Riki Nishimura

Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.

Biallelic DICER1 mutations in sporadic pleuropulmonary blastoma

Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domain was invariably affected. Our results highlight the role of RNase IIIb domain mutations in DICER1 along with TP53 inactivation in PPB pathogenesis.

Aberrant activation of ALK kinase by a novel truncated form ALK protein in neuroblastoma

Anaplastic lymphoma kinase (ALK) was originally identified from a rare subtype of non-Hodgkins lymphomas carrying t(2;5)(p23;q35) translocation, where ALK was constitutively activated as a result of a fusion with nucleophosmin (NPM). Aberrant ALK fusion proteins were also generated in inflammatory fibrosarcoma and a subset of non-small-cell lung cancers, and these proteins are implicated in their pathogenesis. Recently, ALK has been demonstrated to be constitutively activated by gene mutations and/or amplifications in sporadic as well as familial cases of neuroblastoma. Here we describe another mechanism of aberrant ALK activation observed in a neuroblastoma-derived cell line (NB-1), in which a short-form ALK protein (ALKdel2-3) having a truncated extracellular domain is overexpressed because of amplification of an abnormal ALK gene that lacks exons 2 and 3. ALKdel2-3 was autophosphorylated in NB-1 cells as well as in ALKdel2-3-transduced cells and exhibited enhanced in vitro kinase activity compared with the wild-type kinase. ALKdel2-3-transduced NIH3T3 cells exhibited increased colony-forming capacity in soft agar and tumorigenicity in nude mice. RNAi-mediated ALK knockdown resulted in the growth suppression of ALKdel2-3-expressing cells, arguing for the oncogenic role of this mutant. Our findings provide a novel insight into the mechanism of deregulation of the ALK kinase and its roles in neuroblastoma pathogenesis.

IDH1 and IDH2 mutations are rare in pediatric myeloid malignancies

Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993; 73: 703–711. 4 Maciejewski JP, Mufti GJ. Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood 2008; 112: 965–974. 5 Gondek LP, Tiu R, Haddad AS, O’Keefe CL, Sekeres MA, Theil KS et al. Single nucleotide polymorphism arrays complement metaphase cytogenetics in detection of new chromosomal lesions in MDS. Leukemia 2007; 21: 2058–2061. 6 Bessler M, Mason P, Hillmen P, Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. Lancet 1994; 343: 951–953. 7 Endo M, Ware RE, Vreeke TM, Singh SP, Howard TA, Tomita A et al. Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria. Blood 1996; 87: 2546–2557. 8 Young NS, Maciejewski JP. Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes ‘Why? Why? Why?’. J Clin Invest 2000; 106: 637–641.

Aberrations of NEGR1 on 1p31 and MYEOV on 11q13 in neuroblastoma.

MYEOV and NEGR1 are novel candidate gene targets in neuroblastoma that were identified by chromosomal gain in 11q13 and loss in 1p31, respectively, through single nucleotide polymorphism array analysis. In the present study, to assess the involvement of MYEOV and NEGR1 in the pathogenesis of neuroblastoma, we analyzed their mutation status and/or expression profiles in a panel of 55 neuroblastoma samples, including 25 cell lines, followed by additional functional studies. No tumor‐specific mutations of MYEOV or NEGR1 were identified in our case series. Expression of MYEOV was upregulated in 11 of 25 cell lines (44%) and in seven of 20 fresh tumors (35%). The siRNA‐mediated knockdown of MYEOV in NB‐19 cells, which exhibit high expression of MYEOV, resulted in a significant decrease in cell proliferation (P = 0.0027). Conversely, expression studies of NEGR1 revealed significantly lower expression of this gene in neuroblastomas at an advanced stage of the disease. Exogenous NEGR1 expression in neuroblastoma cells induced significant inhibition of cell growth (P = 0.019). The results of these studies provide supporting evidence for MYEOV and NEGR1 as gene targets of 11q13 gains and 1p31 deletions in a neuroblastoma subset. In addition, the findings suggest a possible prognostic value for NEGR1 in neuroblastoma. (Cancer Sci 2011; 102: 1645–1650)

Characterization of genetic lesions in rhabdomyosarcoma using a high-density single nucleotide polymorphism array.

Rhabdomyosarcoma (RMS) is a common solid tumor in childhood divided into two histological subtypes, embryonal (ERMS) and alveolar (ARMS). The ARMS subtype shows aggressive clinical behavior with poor prognosis, while the ERMS subtype has a more favorable outcome. Because of the rarity, diagnostic diversity and heterogeneity of this tumor, its etiology remains to be completely elucidated. Thus, to identify genetic alterations associated with RMS development, we performed single nucleotide polymorphism array analyses of 55 RMS samples including eight RMS‐derived cell lines. The ERMS subtype was characterized by hyperploidy, significantly associated with gains of chromosomes 2, 8 and 12, whereas the majority of ARMS cases exhibited near‐diploid copy number profiles. Loss of heterozygosity of 15q was detected in 45.5% of ARMS that had been unrecognized in RMS to date. Novel amplifications were also detected, including IRS2 locus in two fusion‐positive tumors, and KRAS or NRAS loci in three ERMS cases. Of note, gain of 13q was significantly associated with good patient outcome in ERMS. We also identified possible application of an ALK inhibitor to RMS, as ALK amplification and frequent expression of ALK were detected in our RMS cohort. These findings enhance our understanding of the genetic mechanisms underlying RMS pathogenesis and support further studies for therapeutic development of RMS.

Skin and subcutaneous blood flows of very low birth weight infants during the first 3 postnatal days

Objective. To determine whether skin and subcutaneous blood flow measurements using a novel laser Doppler flow meter are useful for evaluating the cardiovascular status of very low birth weight (VLBW) infants during the early postnatal period. Methods. In eight VLBW infants and eight non-VLBW infants born at Tokyo University Hospital between May 2007 and April 2008, forehead and lower limb skin blood flows were measured continuously for 72 h. Data were averaged every 8 h, and the t-test was used for analysis. Results. In VLBW infants, forehead blood flow started to increase from the start of measurement to 32 h (16.6 ± 3.9 ml/min vs. 24.1 ± 2.1 ml/min; p = 0.002 compared with 8 h) and remained constant thereafter. Lower limb blood flow increased rapidly after 24 h (22.2 ± 5.5 ml/min vs. 29.5 ± 5.0 ml/min; p = 0.002 compared with 8 h) and continued increasing thereafter. In contrast, blood flows remained constant in non-VLBW infants. Conclusions. The results showed that skin and subcutaneous perfusion in VLBW infants increased spontaneously at around 24 h. Differences in blood flow changes between VLBW and non-VLBW infants demonstrate that these parameters successfully identified physiological changes in tissue perfusion in VLBW infants.

Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays

Malignant rhabdoid tumor (MRT) is a rare and highly lethal cancer that mainly affects infants and young children. The majority of MRT are characterized by loss of function of SMARCB1 on chromosome 22q11.2. However, little is known about genetic changes other than SMARCB1 alterations that are responsible for the development and/or progression of MRT. To explore additional gene targets in MRT, we analyzed 21 MRT specimens (12 fresh tumors and 9 MRT‐derived cell lines) using high‐density single nucleotide polymorphism genotyping microarrays. Although MRT genomes are characterized by common 22q11.2 deletions, affecting the SMARCB1 locus with a frequency of 95.2% (20/21 specimens), other genetic changes have been less frequent. Of the 20 specimens with deletions of 22q11.2, eight specimens showed uniparental disomy of the SMARCB1 locus with homozygous deletions or gene mutations. High‐resolution analysis also disclosed the recurrent hemizygous/homozygous deletions of 7q35–q36.1, involving the CNTNAP2 locus in three specimens. Mutations analysis of CNTNAP2 showed a novel R157C missense mutation in a primary case, and methylation analysis showed recurrent hypermethylation of CNTNAP2 in three of nine cell lines. These results demonstrated that CNTNAP2 is one of the additional gene targets, other than SMARCB1, in MRT.

Identification of the genetic and clinical characteristics of neuroblastomas using genome-wide analysis

To provide better insight into the genetic signatures of neuroblastomas, we analyzed 500 neuroblastomas (included specimens from JNBSG) using targeted-deep sequencing for 10 neuroblastoma-related genes and SNP arrays analysis. ALK expression was evaluated using immunohistochemical analysis in 259 samples. Based on genetic alterations, the following 6 subgroups were identified: groups A (ALK abnormalities), B (other gene mutations), C (MYCN amplification), D (11q loss of heterozygosity [LOH]), E (at least 1 copy number variants), and F (no genetic changes). Groups A to D showed advanced disease and poor prognosis, whereas groups E and F showed excellent prognosis. Intriguingly, in group A, MYCN amplification was not a significant prognostic marker, while high ALK expression was a relevant indicator for prognosis (P = 0.033). Notably, the co-existence of MYCN amplification and 1p LOH, and the co-deletion of 3p and 11q were significant predictors of relapse (P = 0.043 and P = 0.040). Additionally, 6q/8p LOH and 17q gain were promising indicators of survival in patients older than 5 years, and 1p, 4p, and 11q LOH potentially contributed to outcome prediction in the intermediate-risk group. Our genetic overview clarifies the clinical impact of genetic signatures and aids in the better understanding of genetic basis of neuroblastoma.

Cytokine Profiles in Pericardial Effusion in a Down Syndrome Infant with Transient Abnormal Myelopoiesis

Infants with Down Syndrome (DS) are at risk of developing a transient abnormal myelopoiesis (TAM). TAM is characterised by increased circulating blast cells but usually self-limiting. DS patients with TAM sometimes show fetal hydrops and effusion in body cavities, but the mechanism remains unclear. We report here a case of infant with DS who had pericardial effusion, TAM, and eosinophilia. In her pericardial effusion, white blood cell count was 6.0 × 103/µL, 41% of which were eosinophils. After administration of prednisolone, pericardial effusion gradually decreased, and TAM and eosinophilia improved. In order to elucidate the immunological mechanism, we measured the levels of 17 cytokines in her pericardial effusion fluid and serum. In her pericardial fluid, there were high levels of 12 cytokines, and they were higher than those in her serum. In particular, IL-6 (44,573 pg/mL), IL-8 (4,865 pg/mL), and IL-13 (579.41 pg/mL) were at extremely high levels in her pericardial fluid. After administration of prednisolone, the levels of 8 of the 12 elevated cytokines in her pericardial fluid decreased and all of the elevated cytokines decreased in her serum. Corticosteroids can be effective to reduce cytokine levels and the amount of effusion in patients with DS. It is presumed that effusion seen in DS with TAM could be related to an abnormal production of cytokines at the effusion site.