Clinical parameter-based prediction of DNA methylation classification generates a prediction model of prognosis in patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare heterogeneous hematological malignancy of early childhood characterized by causative RAS pathway mutations. Classifying patients with JMML using global DNA methylation profiles is useful for risk stratification. We implemented machine learning algorithms (decision tree, support vector machine, and naïve Bayes) to produce a DNA methylation-based classification according to recent international consensus definitions using a well-characterized pooled cohort of patients with JMML (n = 128). DNA methylation was originally categorized into three subgroups: high methylation (HM), intermediate methylation (IM), and low methylation (LM), which is a trichotomized classification. We also dichotomized the subgroups as HM/IM and LM. The decision tree model showed high concordances with 450k-based methylation [82.3% (106/128) for the dichotomized and 83.6% (107/128) for the trichotomized subgroups, respectively]. With an independent cohort (n = 72), we confirmed that these models using both the dichotomized and trichotomized classifications were highly predictive of survival. Our study demonstrates that machine learning algorithms can generate clinical parameter-based models that predict the survival outcomes of patients with JMML and high accuracy. These models enabled us to rapidly and effectively identify candidates for augmented treatment following diagnosis.

Case report: Novel NIPBL-BEND2 fusion gene identified in osteoblastoma-like phosphaturic mesenchymal tumor of the fibula.

Phosphaturic mesenchymal tumor (PMT) is a rare tumor that secretes fibroblast growth factor 23 (FGF23) and causes hypophosphatemia and tumor-induced osteomalacia (TIO). Fusion genes FN1-FGFR1 and FN1-FGF1 have been detected in some PMTs, but the pathogenesis of PMTs without these fusion genes remains unclear. Here, we report a 12-year-old boy with persistent muscle weakness and gait disturbance. Roentgenographic examination revealed a radiolucent lesion with endosteal scalloping in the left fibula, while his serum level of FGF23 was markedly increased. Combined with simple X-ray findings of other body parts, we suspected that TIO was caused by PMT, and resected the tumor. After resection, the serum level of FGF23 started to decrease immediately and normalized within 3 hours after resection, with this being earlier than normalization of the serum phosphorus level. In RNA sequencing, FN1-FGFR1 and FN1-FGF1 were not detected, but a novel NIPBL-BEND2 fusion gene was identified. When we forcedly expressed this fusion gene in HEK293T cells and MG63 cells, cell proliferation was enhanced in both cell lines. Furthermore, Gene set enrichment analysis of HEK293T cells showed significant upregulation of MYC-target genes. Our results suggest that this novel NIPBL-BEND2 fusion gene promotes cell proliferation possibly via the MYC pathway and might be one of the etiologies of PMTs other than FN1-FGFR1 or FN1-FGF1.

Simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following 3 groups: high (HM), intermediate (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML were analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing-based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n = 99) using DREAM data identified HM (HM_DREAM; n = 35) and LM subgroups (LM_DREAM; n = 64). Of the 98 cases that could be compared with the international consensus classification, 90 HM (n = 30) and LM (n = 60) cases had 100% concordance with DREAM clustering results. Of the remaining 8 cases comprising the IM group, 4 were classified as belonging to the HM_DREAM group and 4 to the LM_DREAM group. A machine-learning classifier was successfully constructed using a support vector machine (SVM), which divided the validation cohort (n = 38) into HM (HM_SVM, n = 18) and LM (LM_SVM; n = 20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, we developed a robust methylation test using DREAM for patients with JMML. This simple and straightforward test can be easily incorporated into diagnosis to generate a methylation classification for patients so they can receive risk-adapted treatment in the context of future clinical trials.

Integrated diagnosis based on transcriptome analysis in suspected pediatric sarcomas.

Pediatric solid tumors are a heterogeneous group of neoplasms with over 100 subtypes. Clinical and histopathological diagnosis remains challenging due to the overlapping morphological and immunohistochemical findings and the presence of atypical cases. To evaluate the potential utility of including RNA-sequencing (RNA-seq) in the diagnostic process, we performed RNA-seq in 47 patients with suspected pediatric sarcomas. Histopathologists specialized in pediatric cancer re-evaluated pathological specimens to reach a consensus diagnosis; 42 patients were diagnosed with known subtypes of solid tumors whereas 5 patients were diagnosed with undifferentiated sarcoma. RNA-seq analysis confirmed and refined consensus diagnoses and further identified diagnostic genetic variants in four of the five patients with undifferentiated sarcoma. Genetic lesions were detected in 23 patients, including the novel SMARCA4-THOP1 fusion gene and 22 conventional or recently reported genetic events. Unsupervised clustering analysis of the RNA-seq data identified a distinct cluster defined by the overexpression of rhabdomyosarcoma-associated genes including MYOG and CHRNG. These findings suggest that RNA-seq-based genetic analysis may aid in the diagnosis of suspected pediatric sarcomas, which would be useful for the development of stratified treatment strategies.

Successful treatment of a novel type I interferonopathy due to a de novo PSMB9 gene mutation with a Janus kinase inhibitor.

BACKGROUND: Type I interferonopathies are a recently established subgroup of autoinflammatory diseases caused by mutations in genes associated with proteasome degradation or cytoplasmic RNA- and DNA-sensing pathways. OBJECTIVE: This study aimed to unveil the molecular pathogenesis of a patient with novel type I interferonopathy, for which no known genetic mutations have been identified. METHODS: We performed the whole-exome sequencing of a 1-month-old boy with novel type I interferonopathy. We also investigated proteasome activities using patient-derived B lymphoblastoid cell lines (LCLs) and normal LCLs transduced with the mutant gene. RESULTS: Whole-exome sequencing identified a de novo proteasome 20S subunit beta 9 (PSMB9) p.G156D mutation in the patient who developed fever, a chilblain-like skin rash, myositis, and severe pulmonary hypertension due to the hyperactivation of IFN-α. Patient-derived LCLs revealed reduced proteasome activities, and exogenous transduction of mutant PSMB9 p.G156D into normal LCLs significantly suppressed proteasome activities, and the endogenous PSMB9 protein was lost along with the reduction of other immunoproteasome subunits, PSMB8 and PSMB10 proteins. He responded to the administration of a Janus kinase inhibitor, tofacitinib, and he was successfully withdrawn from venoarterial extracorporeal membranous oxygenation. At age 7 months, he received an unrelated cord blood transplantation. At 2 years posttransplantation, he no longer required tofacitinib and experienced no disease recurrence. CONCLUSIONS: We present the case of a patient with a novel type I interferonopathy caused by a de novo PSMB9 p.G156D mutation that suppressed the wild-type PSMB9 protein expression. Janus kinase inhibitor and stem cell transplantation could be curative therapies in patients with severe interferonopathies.

International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia.

PURPOSE: Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation. EXPERIMENTAL DESIGN: Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients). RESULTS: Analysis of pooled, published data established three DNA methylation subgroups as a de facto standard. Unfavorable prognostic parameters (PTPN11 mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS. CONCLUSIONS: This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.

Publisher Correction: Defective Epstein-Barr virus in chronic active infection and haematological malignancy.

In the version of this Letter originally published, in the sentence beginning "The major driver role of DDX3X mutations...", the citation "Fig. 2a-f" should have been "Fig. 2". In addition, in the sentence beginning "Another finding of interest was the presence of identical driver mutations...", the citation "Fig. 3a,b and Fig. 4" should have been "Fig. 3". This has now been amended in all versions of the Letter.

Defective Epstein-Barr virus in chronic active infection and haematological malignancy.

Epstein-Barr virus (EBV) infection is highly prevalent in humans and is implicated in various diseases, including cancer1,2. Chronic active EBV infection (CAEBV) is an intractable disease classified as a lymphoproliferative disorder in the 2016 World Health Organization lymphoma classification1,2. CAEBV is characterized by EBV-infected T/natural killer (NK) cells and recurrent/persistent infectious mononucleosis-like symptoms3. Here, we show that CAEBV originates from an EBV-infected lymphoid progenitor that acquires DDX3X and other mutations, causing clonal evolution comprising multiple cell lineages. Conspicuously, the EBV genome in CAEBV patients harboured frequent intragenic deletions (27/77) that were also common in various EBV-associated neoplastic disorders (28/61), including extranodal NK/T-cell lymphoma and EBV-positive diffuse large B-cell lymphoma, but were not detected in infectious mononucleosis or post-transplant lymphoproliferative disorders (0/47), which suggests a unique role of these mutations in neoplastic proliferation of EBV-infected cells. These deletions frequently affected BamHI A rightward transcript microRNA clusters (31 cases) and several genes that are essential for producing viral particles (20 cases). The deletions observed in our study are thought to reactivate the lytic cycle by upregulating the expression of two immediate early genes, BZLF1 and BRLF14-7, while averting viral production and subsequent cell lysis. In fact, the deletion of one of the essential genes, BALF5, resulted in upregulation of the lytic cycle and the promotion of lymphomagenesis in a xenograft model. Our findings highlight a pathogenic link between intragenic EBV deletions and EBV-associated neoplastic proliferations.

Ureteral dilatation detected in magnetic resonance imaging predicts vesicoureteral reflux in children with urinary tract infection.

OBJECTIVE: Urinary tract infection (UTI), one of the most common bacterial infections occurring during infancy and early childhood, is frequently associated with vesicoureteral reflux (VUR). Although several guidelines recommend performing ultrasonography as a screening test, its utility is not adequate and appropriate screening tests are strongly desirable. In this study, we evaluate the use of magnetic resonance imaging (MRI) as a screening test for VUR in children with UTI. METHODS: We prospectively studied 108 patients with suspected UTI between April 2014 and March 2016. UTI was diagnosed on the basis of diffusion-weighted MRI (DW-MRI) and urine culture findings. We measured ureteral dilatation using MRI in 96 patients with UTI and assessed the relationship between ureteral dilatation in MRI and VUR in 46 patients who underwent voiding cystourethrography (VCUG). RESULTS: Among 108 patients, 88 and 8 were diagnosed with upper and lower UTI, respectively. Among 46 patients who underwent VCUG, 23 had VUR (14 low grade and 9 high grade). Patients with ureteral dilatation detected on MRI had VUR more frequently than those without ureteral dilatation (any grades VUR, 71% vs. 32%; P = 0.02; high-grade VUR, 38% vs. 2%, P = 0.007). Overall, ureteral dilatation findings on MRI achieved sensitivity 65.2% and specificity 73.9% as a screening test for VUR. In addition, DW-MRI achieved sensitivity 100% and specificity 81.8% in the diagnosis of upper UTI. CONCLUSION: These findings suggested that MRI is a valuable tool for screening of VUR as well as diagnosis of upper UTI.

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.

Integrated molecular profiling of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia.

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow-up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10-year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6-20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

MEF2D-BCL9 Fusion Gene Is Associated With High-Risk Acute B-Cell Precursor Lymphoblastic Leukemia in Adolescents.

PURPOSE: Acute lymphoblastic leukemia (ALL) makes up a significant proportion of all pediatric cancers, and relapsed ALL is a leading cause of cancer-associated deaths in children. Identification of risk factors and druggable molecular targets in ALL can lead to a better stratification of treatments and subsequent improvement in prognosis. PATIENTS AND METHODS: We enrolled 59 children with relapsed or primary refractory ALL who were treated in our institutions. We primarily performed RNA sequencing (RNA-seq) using patients' leukemic cells to comprehensively detect gene fusions and analyze gene expression profiles. On the basis of results obtained by RNA-seq, we performed genetic validation, functional analysis, and in vitro drug sensitivity testing using patients' samples and an exogenous expression model. RESULTS: We identified a total of 26 gene fusions in 22 patients by RNA-seq. Among these, 19 were nonrandom gene fusions already described in ALL, and four of the remaining seven involved identical combination of MEF2D and BCL9. All MEF2D-BCL9-positive patients had B-cell precursor immunophenotype and were characterized as being older in age, being resistant to chemotherapy, having very early relapse, and having leukemic blasts that mimic morphologically mature B-cell leukemia with markedly high expression of HDAC9. Exogenous expression of MEF2D-BCL9 in a B-cell precursor ALL cell line promoted cell growth, increased HDAC9 expression, and induced resistance to dexamethasone. Using a primary culture of leukemic blasts from a patient, we identified several molecular targeted drugs that conferred inhibitory effects in vitro. CONCLUSION: A novel MEF2D-BCL9 fusion we identified characterizes a novel subset of pediatric ALL, predicts poor prognosis, and may be a candidate for novel molecular targeting.