Mitsuteru Hiwatari

Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies

PTPN11 has been identified as a causative gene in Noonan syndrome (NS), responsible for about 50% of cases of NS. Given the association between NS and an increased risk of some malignancies, notably leukemia and probably some solid tumors including neuroblastoma (NB) and rhabdomyosarcoma (RMS), recent studies have reported that gain‐of‐function somatic mutations in PTPN11 occur in some hematological malignancies, especially de novo juvenile myelomonocytic leukemia (JMML) and in some solid tumors such as NB, although at a low frequency. In a screen for mutations of PTPN11 in 7 cell lines and 30 fresh tumors of RMS and in 25 cell lines and 40 fresh tumors of NB, we identified a missense mutation (A72T) in an embryonal RMS patient. In the RMS samples, we also detected mutations of NRAS in 1 cell line and 1 patient; both mutations were in embryonal RMSs and had no PTPN11 mutations. No mutations of PTPN11 were detected in NB. In 95 leukemia cell lines and 261 fresh leukemia samples including 22 JMMLs, 9 kinds of missense mutations were detected in 17 leukemia samples, which included 11 (50.0%) mutations in JMML samples and lower frequencies in other hematological malignancies. Furthermore, we identified 4 (18.2%) NRAS mutations and 1 (4.5%) KRAS mutation in 5 JMML samples, 1 of which had a concomitant PTPN11 mutation. Our data suggest that mutations of PTPN11 as well as RAS play a role in the pathogenesis of not only myeloid hematological malignancies but also a subset of RMS malignancies.

Fusion of an AF4-related gene, LAF4, to MLL in childhood acute lymphoblastic leukemia with t (2 ; 11) (q11 ; q23)

We showed that the LAF4 gene on 2q11.2–12 was fused to the MLL gene on 11q23 in a pediatric patient with CD10 positive acute lymphoblastic leukemia (ALL) having t(2;11)(q11;q23). The LAF4 gene, which encodes a lymphoid nuclear protein of 1227 amino acids with transactivation potential, is thought to have a role in early lymphoid development. The LAF4 protein was homologous to AF4 and AF5q31 proteins that are fused to MLL in infant early pre-B ALL and the breakpoint of LAF4 was located within the region homologous to the transactivation domain of AF4 and AF5q31. Expression of the 8.5-kb LAF4 transcript was detected in the adult heart, brain, and placenta and in the fetal brain. LAF4 expression was found to be higher in ALL cell lines than in AML and Epstein–Barr virus-transformed B-lymphocyte cell lines. These findings suggest that LAF4, AF4 and AF5q31 might define a new family particularly involved in the pathogenesis of 11q23-associated ALL.

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.

Novel missense mutations in the tyrosine kinase domain of the platelet-derived growth factor receptor |[alpha]|(PDGFRA) gene in childhood acute myeloid leukemia with t(8;21)(q22;q22) or inv(16)(p13q22)

Novel missense mutations in the tyrosine kinase domain of the platelet-derived growth factor receptor α ( PDGFRA ) gene in childhood acute myeloid leukemia with t(8;21)(q22;q22) or inv(16)(p13q22)

CBL mutations in infant acute lymphoblastic leukaemia

Infant acute lymphoblastic leukaemia (ALL) is relatively rare, occurring in approximately 2Æ5–5% of cases of childhood ALL (Biondi et al, 2000). Infant ALLs are much more likely to present with high leucocyte counts, hepatosplenomegaly and overt central nervous system (CNS) diseases (Taki et al, 1996). T cell phenotype is much less common in infants, while myeloid antigen co-expression and the absence of CD10 expression are more frequent in infants than in older children with ALL. When molecular techniques [such as fluorescence in situ hybridization (FISH) or Southern blot analysis] are used in addition to karyotype, MLL gene rearrangements (MLL-R) are found in 70–80% of infant ALL compared with only 2–4% of older children with ALL (Taki et al, 1996; Biondi et al, 2000). Thus, infant ALL appears to be biologically distinct from the disease in older children (more than 1 year old). In this regard, recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to result in aberrant tyrosine kinase signalling, which also leads to activation of RAS signalling pathways. So far, we and others have reported that CBL mutations occur in a variety of myeloid neoplasms, including de novo acute myeloid leukaemia (AML) (Caligiuri et al, 2007), myelodysplastic syndrome (MDS), and myeloproliferative neoplasm, especially in chronic myelomonocytic leukaemia (CMML) (Sanada et al, 2009), and juvenile myelomonocytic leukaemia (JMML) (Shiba et al, 2010). The importance of CBL mutations regarding leukaemogenesis is substantially increased. Recently, we found CBL mutation in therapy-related AML with MLL-R (Shiba et al, 2011). Interestingly, the MLL-CBL fusion gene has been reported in a de novo AML case (Fu et al, 2003), and this prompted us to search for possible CBL mutations in infant ALL with MLL-R. Because CBL mutations thus far reported were almost all clustered within exons 8–9 that encode Linker/RING finger domains (Caligiuri et al, 2007; Sanada et al, 2009; Shiba et al, 2010), we confined our mutation analysis to these exons, in which polymerase chain reaction-amplified exons 8–9 were subjected to direct sequencing using an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Branchburg, NJ, USA). The study adhered to the principles of the Helsinki Declaration, and was conducted under the regulations enacted by the Ethics Board of Gunma Children’s Medical Centre. CBL gene analysis was performed in 41 infant ALL patients in which MLL-R was found in 33 patients (80Æ5%), including 15 patients with t(4;11)(q21;q23), 4 with t(9;11)(p22;q23) and 5 with t(11;19)(q23;p13.3). Median age at diagnosis was 4Æ7 months (range, 0–12 months). We also performed CBL gene mutation analysis in 28 B cell precursor (BCP)-ALL patients (age range, 1–14 years). Heterozygous mutations of the CBL gene were identified in 2 (4Æ9%) of 41 infant ALL patients, but not in older children with BCP-ALL. These were located in exon 8 (Fig 1). One patient was a 3-month-old female with t(4;11)(q21;q23) and the other patient was a 6-month-old male with t(11;19)(q23;p13.3). They were registered and treated on two Japanese infant leukaemia protocols, MLL96 and MLL98 respectively (Isoyama et al, 2002; Kosaka et al, 2004). Although strong association between CBL mutations and 11q-acquired uniparental disomy (aUPD) has been reported (Sanada et al, 2009), we did not perform the single nucleotide polymorphism array analysis due to lack of DNA. MLL-R are more frequent in younger infants; up to 90% of infant ALL less than 6 months old at diagnosis have detectable MLL-R compared with 30–50% of infant ALL aged 6–12 months (Taki et al, 1996). MLL-R ALL has a characteristic gene expression profile that significantly differs from that of non-MLL-R BCP-ALL and of AML, confirming that MLL-R ALL is a biologically unique leukaemia subtype.

Novel gain-of-function mutation in the extracellular domain of the PDGFRA gene in infant acute lymphoblastic leukemia with t(4;11)(q21;q23).

Novel gain-of-function mutation in the extracellular domain of the PDGFRA gene in infant acute lymphoblastic leukemia with t(4;11)(q21;q23)

Aggressive transformation of anaplastic large cell lymphoma with increased number of ALK-translocated chromosomes

Pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) usually has a benign clinical course. A small fraction of patients with ALCL develops an aggressive clinical course; however, its etiology remains unclear. Here we report on an ALK-positive ALCL patient, who had complex translocations with TPM3-ALK revealed by RNA sequencing, with a very aggressive clinical course. On admission, the patient had extraordinarily high white blood cell count with double ALK-translocated chromosomes, and subsequently developed a more aggressive transformation with invasion into multiple organs with triple ALK-translocated chromosomes. The aggressive clinical course may have been related to these additional chromosomal aberrations. Our report provides new insights into the clonal evolution in ALCL and suggests the importance of monitoring examinations including fluorescence in situ hybridization analysis.

Neonatal acute megakaryoblastic leukemia mimicking congenital neuroblastoma

We describe a neonate with abdominal distension, massive hepatomegaly, and high serum neuron‐specific enolase level suggestive of congenital neuroblastoma. The patient died of pulmonary hemorrhage after therapy. Autopsy revealed that the tumor cells in the liver indicated acute megakaryocytic leukemia with the RBM15‐MKL1 fusion gene.

Relapsed acute lymphoblastic leukemia with unusual multiple bone invasions: A case report

The present study describes a unique pediatric case with multiple bone invasions of acute lymphoblastic leukemia (ALL) during remission. An eight-year-old male with a history of ALL was admitted complaining of intermittent and migrating pain in the limb 2 years following complete remission. Magnetic resonance imaging and whole-body positron emission tomography with 18F-fluorodeoxyglucose revealed abnormal multifocal involvement in the bones and corresponding soft tissues. Repeated bone marrow (BM) aspiration indicated normal cellular marrow without leukemic cells, and marked leukemic cell infiltration in different sections of the ilium, respectively. These findings suggested isolated bone relapse, and it is probable that systematic BM relapse occurred as a consequence.