Kentaro Ohki

Langerhans cell histiocytosis with multifocal bone lesions: comparative clinical features between single and multi-systems

Langerhans cell histiocytosis (LCH) can be a single system or multi-system disease. Both disease types can be associated with multi-focal bone lesions, but their bone involvement patterns have not been compared systematically. Of the new pediatric LCH cases enrolled into the JLSG-02 study during 2002–2007, 67 cases of single system multifocal bone (SMFB) LCH and 97 cases of multi-system bone (MSB) LCH were analyzed to determine if the bone involvement patterns differ in these two types, and whether these differences correlate with outcome. Statistical analysis was performed with Mann–Whitney U test, Fisher’s exact test, and other measures. Onset ages were higher for SMFB (P < 0.001), but the two types did not differ in the number of bone lesions per patient. The skull was most frequently affected in both types, followed by the spine. Lesions in the temporal bone (P = 0.002), ear-petrous bone (P < 0.001), orbita (P = 0.003), and zygomatic bone (P = 0.016) were significantly more common in MSB. The two types did not differ in response to treatment, but MSB was associated with a significantly higher incidence of diabetes insipidus (DI) (P < 0.001). Novel measures are required in preventing the development of DI in MSB-type LCH patients with “risk” bone lesions.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase–PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

Mutational analyses of the ATP6V1B1 and ATP6V0A4 genes in patients with primary distal renal tubular acidosis

BACKGROUND Mutations in the ATP6V1B1 and the ATP6V0A4 genes cause primary autosomal-recessive distal renal tubular acidosis (dRTA). Large deletions of either gene in patients with dRTA have not been described. METHODS The ATP6V1B1 and ATP6V0A4 genes were directly sequenced in 11 Japanese patients with primary dRTA from nine unrelated kindreds. Large heterozygous deletions were analyzed by quantitative real-time polymerase chain reaction (PCR). The clinical features of the 11 patients were also investigated. RESULTS Novel mutations in the ATP6V1B1 gene were identified in two kindreds, including frameshift, in-frame insertion and nonsense mutations. Large deletions in the ATP6V0A4 gene were identified in two kindreds. Exon 15 of ATP6V0A4 was not amplified in one patient, with a long PCR confirming compound heterozygous deletions of 3.7- and 6.9-kb nucleotides, including all of exon 15. Direct DNA sequencing revealed a heterozygous frameshift mutation in ATP6V0A4 in another patient, with quantitative real-time PCR indicating that all exons up to exon 8 were deleted in one allele. Clinical investigation showed that four of the six patients with available clinical data presented with hyperammonemia at onset. CONCLUSIONS To our knowledge, these dRTA patients are the first to show large deletions involving one or more entire exons of the ATP6V0A4 gene. Quantitative PCR amplification may be useful in detecting heterozygous large deletions. These results expand the spectrum of mutations in the ATP6V0A4 and ATP6V1B1 genes associated with primary dRTA and provide insight into possible structure-function relationships.

ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384. Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

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)

Recurrent SPI1 (PU.1) fusions in high-risk pediatric T cell acute lymphoblastic leukemia

The outcome of treatment-refractory and/or relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis for this is not well understood. Here we report comprehensive profiling of 121 cases of pediatric T-ALL using transcriptome and/or targeted capture sequencing, through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), accounting for 3.9% (7/181) of the examined pediatric T-ALL cases, showed a double-negative (DN; CD4−CD8−) or CD8+ single-positive (SP) phenotype and had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell precommitment, establishment of T cell identity, and post-β-selection maturation and with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and, when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in a maturation block. Our findings highlight a unique role of SPI1 fusions in high-risk pediatric T-ALL.

Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole‐exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.

EVI1 overexpression is a poor prognostic factor in pediatric patients with mixed lineage leukemia-AF9 rearranged acute myeloid leukemia

The ecotropic viral integration site-1 gene (EVI1) encodes a zinc finger protein that functions as a transcriptional regulator of hematopoietic stem cell self-renewal and long-term multilineage repopulating activity.1,2 The mixed lineage leukemia gene (MLL) rearrangements [i.e. t(11q23)] occur at high frequency in pediatric acute myeloid leukemia (AML) patients with EVI1 overexpression,3 and EVI1 is a transcriptional target of MLL oncoproteins.4 EVI1 overexpression has been reported in up to 10% of patients with AML and is associated with an adverse prognosis. However, the prognostic value of EVI1 overexpression has been studied mostly in adult AML.5–9 Only two studies have examined EVI1 overexpression in pediatric AML, but a detailed analysis according to the type of leukemia was not performed because of the small sample size.3,10 Recent data from an international consortium, including those from our group, suggest that pediatric MLL-rearranged AML can be divided into certain risk groups on the basis of different translocation partners.11 However, clinical outcome data leading to risk stratification of the MLL-rearranged subgroups are still scarce and further investigation is necessary to identify new prognostic factors. Here, we retrospectively examined EVI1 expression levels and clinical outcomes of pediatric MLL-rearranged AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study. After excluding patients with acute promyelocytic leukemia, Down syndrome, secondary AML, myeloid/natural killer cell leukemia and myeloid sarcoma, 485 AML patients were enrolled in the AML-05 study. Overall, 42 patients were excluded, mainly because of misdiagnosis. Details of the treatment schedules and risk stratification were described in previous publication.12 This study was conducted in accordance with the principles set down in the Declaration of Helsinki and was approved by the Ethics Committees of all participating institutions. All patients, or the patients’ parents/guardians, provided written informed consent. RNA obtained from diagnostic bone marrow samples was used to analyze the expression of EVI1 using a previously established EVI1 quantitative real-time polymerase chain reaction assay that covers the various EVI1 splice variants.7 Event-free survival (EFS) was defined as the time from the diagnosis of AML to the last follow up or the first event (failure to achieve remission, relapse, secondary malignancy, or any cause of death). In this study, most of the events were relapses (n=23) and the rest were deaths with sepsis (n=1) and acute respiratory distress syndrome (n=1). Overall survival (OS) was defined as the time from the diagnosis of AML to any cause of death. All tests were two-tailed and P<0.05 was considered statistically significant. Among 443 eligible AML patients, 69 were diagnosed as MLL-rearranged AML and diagnostic samples from 50 patients were analyzed for EVI1 mRNA expression. No significant differences in the characteristics and clinical outcomes were observed between these 50 patients and the 19 patients who did not have EVI1 data [EFS (P=0.20), OS (P=0.45)]. EVI1 expression levels were dichotomized based on a cut off of 0.1 relative to SKOV3, an ovarian carcinoma cell line overexpressing EVI1: values higher than 0.1 were defined as EVI1+ and those lower than 0.1 or undetectable were defined as EVI1−, as described in a previous study.7 EVI1+ was present in 18 patients (36%). EVI1 expression levels in different MLL translocation partners relative to that in SKOV3 cells are shown in Online Supplementary Figure S1. The clinical features of EVI1+ and EVI1− patients are summarized in Table 1. EVI1+ patients were significantly older (P=0.03) and had a higher WBC count (P=0.01) at the time of diagnosis than EVI1− patients. Most of the MLL-rearranged AML cases were classified as FAB-M5 or FAB-M4. Specifically, most EVI1− patients (84%) presented with FAB-M5 morphology, which was less frequent in EVI1+ patients (22%), consistent with the findings of a previous study.8 EVI1+ was not correlated with sex or MLL translocation partners. The frequency of FLT3-ITD was significantly higher in EVI1+ patients (P=0.04). We also analyzed CEBPA and NPM1 mutations, which are established favorable prognostic factors; however, none of the patients harbored these mutations, except for one EVI1− patient harboring double CEBPA mutations. Table 1. Characteristics of patients categorized according to EVI1 expression status. Next, clinical outcomes were compared between EVI1+ patients and EVI1− patients (Figure 1). In the MLL-rearranged AML cohort (n=50), EVI1+ patients had a significantly worse EFS than EVI1− patients (P<0.0001) (Figure 1A). However, OS did not differ significantly between the two groups (P=0.054) (Figure 1B). Among several types of MLL-rearrangements, MLL-AF9 was the most common translocation (n=29, 58%) (Table 1). Therefore, clinical outcomes in the cohort of MLL-AF9 positive patients were compared between EVI1+ patients (n=11) and EVI1−patients (n=18). The results showed significant differences in EFS (P<0.0001) and OS (P=0.0008) (Figure 1C and D). By contrast, no differences in EFS (P=0.36) or OS (P=0.57) were observed among patients with MLL-rearranged AML after excluding MLL-AF9 positive patients (Figure 1E and F). The clinical outcomes associated with each type of MLL-rearrangement could not be analyzed because of the small sample size. Multivariate Cox regression analysis, including FLT3-ITD, WBC count, and age identified EVI1+ as the only prognostic factor predicting poor EFS in the total cohort of MLL-rearranged AML (hazard ratio (HR), 4.94; P<0.01) and in the MLL-AF9 positive cohort (HR, 33.81; P<0.01), but not OS (Online Supplementary Table S1). Figure 1. Kaplan-Meier survival curves of event-free survival (EFS) and overall survival (OS) from the time of diagnosis according to EVI1 expression status. (A) Kaplan-Meier estimates of EFS in the cohort of MLL-rearranged AML in EVI1+ and EVI1− patients. ... These results suggest that EVI1 overexpression is an independent adverse prognostic factor because of its association with reduced remission duration in pediatric patients with MLL-rearranged AML, especially in patients harboring MLL-AF9. A recent large study identified several novel prognostic MLL-rearranged subgroups, including a favorable-risk MLL-AF1q positive subgroup and a poor-risk MLL-AF6 positive subgroup.11 However, MLL-AF9 positive patients are categorized as an intermediate risk group, and this subgroup may be dichotomized as a favorable and poor-risk subgroup based on EVI1 expression levels. Pretreatment screening for EVI1 expression should be considered in patients with MLL-rearranged AML to enable better risk assessment and alternative consolidation therapies to be considered. Our results need to be confirmed in larger studies because of the limited case numbers. From a biological viewpoint, the ‘evil’-like adverse effects of EVI1 in patients with MLL-AF9-positive AML were partially elucidated in a recent study in which EVI1 positive cells harboring MLL-AF9 showed distinct morphological, molecular, and mechanistic differences from EVI1 negative cells.13 Moreover, EVI1 overexpression has been linked to CD52 overexpression, which could be a therapeutic target for monoclonal antibody treatment.14 Further investigation is required to identify novel prognostic factors in the various subgroups of MLL-rearranged AML and to develop therapeutic strategies effective for patients with EVI1 overexpression.

CSF3R and CALR mutations in paediatric myeloid disorders and the association of CSF3R mutations with translocations, including t(8; 21)

Mutations in the colony‐stimulating factor 3 receptor (CSF3R) and calreticulin (CALR) genes have been reported in a proportion of adults with myeloproliferative disease. However, little is known about CSF3R or CALR mutations in paediatric myeloid disorders. We analysed CSF3R exons 14 and 17, and CALR exon 9, using direct sequencing in samples of paediatric acute myeloid leukaemia (AML; n = 521), juvenile myelomonocytic leukaemia (JMML; n = 40), myelodysplastic syndrome (MDS; n = 20) and essential thrombocythaemia (ET; n = 21). CSF3R mutations were found in 10 (1·92%) of 521 patients with AML; two in exon 14 (both missense mutations resulting in p.T618I) and eight in exon 17 (three frameshift mutations: p.S715X, p.Q774R, and p.S783Q; and five novel missense mutations: p.Q754K, p.R769H, p.L777F, p.T781I, and S795R). All of the patients with mutations in CSF3R exon 17 had chromosomal translocations, including four with t(8;21). At the time of reporting, seven of these ten patients are alive; three have died, due to side effects of chemotherapy. No CSF3R mutations were found in cases of MDS, JMML or ET. The only mutation found in the CALR gene was a frameshift (p.L367 fs) in one ET patient. We discuss the potential impact of these findings for the leukaemogenesis and clinical features of paediatric myeloid disorders.

SETBP1 mutations in juvenile myelomonocytic leukaemia and myelodysplastic syndrome but not in paediatric acute myeloid leukaemia.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative disorder that is characterized by excessive myelomonocytic proliferation (Loh, 2011). Gene mutations in the components of the RAS signalling pathways are a hallmark of JMML and are considered to be central to the pathogenesis of JMML. Mutations in NRAS, KRAS, PTPN11, NF1, and CBL genes are found in approximately 75–85% of patients with JMML and are implicated in the aberrant RAS signalling (Loh, 2011). These mutations are also associated with congenital abnormalities, such as cardio–facio–cutaneous syndrome (KRAS), Noonan syndrome (PTPN11), neurofibromatosis (NF1), and Noonan-like syndrome (CBL). However, no other mutations have been identified in the remaining approximately 20% of patients with JMML. In this regard, massively parallel sequencing technology has recently identified recurrent somatic mutations in SETBP1 in atypical chronic myeloid leukaemia (aCML) (Piazza et al, 2012). Of the 70 patients with aCML that were examined, 17 (24%) were found to carry SETBP1 mutations. These mutations clustered between codons 858 and 871, all located in the SKI-homologous region of SETBP1. Identical nucleotide alterations have been reported in Schinzel–Giedion syndrome (Hoischen et al, 2010), a rare congenital disorder that is characterized by severe mental retardation, distinctive facial features, and higher than normal prevalence of tumours, notably neuroepithelial neoplasia (Schinzel & Giedion, 1978). This report prompted us to search for possible SETBP1 mutations in JMML or other paediatric haematological malignancies. To assess the clinical significance of SETBP1 mutations in paediatric leukaemias, we analysed a total of 414 patients with paediatric leukaemia/myelodysplastic syndrome (MDS) that comprised 42 patients with primary JMML, 24 with MDS, 22 with therapy-related leukaemia, 68 with infant acute lymphoblastic leukaemia (ALL), and 258 with de novo acute myeloid leukaemia (AML), including 10 patients with acute promyelocytic leukaemia (APL) and 22 with acute megakaryoblastic leukaemia (AMKL). The median age at diagnosis of JMML was 1 year and 10 months (range, 2 months to 8 years and 4 months), with 27 males and 15 females. MDS included 9 patients with refractory anaemia (RA), 14 with RA with an excess of blasts, and 1 with secondary MDS. The genomic region of the SETBP1 gene, containing codons 858–871 with the mutation hotspots D868 and G870 in the SKI-homologous region, was amplified using polymerase chain reaction (PCR) with the following primer sequences: forward, 5′-ACCAAAACCCAAAAGGGAAT3′; reverse, 5′-CGGTTTTGCAGGCTTTTC-3′. Purified PCR products were sequenced using an ABI PRISM 3130 Genetic Analyser (Applied Biosystems, Branchburg, NJ). Mutations in RAS, PTPN11, and CBL have been previously reported in JMML (Shiba et al, 2010). The present study adhered to the principles of the Helsinki Declaration and was conducted under the regulations outlined by the Ethics Board of Gunma Children’s Medical Centre. SETBP1 mutations were found in 2 of the 42 patients with JMML (4 8%; Gly870Arg in JMML 2, Ser869Arg in JMML 24) and one of the 24 patients with MDS (4 2%; Ile871Thr in MDS 3) but not in the 22 patients with secondary AML, 68 with infant ALL, or 258 with de novo paediatric AML, including 10 patients with APL and 22 with AMKL (Fig 1A). The origin of the mutations was not determined due to the lack of appropriate normal tissue samples. In all 3 patients with SETBP1 mutations, a chromatogram exclusively showed a mutated sequence, indicating that the mutations were heterozygous (Fig 1A). Although one of the 2 JMML patients with an SETBP1 mutation survived after unrelated cord blood transplantation, the other died following relapse 4 months after undergoing related peripheral blood stem cell transplantation (Table I). In contrast, the MDS patient who had an SETBP1 mutation was initially diagnosed with neuroblastoma at the age of 6 years. He was subsequently treated with chemotherapy and autologous bone marrow transplantation and achieved complete remission (CR). However, 3 years after the initial diagnosis, blast cells appeared in his peripheral blood and he was diagnosed with secondary MDS. Chromosomal analysis of the bone marrow cells revealed 45, XY, 15, der(7)t(7;15)(p13;q15), add(18)(q21) and add(20)(p13). He received chemotherapy with etoposide and cytarabine; however, he did not achieve CR. He died of haemorrhagic shock 18 months after being diagnosed with secondary MDS. Mutations in NRAS, KRAS, PTPN11 and CBL genes were found in 21%, 4 8%, 38% and 12% of patients with JMML respectively, in our study (Fig 1B) (Shiba et al, 2010). Although almost all of the NRAS, KRAS, PTPN11 and CBL mutations occurred in a mutually exclusive manner, SETBP1 mutations were found in patients with PTPN11 or NRAS mutations (Table I and Fig 1B). This finding suggests that both gene mutations associated with the RAS pathway and SETBP1 mutations can cooperate in the pathogenesis of JMML. Correspondence