilateral Phrenic Nerve Plasy: A Rare Manifestation of Vincristine Neurotoxicity: Correspondence

To the Editor: We read with interest the article written by Dhingra et al. entitled “Unilateral phrenic nerve palsy: A rare manifestation of vincristine neurotoxicity” [1]. Here we present a patient with mesenchymal chondrosarcoma (MC) who developed hiccups repeatedly after receiving vincristine infusion. In addition to our case, several agents such as corticosteroid and cyclophosphamide infusion are also known to cause drug-induced hiccups [2–4]. A 9-y-old boy presented with pain and swelling in the right knee for 1 mo. Limb-sparing operation with wide excision of the tumor and pasteurized autologous bone graft was performed. He was given six courses of adjuvant chemotherapy, of alternative course of combination of vincristine, doxorubicin, cyclophosphamide (VDC), combination of ifosfamide, etoposide (IE), every 3 wk intervals. When he received 1.5 mg/m of vincristine and granisetron as an anti-emesis, intravenously on the first day of the VDC regimen, he developed hiccups for half an hour after vincristine infusion. The event occurred reproducibly, never occurred at IE regimen, when the patient also received granisetron. The hiccups improved spontaneously later without any treatment. The temporal profile of hiccup disappearance, 6 h after vincristine administration spontaneously, might be explained exponential regression curve of blood concentration of vincristine, although the mechanism still remains unknown [5]. Impaired peripheral nerve functions and bowel movement are well known adverse effects caused by vincristine. Vincristine could cause hiccups as a part of combination chemotherapy.

Activation of Notch1 promotes development of human CD8(+) single positive T cells in humanized mice.

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ(null) (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8(+) single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8(+) SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.