Atsushi Fujiki

Intestinal pseudo-obstruction in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) associated with phenytoin therapy

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is most commonly associated with a mitochondrial DNA A to G point mutation at nucleotide 3243 (A3243G) and individuals with the disorder present a wide range of multisystemic symptoms. Seizures in MELAS patients are often intractable and require multiple antiepileptic drugs. Here we report a MELAS patient who presented with acute intestinal pseudo-obstruction following the administration of phenytoin (PHT) as an antiepileptic treatment. She presented with the first stroke-like episode at the age of 6 years and mitochondrial DNA analysis revealed A3243G with 94% mutation load in skeletal muscle. Despite treatment with phenobarbital and clobazam at the age of 16 years, she developed status epilepticus which ceased following PHT infusion. Thereafter, she was started on PHT treatment. One month later, however, she was readmitted because of remarkable abdominal distention. Although abdominal CT showed acute ileus with hepatic portal venous gas mimicking surgical emergency, the abdominal distention gradually recovered over several days following the discontinuation of PHT. Our clinical observations suggest the possibility that intestinal pseudo-obstruction in this patient related to PHT therapy. Careful clinical observation including gastrointestinal symptoms is required in the management of epilepsy in MELAS patients.

Sensitivity of MLL -rearranged AML cells to all- trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region

All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML–RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPɛ and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.

Post-transcriptional modulation of C/EBPα prompts monocytic differentiation and apoptosis in acute myelomonocytic leukaemia cells.

CCAAT/enhancer binding protein alpha (C/EBPα) induction induces monocytic differentiation even in acute myeloid leukaemia (AML). In this study, the induction/activation of C/EBPα in myelomonocytic AML was investigated using a combination of all-trans retinoic acid (ATRA) and RAD001 (Everolimus), a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Combining these agents increased PU.1, C/EBPε and C/EBPα expression, increased the p42/p30 C/EBPα ratio, and decreased C/EBPα phosphorylation at serine 21, and was accompanied by growth inhibition, induction of CD11b expression and apoptosis in AML cell lines. Thus, agents that induce sufficient levels of C/EBPα expression might be useful in treating AML.

Late appearance of a Philadelphia chromosome in a patient with therapy-related acute myeloid leukemia and high expression of EVI1

A 17-year-old boy developed therapy-related acute myeloid leukemia (t-AML) 3 years after the cessation of chemo- and radiotherapy for undifferentiated sarcoma of the liver. At the onset of the t-AML, his white blood cell count was 900/microL with a 46,XY,t(2;3)(p21;q26),del(5)(q?) karyotype. Despite intensive chemotherapy and two hematopoietic stem cell transplants, he died of the leukemia. At the terminal phase, his white blood cell count surpassed 30,000/microL and the Philadelphia (Ph) chromosome appeared. Expression of EVI1 in bone marrow cells was remarkably high at the onset of t-AML, although it was not detected at the end of therapy for the sarcoma. Polymerase chain reaction analysis of bone marrow cells revealed that mRNA for the bcr-abl chimera was negative at the onset of t-AML and positive at the terminal phase. These results suggest that EVI1 overexpression was the major factor contributing to leukemogenesis, and the late appearance of the Ph chromosome is closely associated with the progression to an aggressive form of leukemia.

BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?

Early T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as a high-risk subtype of pediatric T-cell acute lymphoblastic leukemia (T-ALL). Conventional chemotherapy is not fully effective for this subtype of leukemia; therefore, potential therapeutic targets need to be explored. Analysis of the gene expression patterns of the transcription factors in pediatric T-ALL revealed that MEF2C and FLT3 were expressed at higher levels in ETP-ALL than typical T-ALL. Using human T-ALL and BaF3 cell lines with high expression levels of MEF2C, the present study tested whether the BCL2 inhibitor (ABT-737) restores the sensitivity to prednisolone (PSL), because MEF2C causes PSL resistance, possibly by augmenting the anti-apoptotic activity of BCL2. Treatment with PSL and ABT-737 caused a significant reduction in the IC50 of PSL in the MEF2C-expressing LOUCY cells, in addition to the MEF2C-transduced BaF3 cells, but not in the non-MEF2C-expressing Jurkat cells. The combination treatment significantly accelerated the killing of primary leukemic blast cells of ETP-ALL with high expression levels of MEF2C, which were co-cultured with murine stromal cells. These findings suggest that BCL2 inhibitors may be a therapeutic candidate in vivo for patients with ETP-ALL with high expression levels of MEF2C.

Atopic dermatitis and transient hypogammaglobulinemia of infancy improved simultaneously

The serum level of IgG in an infant reduces with the decline of the maternally derived IgG antibody, reaching a nadir at 3 – 4 months of age, and then rises as the infant’s own IgG production gradually increases. 1 Transient hypogammaglobulinemia of infancy (THI) is an extension of this physiological hypogammaglobulinemia. 2,3 Atopic dermatitis (AD) is a common chronic eczematous dermatitis that typically begins during the fi rst 6 months of life. 4 In both THI and infantile AD, many affected patients experience resolution before early childhood. Atopic disease has been suggested to be one of the features of THI in a few reports. Recently, it has been reported that i.v. gammaglobulin treatment for resistant AD was clinically effective and that AD worsened on bacterial infection due to hypogammaglobulinemia. 5 – 8 Although infections are not lifethreatening, in some infants THI may be associated with recurrent infections. 1 – 3 Various studies indicate that bacterial infection, such as Staphylococcus aureus , is one exacerbating factor of AD. 4,9 Therefore an etiological relationship between AD and THI is predictable but no evidence has been produced. In this paper we present fi ve case studies of 4-month-old infants with AD who subsequently improved simultaneously with resolution of concomitant THI.

Successful treatment of relapsed anaplastic large cell lymphoma with vinblastine monotherapy and allo‐HSCT with reduced intensity conditioning regimen

Relapsed anaplastic large cell lymphoma (ALCL) is chemosensitive, but recurrence is common. Although vinblastine (VLB) monotherapy is an effective treatment for relapsed ALCL, the optimal treatment duration is unknown, and some patients experience further relapse after completing the treatment. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is also an effective treatment for relapsed ALCL, although transplant‐related toxicity is a problem. Here, we report an 11‐year‐old patient with relapsed ALCL who underwent induction therapy with VLB monotherapy and achieved complete remission (CR) after 12 courses. CR was confirmed on positron emission tomography–computed tomography. The patient then underwent allo‐HSCT with reduced intensity conditioning (fludarabine, melphalan, and low‐dose total body irradiation). He developed grade II acute graft‐versus‐host disease (GVHD), which was successfully treated with methylprednisolone. There was no evidence of chronic GVHD. He has remained in CR without any complications for 19 months after allo‐HSCT.

Identification of a homozygous JAK3 V674A mutation caused by acquired uniparental disomy in a relapsed early T-cell precursor ALL patient

Investigation of genetic alterations associated with relapse in acute lymphoblastic leukemia (ALL) may help to identify druggable targets for specific therapies. Early T-cell precursor ALL (ETP-ALL) is a subtype of T-ALL with poor prognosis. Although the genetic landscape of ETP-ALL has been determined, genetic alterations related to the relapse of ETP-ALL have not been fully investigated. Here, we report the first patient with relapsed pediatric ETP-ALL to exhibit a homozygous JAK3 activating mutation, V674A, caused by acquired uniparental disomy (UPD). Single nucleotide polymorphism array analysis revealed acquired UPD (aUPD) at the 19p13.3-p12 locus only in leukemic cells at relapse. Sanger sequence of the JAK3 gene, which was located at 19p13.1 and frequently mutated in ETP-ALL, was performed in paired leukemic samples to determine homozygous JAK3 V674A mutation only in relapsed leukemic cells. In contrast, leukemic cells at initial diagnosis harbored hemizygous JAK3 V674A mutation. Further, whole-exome sequencing revealed mutations in 18 genes only in relapsed samples, although none of these was recurrent in T-ALL. These findings suggest that aUPD at 19p13.1 is partly associated with relapse in this patient. Pharmacological inhibition of JAK3 may be therapeutic in such cases.

Acute lymphoblastic leukemia developing in a patient with Noonan syndrome harboring a PTPN11 germline mutation.

Noonan syndrome (NS) is a congenital genetic disorder characterized by certain facial features, short stature, and congenital heart disease. The disorder is caused by genetic alterations in the RAS/MAPK signal pathway. NS patients show a predisposition to malignancy; however, acute lymphoblastic leukemia (ALL) is rarely reported. Here, we describe a NS patient with B-cell precursor ALL (BCP-ALL) harboring a hyperdiploid karyotype and a PTPN11 germline mutation (c.922A>G; p.N308D). We also discuss the relationship between the hyperdiploid karyotype and genetic alterations in the RAS/MAPK pathway in BCP-ALL.

Abstract 5346: Dual role of PAX3-NCOA2 in tumorigenesis of rhabdomyosarcoma

Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults, and can be classified into two major histological subtypes, embryonal RMS and alveolar RMS. Recently, two novel fusion genes, PAX3-NCOA1 and PAX3-NCOA2, were identified in the embryonal and alveolar subtypes. We previously analyzed the complex chromosomal translocation in one case with embryonal RMS by means of spectral karyotyping (SKY) and identified a translocation involving chromosome band 2q35, which is the locus of the PAX3 gene (Hosoi H, Kakazu N, et al. Cancer Genetics and Cytogenetics 189 (2009) 98-104). The patient is alive and in remission at nine years after treatment. We later identified a PAX3 partner gene as NCOA2 using fluorescence in situ hybridization (FISH) and cDNA sequence analysis. Because the role of PAX3-NCOA2 in rhabdomyosarcoma tumorigenesis is unknown, we investigated the biological function of PAX3-NCOA2 in this study. Methods: Mouse myoblast C2C12 cells and human embryonic kidney HEK293 cells were cultured in DMEM containing 10% fetal bovine serum. HEK293 cells were transfected with GFP-PAX3-NCOA2 expression vector and the fusion protein was localized by confocal microscopy. C2C12 cell lines expressing wild type PAX3, PAX3-FOXO1, PAX3-NCOA2 and a C-terminal activation domain deletion mutant of PAX3 were established using a murine stem cell virus (MSCV) retrovirus expression system. Anchorage-independent growth was assessed using soft agar colony formation. Myosin Heavy Chain (MHC), a marker of fully differentiated myocytes, was identified immunohistochemically. Myogenic differentiation was induced by switching the medium to DMEM containing 2% horse serum. Results and Discussion: In transfected HEK293 cells, GFP-PAX3-NCOA2 was observed in the nucleus, consistent with the hypothesis that PAX3-NCOA2 is involved in transcriptional regulation. Expression of PAX3-NCOA2 protein promoted anchorage-independent growth in C2C12 myoblasts. The number of colonies of the PAX3-NCOA2 stable cell line was half the number of colonies of the PAX3-FOXO1 stable cell line. Negligible colony formation was observed in wild-type PAX3 and PAX3 deletion mutant stable cell lines. Expression of PAX3-NCOA2, wild-type PAX3 and PAX3-FOXO1 each blocked the fusion of myoblasts to myotubes in differentiation medium. MHC was not detected in any of the PAX3-NCOA2, wild-type PAX3 or PAX3-FOXO1 stable cell lines. The PAX3-NCOA2 and PAX3-FOXO1 stable cell lines had the comparable phenotype. However, the finding that the PAX3-NCOA2 stable cell line produced fewer colonies than the PAX3-FOXO1 stable cell line may indicate that RMS with PAX3-NCOA2 fusion gene has a less aggressive phenotype and a better prognosis. Conclusions: The PAX3-NCOA2 fusion gene promoted anchorage-independent growth and inhibited myogenic differentiation in mouse myoblasts. These data suggest that PAX3-NCOA2 has a dual role in the tumorigenesis of RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2011-5346