International Consensus Definition of DNA Methylation Subgroups in 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 three groups: high methylation (HM), intermediate methylation (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML have been 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 the DREAM data has identified HM and LM subgroups (HM_DREAM, n=35; LM_DREAM; n=64). Of the 98 cases that could be compared with the international consensus classification, 90 cases of HM (n=30) and LM (n=60) had 100% concordance with the DREAM clustering results. For the remaining eight cases classified as the IM group, four cases were classified into the HM_DREAM group and four cases into the LM_DREAM group. A machine-learning classifier has been 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, a robust methylation test has been developed using the DREAM analysis for patients with JMML. This simple and straightforward test can be easily incorporated in diagnosis to generate a methylation classification for patients so that they can receive risk-adapted treatment in the context of future clinical trials.

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MEK Inhibition Demonstrates Activity in Relapsed, Refractory Patients with Juvenile Myelomonocytic Leukemia: Results from COG Study ADVL1521

Blood ◽

10.1182/blood-2021-144850 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3679-3679

Author(s):

Elliot Stieglitz ◽

Mignon L. Loh ◽

Julia Meyer ◽

Chujing Zhang ◽

Donald A Barkauskas ◽

...

Keyword(s):

United States ◽

Clinical Trial ◽

Dna Methylation ◽

Objective Response ◽

The United States ◽

Juvenile Myelomonocytic Leukemia ◽

International Consensus ◽

Molecular Responses ◽

Mek Inhibition ◽

Myelomonocytic Leukemia

Abstract Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response. Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling. Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

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Genetic Alterations Precede DNA Methylation Changes in Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2020-143443 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 19-20

Author(s):

Astrid Behnert ◽

Julia Meyer ◽

Jahan-Yar Parsa ◽

Aaron Hechmer ◽

Mignon L. Loh ◽

...

Keyword(s):

Dna Methylation ◽

Somatic Mutation ◽

Methylation Status ◽

Juvenile Myelomonocytic Leukemia ◽

Genetic Mutations ◽

P Value ◽

International Consensus ◽

Consensus Definition ◽

Dna Methylation Profiling ◽

Methylation Profiling

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid malignancy of early childhood. Approximately 95% of patients have at least one mutation that leads to hyperactive RAS signaling. Both the presence of multiple mutations at diagnosis as well as altered DNA methylation are associated with a poor prognosis. Whether altered DNA methylation leads to the acquisition of additional genetic mutations or is a secondary event to genetic mutations is unknown. In this study, we analyzed a total of 33 newborn blood screening (NBS) cards from children who went on to develop JMML later in childhood. Using next generation sequencing to detect both genetic mutations and DNA methylation status we sought to determine the sequence of events that lead to the development of JMML. Patients and Methods We identified 33 patients that were born in the state of California from 1990 to 2017 who were confirmed to have JMML and were previously consented to participate in a JMML tissue bank study. Guthrie cards from these 33 patients as well as 12 healthy controls were obtained from the California Biobank Program. DNA samples (20 ng) were sequenced for mutations using a custom amplicon-based sequencing approach (Paragon Genomics, Hayward, CA) targeting 26 genes that are recurrently mutated in JMML. For DNA methylation profiling, 300ng of genomic DNA was processed for bisulfite conversion using the TrueMethyl oxBS Module (Tecan Genomics Inc., Redwood City, CA.) according to manufacturer's guidelines and next generetation sequencing (NGS) libraries were prepared by targeting 3000 CpG loci using custom probes for Targeted Methyl-Seq assay (Tecan Genomics Inc., Redwood City, CA). Sequence reads were trimmed using Cutadapt to remove adapters and methylation status called using Bismark. Samples were classified into one of three methylation subgroups: Low, Intermediate, or High using 1386 probes according to an international consensus definition. Results At diagnosis of JMML, somatic mutations were identified in 32 of 33 patients. Of the 32 patients with a somatic mutation present upon diagnosis, the same mutations were found in NBS cards in 13 (41%) patients using a mutant allele fraction (MAF) cut-off of 1%. Clonal mutations (MAF >15%) were found in 9 of 32 (28%) patients. Patients who had a somatic mutation detected at birth were significantly younger at diagnosis with a median age of 7.1 months (range 2.5-91.6 months) compared to patients who had none (19.8 months; p-value = 0.042). However, no difference was observed in overall or event-free survival for patients with or without somatic mutations at birth. Methylation profiling classified all NBS cards as having "low" DNA methylation using an international, consensus definition (Figure 1A). Of the 33 patients, ten also had DNA methylation profiling performed on their diagnostic JMML sample which were classified as low (LM), intermediate (IM) or high methylation (HM) (Figure 1B). Amongst patients with both NBS and JMML methylation data available, 5 had mutations present in the NBS card and all 5 had lower methylation (β values) in their NBS card compared to their JMML sample (mean 0.38 vs 0.29, p-value = 0.002). Discussion We identified somatic Ras pathway mutations at birth in 13/32 (41%) of newborns that developed JMML later in their childhood. We found that patients with a somatic mutation detected at birth were significantly younger at diagnosis compared to patients who had no mutations at birth. DNA methylation profiling of NBS cards from children who went on to develop JMML revealed methylation signatures that were similar to normal age-matched controls. All newborn cards were classified as low methylation at birth. Specifically, amongst patients who had genetic mutations present at birth, methylation changes that were eventually detected in JMML samples were not present in the NBS cards. Therefore, we conclude that aberrant DNA methylation is a secondary event to genetic changes in JMML. Figure 1: Panel A: Methylation profiling of JMML NBS and control NBS samples, classified according to the international JMML methylation consensus signature. Panel B: Methylation profiling of paired NBS and diagnosis samples from JMML patients. The star indicates the NBS methylation profile. Heatmaps (A and B) show the beta values of 1386 CpG loci used for methylation classification. Figure 1 Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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International Consensus Definition of a Serious Infection in a Geriatric Patient Presenting to Ambulatory Care

Journal of the American Medical Directors Association ◽

10.1016/j.jamda.2020.01.015 ◽

2020 ◽

Vol 21 (5) ◽

pp. 578-582.e1 ◽

Cited By ~ 2

Author(s):

Thomas Struyf ◽

Jos Tournoy ◽

Jan Y. Verbakel ◽

Ann Van den Bruel

Keyword(s):

Ambulatory Care ◽

Geriatric Patient ◽

International Consensus ◽

Consensus Definition ◽

Serious Infection ◽

Definition Of

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Interrater reliability of the international consensus definition of drug-resistant epilepsy: A pilot study

Epilepsy & Behavior ◽

10.1016/j.yebeh.2011.06.035 ◽

2011 ◽

Vol 22 (2) ◽

pp. 388-390 ◽

Cited By ~ 18

Author(s):

Xiao-ting Hao ◽

Irina S.M. Wong ◽

Patrick Kwan

Keyword(s):

Pilot Study ◽

Interrater Reliability ◽

Drug Resistant ◽

International Consensus ◽

Consensus Definition ◽

Drug Resistant Epilepsy ◽

Definition Of

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Aberrant DNA Methylation Is Associated With Poor Outcomes In Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v122.21.517.517 ◽

2013 ◽

Vol 122 (21) ◽

pp. 517-517

Author(s):

Hirotoshi Sakaguchi ◽

Hideki Muramatsu ◽

Xinan Wang ◽

Yinyan Xu ◽

Yoko Hibi ◽

...

Keyword(s):

Dna Methylation ◽

Chromosomal Aberration ◽

Research Funding ◽

Methylation Pattern ◽

World Health ◽

Juvenile Myelomonocytic Leukemia ◽

Aberrant Methylation ◽

Aberrant Dna Methylation ◽

Poor Outcomes ◽

Myelomonocytic Leukemia

Abstract Recent studies suggest that aberrant methylation plays a fundamental role in the development of a variety of cancers, including myeloid malignancies. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is characterized by both excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor. It is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) according to the World Health Organization classification. We recently reported that somatic mutations in SETBP1 and JAK3 were identified in JMML patients and were associated with poor outcomes (Nat Genet 2013;45:937–41). The goal of this study was to clarify the clinical significance of aberrant DNA methylation in JMML. We studied 92 children (61 boys and 31 girls) who were diagnosed with JMML in institutions throughout Japan. A diagnosis of JMML was made based on internationally accepted criteria. We quantitatively evaluated the CpG methylation pattern in the promoter regions of 16 candidate genes (APC, BMP4, CALCA, CDH13, CDKN2A, CDKN2B, CHFR, DAPK, DMR-H19, ER, IGF2, MGMT, MLH1, RARB, RASSF1, TP73) from genomic DNA derived from bone marrow specimens at the time of diagnosis. This was accomplished by bisulfite conversion and the pryosequencing technique. We defined aberrant methylation as >3 standard deviations from the mean methylation level derived from 8 healthy individuals. The median age at diagnosis was 16 months (range, 0.3–160). By genetic analysis, PTPN11, NF1, NRAS, KRAS, and CBL mutations were found in 39 (42%), 7 (8%), 12 (13%), 13 (14%), and 11 (12%) patients, respectively. In addition, 16 patients had SETBP1 or JAK3 mutations. Karyotypic abnormalities were detected in 15 patients, including 8 with monosomy 7. The median monocyte count, percentage of hemoglobin F, and platelet count at the time of diagnosis were 4.6x109/L (range, 0.2–31.6), 21% (range, 0–68), and 61.0x109/L (range, 1.4–483), respectively. The median observation period was 18 months (range, 1–287). During observation, 56 of the 92 patients received allogeneic hematopoietic stem cell transplantation (HSCT), and 30 of 92 patients died. Outcomes were assessed according to transplantation-free survival (TFS), in which HSCT and death were censored, and overall survival (OS) by the Kaplan-Meier method. Aberrant methylation of BMP4, CALCA, CDKN2A, CDKN2B, DMR-H19 and RARB were detected, of which hypermethylation of BMP4, CALCA, CDKN2A, and RARB were associated with poor TFS according to univariate analyses (P<0.10). We integrated the number of aberrant methylation of these four genes to arrive at an aberrant methylation score (AMS). An AMS of 0, 1, 2, 3, and 4 was seen in 36, 29, 19, 7, and 1 of the 92 patients, respectively. The AMS was significantly higher in patients with SETBP1 or JAK3 mutations than in other patients (P=0.03): 1, 8, 3, 3, and 1 of the 16 patients showed an AMS of 0, 1, 2, 3, and 4, respectively. The probability of 5-year TFS was 42% in the AMS = 0 cohort and 4% in the AMS = 1 to 4 cohort, respectively (log-rank, P<0.001). Moreover, the probability of 5-year OS was 65% in the AMS = 0 to 2 cohort and 8% in the AMS = 3 and 4 cohort, respectively (log-rank, P=0.004). In multivariable analysis using the Cox-proportional hazard model, AMS = 1 to 4 (hazard ratio [HR], 2.6; 95% confidential interval [CI], 1.2–5.5; P=0.013), mutations of PTPN11 or NF1 (HR, 2.7; 95% CI, 1.3–5.5; P=0.010), and chromosomal aberration (HR, 3.3; 95% CI, 1.7–6.5; P=0.001) were independent predictors of poor TFS. Further, chromosomal aberration (HR, 4.4; 95% CI, 1.6–11.8; P=0.004) and platelet counts <33x109/L (HR, 2.8; 95% CI, 1.3–6.4; P=0.013) were independent predictors of poor OS. The present study shows that aberrant methylation of four genes (BMP4, CALCA, CDKN2A, and RARB) is associated with poor outcomes in JMML patients. Patients with SETBP1/JAK3 mutations frequently show the hypermethylation of these genes. Further, allogeneic HSCT is associated with improved outcomes for patients with AMS = 1 and 2. Therefore, these results suggest that examination of the methylation pattern of these four genes may help guide clinical decisions for the management of patients with JMML. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome

Blood ◽

10.1182/blood-2010-08-298968 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4871-4880 ◽

Cited By ~ 60

Author(s):

Christiane Olk-Batz ◽

Anna R. Poetsch ◽

Peter Nöllke ◽

Rainer Claus ◽

Manuela Zucknick ◽

...

Keyword(s):

Dna Methylation ◽

Cox Regression ◽

Cpg Island ◽

Cpg Islands ◽

Prognostic Scores ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Prognostic Features ◽

Aberrant Dna Methylation ◽

Myelomonocytic Leukemia

Abstract Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

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Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia

Nature Communications ◽

10.1038/s41467-017-02178-9 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 26

Author(s):

Elliot Stieglitz ◽

Tali Mazor ◽

Adam B. Olshen ◽

Huimin Geng ◽

Laura C. Gelston ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

Genome Wide ◽

Myelomonocytic Leukemia

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Malnutrition assessment methods in adult patients with tuberculosis: a systematic review

BMJ Open ◽

10.1136/bmjopen-2021-049777 ◽

2021 ◽

Vol 11 (12) ◽

pp. e049777

Author(s):

Lies ter Beek ◽

Mathieu S Bolhuis ◽

Harriët Jager-Wittenaar ◽

René X D Brijan ◽

Marieke G G Sturkenboom ◽

...

Keyword(s):

Systematic Review ◽

Body Composition ◽

Cognitive Function ◽

Assessment Method ◽

Assessment Methods ◽

International Consensus ◽

Consensus Definition ◽

Tb Treatment ◽

Registration Number ◽

Definition Of

ObjectivesMalnutrition is associated with a twofold higher risk of dying in patients with tuberculosis (TB) and considered an important potentially reversible risk factor for failure of TB treatment. The construct of malnutrition has three domains: intake or uptake of nutrition; body composition and physical and cognitive function. The objectives of this systematic review are to identify malnutrition assessment methods, and to quantify how malnutrition assessment methods capture the international consensus definition for malnutrition, in patients with TB.DesignDifferent assessment methods were identified. We determined the extent of capturing of the three domains of malnutrition, that is, intake or uptake of nutrition, body composition and physical and cognitive function.ResultsSeventeen malnutrition assessment methods were identified in 69 included studies. In 53/69 (77%) of studies, body mass index was used as the only malnutrition assessment method. Three out of 69 studies (4%) used a method that captured all three domains of malnutrition.ConclusionsOur study focused on published articles. Implementation of new criteria takes time, which may take longer than the period covered by this review. Most patients with TB are assessed for only one aspect of the conceptual definition of malnutrition. The use of international consensus criteria is recommended to establish uniform diagnostics and treatment of malnutrition.PROSPERO registration numberCRD42019122832.

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