1 4601 157 NDRG2 MRNA LEVELS AND MIR-28-5P AND MIR-650 ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: NDRG2 IS IDENTIFIED AS A TUMOR SUPPRESSOR GENE IN MANY TUMORS, AND FUNCTIONS IN CELL PROLIFERATION, DIFFERENTIATION AND APOPTOSIS. RECENT DATA INDICATE THAT NDRG2 EXPRESSION IS UP-REGULATED BY TP53. MOREOVER, PROPOSED MECHANISMS OF NDRG2 INACTIVATION INCLUDE EPIGENETIC SILENCING OF THE NDRG2 PROMOTER AND DOWN-REGULATION BY MICRORNAS (MIRNAS). HOWEVER, FEW STUDIES HAVE EVER BEEN DONE ON THE ROLE OF NDRG2 AND THE NDRG2-REGULATING MIRNAS INTERFERENCE IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). METHODS: NDRG2 AND MICRORNAS MRNA LEVELS IN CLL SUBJECTS WERE ASSESSED BY QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QRT-PCR). THE DUAL-LUCIFERASE REPORTER ASSAY WAS PERFORMED TO DETERMINE NDRG2-RELATED MIRNAS. LOW EXPRESSION OF MATURE EXOGENOUS MIRNAS IN CLL CELLS WAS ESTABLISHED BY TRANSIENT TRANSFECTION. NDRG2 PROTEIN LEVELS IN CLL CELLS WERE DETECTED BY WESTERN BLOT. IN ADDITION, FLOW CYTOMETRY WAS CONDUCTED TO EXAMINE THE APOPTOSIS OF CLL CELLS. RESULTS: LOWER EXPRESSION OF NDRG2 WAS FOUND IN THE B-CELLS FROM 102 CLL PATIENTS COMPARED THE 40 NORMAL SUBJECTS (P < 0.001). PATIENTS WITH ADVANCED BINET STAGE (P = 0.001), HIGH LACTATE DEHYDROGENASE (LDH) LEVEL (P = 0.036), UN-MUTATED IMMUNOGLOBULIN HEAVY CHAIN VARIABLE REGION GENE (IGHV) (P = 0.004) AND THOSE WITH P53 ABERRATIONS (P < 0.001) HAD A MARKEDLY LOWER LEVELS OF NDRG2 MRNA. THIS DECREASE WAS ASSOCIATED WITH BRIEFER TIME-TO-TREATMENT (P = 0.001) AND POORER SURVIVAL (P < 0.001). HIGH EXPRESSION OF MIR-28-5P AND MIR-650 WAS ASSOCIATED WITH BINET B/C STAGE (P = 0.044) AND IGHV UN-MUTATED (P = 0.011), AS WELL AS BINET B/C STAGE (P = 0.013) AND P53 ABERRATIONS (P = 0.037), RESPECTIVELY. INHIBITION OF MIR-28-5P OR MIR-650 COULD INDUCE MORE APOPTOSIS IN CLL CELLS WITH GERMLINE TP53. CONCLUSIONS: NDRG2 MRNA LEVELS MIGHT BE A USEFUL PROGNOSTIC VARIABLE FOR PATIENTS OF CLL AND UP-REGULATING NDRG2 TRANSCRIPTION MAY BE A THERAPY APPROACH IN CLL WITHOUT P53 ABERRATIONS. 2018 2 25 41 A 3' TRNA-DERIVED FRAGMENT PRODUCED BY TRNA(LEUAAG) AND TRNA(LEUTAG) IS ASSOCIATED WITH POOR PROGNOSIS IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA, INDEPENDENTLY OF CLASSICAL PROGNOSTIC FACTORS. OBJECTIVE: 3' TRNA-DERIVED FRAGMENTS (3' TRFS) ARE IMPORTANT EPIGENETIC REGULATORS IN NORMAL AND PATHOLOGICAL CONDITIONS. IN THIS STUDY, WE AIMED TO EXPLORE THE POTENTIAL VALUE OF A 3' TRF AS A PROGNOSTIC AND/OR SCREENING BIOMARKER FOR B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL). METHODS: PUBLICLY AVAILABLE NEXT-GENERATION SEQUENCING DATA FROM 20 B-CLL CASES WERE ANALYZED, FOLLOWED BY PREDICTION OF TARGETS OF THE MOST ABUNDANTLY AND UBIQUITOUSLY EXPRESSED 3' TRFS, LEADING TO SELECTION OF TRF-LEU(AAG/TAG) . PBMCS WERE ISOLATED FROM BLOOD SAMPLES OF 91 B-CLL PATIENTS AND 43 NON-LEUKEMIC DONORS, FOLLOWED BY TOTAL RNA EXTRACTION, IN-VITRO POLYADENYLATION, AND FIRST-STRAND CDNA SYNTHESIS. NEXT, A REAL-TIME QUANTITATIVE PCR (QPCR) ASSAY WAS DEVELOPED FOR THE ACCURATE QUANTIFICATION OF TRF-LEU(AAG/TAG) AND APPLIED IN ALL SAMPLES, PRIOR TO BIOSTATISTICAL ANALYSIS. RESULTS: HIGH TRF-LEU(AAG/TAG) LEVELS ARE ASSOCIATED WITH INFERIOR OVERALL SURVIVAL (OS) OF B-CLL PATIENTS. THE UNFAVORABLE SIGNIFICANCE OF TRF-LEU(AAG/TAG) WAS INDEPENDENT OF ESTABLISHED PROGNOSTIC FACTORS IN B-CLL. STRATIFIED KAPLAN-MEIER OS ANALYSIS UNCOVERED THE UNFAVORABLE PROGNOSTIC ROLE OF HIGH TRF-LEU(AAG/TAG) LEVELS FOR PATIENTS IN BINET A OR RAI I STAGE, NEGATIVE CD38 EXPRESSION, MUTATED, OR UNMUTATED IGHV GENOMIC LOCUS. CONCLUSION: OUR APPROACH REVEALED THE INDEPENDENT PROGNOSTIC VALUE OF A PARTICULAR 3' TRF, DERIVED FROM TRNA(LEUAAG) AND TRNA(LEUTAG) (TRF-LEU(AAG/TAG) ) IN B-CLL. 2021 3 2678 32 EVALUATION OF A PROGNOSTIC EPIGENETIC CLASSIFICATION SYSTEM IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS. BACKGROUND: METHYLATION AT 5 CPG SITES WAS PREVIOUSLY SHOWN TO CLASSIFY CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) INTO 3 PROGNOSTIC SUBGROUPS. HERE, WE AIMED TO VALIDATE THE MARKER SET IN AN ADDITIONAL COHORT AND TO EVALUATE ITS CLINICAL UTILITY FOR CLL PATIENT STRATIFICATION. METHODS: WE EVALUATED THIS EPIGENETIC MARKER SET IN 79 GERMAN PATIENTS USING BISULFITE TREATMENT FOLLOWED BY PYROSEQUENCING AND CLASSIFICATION USING A SUPPORT VECTOR MACHINE-LEARNING TOOL. RESULTS: THE N-CLL, I-CLL, AND M-CLL CLASSIFICATION WAS DETECTED IN 28 (35%), 10 (13%), AND 41 (51%) PATIENTS, RESPECTIVELY. EPIGENETIC GROUPING WAS ASSOCIATED WITH IGHV MUTATIONAL STATUS (P = 2 X 10(-12)), ISOLATED DEL13Q (P = 9 X 10(-6)), DEL17P (P = .015), COMPLEX KARYOTYPE (P = .005), VH-USAGE, AND CLINICAL OUTCOME AS TIME TO FIRST TREATMENT (P = 1.4 X 10(-12)) AND OVERALL SURVIVAL (P = .003). MULTIVARIATE COX REGRESSION ANALYSIS IDENTIFIED N-CLL AS A FACTOR FOR EARLIER TREATMENT HAZARD RATIO (HR), 6.3 (95% CONFIDENCE INTERVAL [CI] 2.4-16.4; P = .0002) COMPARED TO IGHV MUTATIONAL STATUS (HR 4.6, 95% CI 1.9-11.3, P = .0008). IN ADDITION, WHEN COMPARING THE PROGNOSTIC VALUE OF THE EPIGENETIC CLASSIFICATION SYSTEM WITH THE IGHV CLASSIFICATION, EPIGENETIC GROUPING PERFORMED BETTER COMPARED TO IGHV MUTATIONAL STATUS USING KAPLAN-MEIER ESTIMATION AND ALLOWED THE IDENTIFICATION OF A THIRD, INTERMEDIATE (I-CLL) GROUP. THUS, OUR STUDY CONFIRMED THE PROGNOSTIC VALUE OF THE EPIGENETIC MARKER SET FOR PATIENT STRATIFICATION IN ROUTINE CLINICAL DIAGNOSTICS. 2022 4 5606 33 RPPA-BASED PROTEOMICS RECOGNIZES DISTINCT EPIGENETIC SIGNATURES IN CHRONIC LYMPHOCYTIC LEUKEMIA WITH CLINICAL CONSEQUENCES. THE CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) ARMAMENTARIUM HAS EVOLVED SIGNIFICANTLY, WITH NOVEL THERAPIES THAT INHIBIT BRUTON TYROSINE KINASE, PI3K DELTA AND/OR THE BCL2 PROTEIN IMPROVING OUTCOMES. STILL, THE CLINICAL COURSE OF CLL PATIENTS IS HIGHLY VARIABLE AND MOST PREVIOUSLY RECOGNIZED PROGNOSTIC FEATURES LACK THE CAPACITY TO PREDICT RESPONSE TO MODERN TREATMENTS INDICATING THE NEED FOR NEW PROGNOSTIC MARKERS. IN THIS STUDY, WE IDENTIFIED FOUR EPIGENETICALLY DISTINCT PROTEOMIC SIGNATURES OF A LARGE COHORT OF CLL AND RELATED DISEASES DERIVED SAMPLES (N = 871) USING REVERSE PHASE PROTEIN ARRAY TECHNOLOGY. THESE SIGNATURES ARE ASSOCIATED WITH CLINICAL FEATURES INCLUDING AGE, CYTOGENETIC ABNORMALITIES [TRISOMY 12, DEL(13Q) AND DEL(17P)], IMMUNOGLOBULIN HEAVY-CHAIN LOCUS (IGHV) MUTATIONAL LOAD, ZAP-70 STATUS, BINET AND RAI STAGING AS WELL AS WITH THE OUTCOME MEASURES OF TIME TO TREATMENT AND OVERALL SURVIVAL. PROTEIN SIGNATURE MEMBERSHIP WAS IDENTIFIED AS PREDICTIVE MARKER FOR OVERALL SURVIVAL REGARDLESS OF OTHER CLINICAL FEATURES. AMONG THE ANALYZED EPIGENETIC PROTEINS, EZH2, HDAC6, AND LOSS OF H3K27ME3 LEVELS WERE THE MOST INDEPENDENTLY ASSOCIATED WITH POOR SURVIVAL. THESE FINDINGS DEMONSTRATE THAT PROTEOMIC BASED EPIGENETIC BIOMARKERS CAN BE USED TO BETTER CLASSIFY CLL PATIENTS AND PROVIDE THERAPEUTIC GUIDANCE. 2022 5 5243 30 PROGNOSTIC IMPACT OF EPIGENETIC CLASSIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE CASE OF SUBSET #2. BASED ON THE METHYLATION STATUS OF 5 SINGLE CPG SITES, A NOVEL EPIGENETIC CLASSIFICATION OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) WAS RECENTLY PROPOSED, CLASSIFYING CLL PATIENTS INTO 3 CLINICO-BIOLOGICAL SUBGROUPS WITH DIFFERENT OUTCOME, TERMED MEMORY LIKE CLL (M-CLL), NAIVE LIKE CLL (N-CLL), AND A THIRD INTERMEDIATE CLL SUBGROUP (I-CLL). WHILE M-CLL AND N-CLL PATIENTS AT LARGE CORRESPONDED TO PATIENTS CARRYING MUTATED AND UNMUTATED IGHV GENES, RESPECTIVELY, LIMITED INFORMATION EXISTS REGARDING THE LESS DEFINED I-CLL GROUP. USING PYROSEQUENCING, WE INVESTIGATED THE PROGNOSTIC IMPACT OF THE PROPOSED 5 CPG SIGNATURE IN A WELL-CHARACTERIZED CLL COHORT (135 CASES), INCLUDING IGHV-MUTATED AND UNMUTATED PATIENTS AS WELL AS CLINICALLY AGGRESSIVE STEREOTYPED SUBSET #2 PATIENTS. OVERALL, WE CONFIRMED THE SIGNATURE'S ASSOCIATION WITH ESTABLISHED PROGNOSTIC MARKERS. MOREOVER, IN THE PRESENCE OF THE IGHV MUTATIONAL STATUS, THE EPIGENETIC SIGNATURE REMAINED INDEPENDENTLY ASSOCIATED WITH BOTH TIME-TO-FIRST-TREATMENT AND OVERALL SURVIVAL IN MULTIVARIATE ANALYSES. AS A PRIME FINDING, WE OBSERVED THAT SUBSET #2 PATIENTS WERE PREDOMINANTLY CLASSIFIED AS I-CLL, PROBABLY REFLECTING THEIR BORDERLINE IGHV MUTATIONAL STATUS (97-99% GERMLINE IDENTITY), THOUGH HAVING A SIMILARLY POOR PROGNOSIS AS N-CLL PATIENTS. IN SUMMARY, WE VALIDATED THE EPIGENETIC CLASSIFIER AS AN INDEPENDENT FACTOR IN CLL PROGNOSTICATION AND PROVIDE FURTHER EVIDENCE THAT SUBSET #2 IS A MEMBER OF THE I-CLL GROUP, HENCE SUPPORTING THE EXISTENCE OF A THIRD, INTERMEDIATE EPIGENETIC SUBGROUP. 2016 6 1062 31 CLINICAL SIGNIFICANCE OF DNA METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: RESULTS FROM 3 UK CLINICAL TRIALS. CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH MUTATED IMMUNOGLOBULIN HEAVY-CHAIN GENES (IGHV-M), PARTICULARLY THOSE LACKING POOR-RISK GENOMIC LESIONS, OFTEN RESPOND WELL TO CHEMOIMMUNOTHERAPY (CIT). DNA METHYLATION PROFILING CAN SUBDIVIDE EARLY-STAGE PATIENTS INTO NAIVE B-CELL-LIKE CLL (N-CLL), MEMORY B-CELL-LIKE CLL (M-CLL), AND INTERMEDIATE CLL (I-CLL), WITH DIFFERING TIMES TO FIRST TREATMENT AND OVERALL SURVIVAL. HOWEVER, WHETHER DNA METHYLATION CAN IDENTIFY PATIENTS DESTINED TO RESPOND FAVORABLY TO CIT HAS NOT BEEN ASCERTAINED. WE CLASSIFIED TREATMENT-NAIVE PATIENTS (N = 605) FROM 3 UK CHEMO AND CIT CLINICAL TRIALS INTO THE 3 EPIGENETIC SUBGROUPS, USING PYROSEQUENCING AND MICROARRAY ANALYSIS, AND PERFORMED EXPANSIVE SURVIVAL ANALYSIS. THE N-CLL, I-CLL, AND M-CLL SIGNATURES WERE FOUND IN 80% (N = 245/305), 17% (53/305), AND 2% (7/305) OF IGHV-UNMUTATED (IGHV-U) CASES, RESPECTIVELY, AND IN 9%, (19/216), 50% (108/216), AND 41% (89/216) OF IGHV-M CASES, RESPECTIVELY. MULTIVARIATE COX PROPORTIONAL ANALYSIS IDENTIFIED M-CLL AS AN INDEPENDENT PROGNOSTIC FACTOR FOR OVERALL SURVIVAL (HAZARD RATIO [HR], 0.46; 95% CONFIDENCE INTERVAL [CI], 0.24-0.87; P = .018) IN CLL4, AND FOR PROGRESSION-FREE SURVIVAL (HR, 0.25; 95% CI, 0.10-0.57; P = .002) IN ARCTIC AND ADMIRE PATIENTS. THE ANALYSIS OF EPIGENETIC SUBGROUPS IN PATIENTS ENTERED INTO 3 FIRST-LINE UK CLL TRIALS IDENTIFIES M-CLL AS AN INDEPENDENT MARKER OF PROLONGED SURVIVAL AND MAY AID IN THE IDENTIFICATION OF PATIENTS DESTINED TO DEMONSTRATE PROLONGED SURVIVAL AFTER CIT. 2019 7 27 31 A B-CELL EPIGENETIC SIGNATURE DEFINES THREE BIOLOGIC SUBGROUPS OF CHRONIC LYMPHOCYTIC LEUKEMIA WITH CLINICAL IMPACT. PROSPECTIVE IDENTIFICATION OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) DESTINED TO PROGRESS WOULD GREATLY FACILITATE THEIR CLINICAL MANAGEMENT. RECENTLY, WHOLE-GENOME DNA METHYLATION ANALYSES IDENTIFIED THREE CLINICOBIOLOGIC CLL SUBGROUPS WITH AN EPIGENETIC SIGNATURE RELATED TO DIFFERENT NORMAL B-CELL COUNTERPARTS. HERE, WE DEVELOPED A CLINICALLY APPLICABLE METHOD TO IDENTIFY THESE SUBGROUPS AND TO STUDY THEIR CLINICAL RELEVANCE. USING A SUPPORT VECTOR MACHINE APPROACH, WE BUILT A PREDICTION MODEL USING FIVE EPIGENETIC BIOMARKERS THAT WAS ABLE TO CLASSIFY CLL PATIENTS ACCURATELY INTO THE THREE SUBGROUPS, NAMELY NAIVE B-CELL-LIKE, INTERMEDIATE AND MEMORY B-CELL-LIKE CLL. DNA METHYLATION WAS QUANTIFIED BY HIGHLY REPRODUCIBLE BISULFITE PYROSEQUENCING ASSAYS IN TWO INDEPENDENT CLL SERIES. IN THE INITIAL SERIES (N=211), THE THREE SUBGROUPS SHOWED DIFFERENTIAL LEVELS OF IGHV (IMMUNOGLOBULIN HEAVY-CHAIN LOCUS) MUTATION (P<0.001) AND VH USAGE (P<0.03), AS WELL AS DIFFERENT CLINICAL FEATURES AND OUTCOME IN TERMS OF TIME TO FIRST TREATMENT (TTT) AND OVERALL SURVIVAL (P<0.001). A MULTIVARIATE COX MODEL SHOWED THAT EPIGENETIC CLASSIFICATION WAS THE STRONGEST PREDICTOR OF TTT (P<0.001) ALONG WITH BINET STAGE (P<0.001). THESE FINDINGS WERE CORROBORATED IN A VALIDATION SERIES (N=97). IN THIS STUDY, WE DEVELOPED A SIMPLE AND ROBUST METHOD USING EPIGENETIC BIOMARKERS TO CATEGORIZE CLLS INTO THREE SUBGROUPS WITH DIFFERENT CLINICOBIOLOGIC FEATURES AND OUTCOME. 2015 8 1473 39 DISTINCT PATTERNS OF GLOBAL PROMOTER METHYLATION IN EARLY STAGE CHRONIC LYMPHOCYTIC LEUKEMIA. GENOMIC AND EPIGENOMIC STUDIES OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) ARE RESHAPING OUR UNDERSTANDING OF THE DISEASE AND HAVE PROVIDED NEW PERSPECTIVES FOR A MORE INDIVIDUALIZED DIAGNOSIS AND NEW POTENTIAL THERAPEUTIC TARGETS. IN THIS STUDY, THE GLOBAL PROMOTER METHYLATION PROFILE WAS DETERMINED IN HIGHLY PURIFIED B-CELLS FROM 37 (BINET STAGE A) CLL PATIENTS, USING HIGH-RESOLUTION METHYLATION MICROARRAYS (27,578 CPG). OVERALL, THE METHYLATION PATTERN CORRELATED WITH THE MAJOR BIOLOGICAL (ZAP-70 AND CD38), AND MOLECULAR (IGHV MUTATION) MARKERS, DISTINGUISHING CLL CASES ACCORDING TO IGHV MUTATIONAL STATUS. CELL ADHESION MOLECULES WERE ENRICHED IN THE SIGNATURE OF UNMUTATED (UM) VERSUS MUTATED (M-) CLL. MOREOVER, IN M-CLL CPG HYPER-METHYLATION IN THREE GENES, INCLUDING SPG20, WAS SIGNIFICANTLY ANTI-CORRELATED WITH THE CORRESPONDING GENE EXPRESSION LEVEL. FINALLY, THE CORRELATION BETWEEN THE METHYLATION PATTERN AND CLINICAL PARAMETERS WAS INVESTIGATED. NOTABLY, OUT OF 42 METHYL-PROBES THAT WERE SIGNIFICANTLY ASSOCIATED WITH PROGRESSION FREE SURVIVAL (PFS), HYPER-METHYLATION OF SPG20 WAS ALSO POSITIVELY ASSOCIATED WITH PFS. THESE DATA SUPPORT THE NOTION THAT EPIGENETIC CHANGES HAVE CLINICAL IMPACT IN CLL AND MAY CONTRIBUTE TO THE IDENTIFICATION OF NOVEL CANDIDATE DISEASE-ASSOCIATED GENES POTENTIALLY USEFUL TO PREDICT THE CLINICAL OUTCOME OF EARLY STAGE CLL PATIENTS. 2014 9 1107 46 COMBINING CYTOGENETIC AND EPIGENETIC APPROACHES IN CHRONIC LYMPHOCYTIC LEUKEMIA IMPROVES PROGNOSIS PREDICTION FOR PATIENTS WITH ISOLATED 13Q DELETION. BACKGROUND: BOTH DEFECTIVE DNA METHYLATION AND ACTIVE DNA DEMETHYLATION PROCESSES ARE EMERGING AS IMPORTANT RISK FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). HOWEVER, ASSOCIATIONS BETWEEN 5-CYTOSINE EPIGENETIC MARKERS AND THE MOST FREQUENT CHROMOSOMAL ABNORMALITIES DETECTED IN CLL REMAIN TO BE ESTABLISHED. METHODS: CLL PATIENTS WERE RETROSPECTIVELY CLASSIFIED INTO A CYTOGENETIC LOW-RISK GROUP (ISOLATED 13Q DELETION), AN INTERMEDIATE-RISK GROUP (NORMAL KARYOTYPE OR TRISOMY 12), AND A HIGH-RISK GROUP (11Q DELETION, 17P DELETION, OR COMPLEX KARYOTYPE [>/= 3 BREAKPOINTS]). THE TWO 5-CYTOSINE DERIVATIVES, 5-METHYLCYTOSINE (5-MCYT) AND 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), WERE TESTED BY ELISA (N = 60), WHILE REAL-TIME QUANTITATIVE PCR WAS USED FOR DETERMINING TRANSCRIPTIONAL EXPRESSION LEVELS OF DNMT AND TET (N = 24). RESULTS: BY USING GLOBAL DNA METHYLATION/DEMETHYLATION LEVELS, IN THE LOW-RISK DISEASE GROUP, TWO SUBGROUPS WITH SIGNIFICANTLY DIFFERENT CLINICAL OUTCOMES HAVE BEEN IDENTIFIED (MEDIAN TREATMENT-FREE SURVIVAL [TFS] 45 VERSUS > 120 MONTHS FOR 5-MCYT, P = 0.0008, AND 63 VERSUS > 120 MONTHS FOR 5-HMCYT, P = 0.04). A DEFECTIVE 5-MCYT STATUS WAS FURTHER ASSOCIATED WITH A HIGHER PERCENTAGE OF 13Q DELETED NUCLEI (> 80%), THUS SUGGESTING AN ACQUIRED PROCESS. WHEN CONSIDERING THE CYTOGENETIC INTERMEDIATE/HIGH-RISK DISEASE GROUPS, AN ASSOCIATION OF 5-MCYT STATUS WITH LYMPHOCYTOSIS (P = 0.0008) AND THE LYMPHOCYTE DOUBLING TIME (P = 0.04) BUT NOT WITH TFS WAS OBSERVED, AS WELL AS A REDUCTION OF DNMT3A, TET1, AND TET2 TRANSCRIPTS. CONCLUSIONS: COMBINING CYTOGENETIC STUDIES WITH 5-MCYT ASSESSMENT ADDS ACCURACY TO CLL PATIENTS' PROGNOSES AND PARTICULARLY FOR THOSE WITH 13Q DELETION AS A SOLE CYTOGENETIC ABNORMALITY. 2017 10 1433 44 DIFFERENTIAL GENOME-WIDE ARRAY-BASED METHYLATION PROFILES IN PROGNOSTIC SUBSETS OF CHRONIC LYMPHOCYTIC LEUKEMIA. GLOBAL HYPOMETHYLATION AND REGIONAL HYPERMETHYLATION ARE WELL-KNOWN EPIGENETIC FEATURES OF CANCER; HOWEVER, IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), STUDIES ON GENOME-WIDE EPIGENETIC MODIFICATIONS ARE LIMITED. HERE, WE ANALYZED THE GLOBAL METHYLATION PROFILES IN CLL, BY APPLYING HIGH-RESOLUTION METHYLATION MICROARRAYS (27,578 CPG SITES) TO 23 CLL SAMPLES, BELONGING TO THE IMMUNOGLOBULIN HEAVY-CHAIN VARIABLE (IGHV) MUTATED (FAVORABLE) AND IGHV UNMUTATED/IGHV3-21 (POOR-PROGNOSTIC) SUBSETS. OVERALL, RESULTS DEMONSTRATED SIGNIFICANT DIFFERENCES IN METHYLATION PATTERNS BETWEEN THESE SUBGROUPS. SPECIFICALLY, IN IGHV UNMUTATED CLL, WE IDENTIFIED METHYLATION OF 7 KNOWN OR CANDIDATE TUMOR SUPPRESSOR GENES (EG, VHL, ABI3, AND IGSF4) AS WELL AS 8 UNMETHYLATED GENES INVOLVED IN CELL PROLIFERATION AND TUMOR PROGRESSION (EG, ADORA3 AND PRF1 ENHANCING THE NUCLEAR FACTOR-KAPPAB AND MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS, RESPECTIVELY). IN CONTRAST, THESE LATTER GENES WERE SILENCED BY METHYLATION IN IGHV MUTATED PATIENTS. THE ARRAY DATA WERE VALIDATED FOR SELECTED GENES USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION, QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION, AND BISULFITE SEQUENCING. FINALLY, THE SIGNIFICANCE OF DNA METHYLATION IN REGULATING GENE PROMOTERS WAS SHOWN BY REINDUCING 4 METHYLATED TUMOR SUPPRESSOR GENES (EG, VHL AND ABI3) IN IGHV UNMUTATED SAMPLES USING THE METHYL-INHIBITOR 5-AZA-2'-DEOXYCYTIDINE. TAKEN TOGETHER, OUR DATA FOR THE FIRST TIME REVEAL DIFFERENCES IN GLOBAL METHYLATION PROFILES BETWEEN PROGNOSTIC SUBSETS OF CLL, WHICH MAY UNFOLD EPIGENETIC SILENCING MECHANISMS INVOLVED IN CLL PATHOGENESIS. 2010 11 2438 42 EPIGENETIC SILENCING OF THE CIRCADIAN CLOCK GENE CRY1 IS ASSOCIATED WITH AN INDOLENT CLINICAL COURSE IN CHRONIC LYMPHOCYTIC LEUKEMIA. DISRUPTION OF CIRCADIAN RHYTHM IS BELIEVED TO PLAY A CRITICAL ROLE IN CANCER DEVELOPMENT. CRYPTOCHROME 1 (CRY1) IS A CORE COMPONENT OF THE MAMMALIAN CIRCADIAN CLOCK AND WE HAVE PREVIOUSLY SHOWN ITS DEREGULATED EXPRESSION IN A SUBGROUP OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). USING REAL-TIME RT-PCR IN A COHORT OF 76 CLL PATIENTS AND 35 NORMAL BLOOD DONORS WE NOW DEMONSTRATE THAT DIFFERENTIAL CRY1 MRNA EXPRESSION IN HIGH-RISK (HR) CD38+/IMMUNOGLOBULIN VARIABLE HEAVY CHAIN GENE (IGVH) UNMUTATED PATIENTS AS COMPARED TO LOW-RISK (LR) CD38-/IGVH MUTATED PATIENTS CAN BE ATTRIBUTED TO DOWN-MODULATION OF CRY1 IN LR CLL CASES. ANALYSIS OF THE DNA METHYLATION PROFILE OF THE CRY1 PROMOTER IN A SUBGROUP OF 57 PATIENTS REVEALED THAT CRY1 EXPRESSION IN LR CLL CELLS IS SILENCED BY ABERRANT PROMOTER CPG ISLAND HYPERMETHYLATION. THE METHYLATION PATTERN OF THE CRY1 PROMOTER PROVED TO HAVE HIGH PROGNOSTIC IMPACT IN CLL WHERE ABERRANT PROMOTER METHYLATION PREDICTED A FAVOURABLE OUTCOME. CRY1 MRNA TRANSCRIPT LEVELS DID NOT CHANGE OVER TIME IN THE MAJORITY OF PATIENTS WHERE SEQUENTIAL SAMPLES WERE AVAILABLE FOR ANALYSIS. WE ALSO COMPARED THE CRY1 EXPRESSION IN CLL WITH OTHER LYMPHOID MALIGNANCIES AND OBSERVED EPIGENETIC SILENCING OF CRY1 IN A PATIENT WITH B CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL). 2012 12 2133 31 EPIGENETIC INACTIVATION OF THE MIR-34A IN HEMATOLOGICAL MALIGNANCIES. MIR-34A IS A TRANSCRIPTIONAL TARGET OF P53 AND IMPLICATED IN CARCINOGENESIS. WE STUDIED THE ROLE OF MIR-34A METHYLATION IN A PANEL OF HEMATOLOGICAL MALIGNANCIES INCLUDING ACUTE LEUKEMIA [ACUTE MYELOID LEUKEMIA (AML) AND ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)], CHRONIC LEUKEMIA [CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND CHRONIC MYELOID LEUKEMIA (CML)], MULTIPLE MYELOMA (MM) AND NON-HODGKIN'S LYMPHOMA (NHL). THE METHYLATION STATUS OF MIR-34A PROMOTER WAS STUDIED IN 12 CELL LINES AND 188 DIAGNOSTIC SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. MIR-34A PROMOTER WAS UNMETHYLATED IN NORMAL CONTROLS BUT METHYLATED IN 75% LYMPHOMA AND 37% MYELOMA CELL LINES. HYPOMETHYLATING TREATMENT LED TO RE-EXPRESSION OF PRI-MIR-34A TRANSCRIPT IN LYMPHOMA CELLS WITH HOMOZYGOUS MIR-34A METHYLATION. IN PRIMARY SAMPLES AT DIAGNOSIS, MIR-34A METHYLATION WAS DETECTED IN 4% CLL, 5.5% MM SAMPLES AND 18.8% OF NHL AT DIAGNOSIS BUT NONE OF ALL, AML AND CML (P = 0.011). IN MM PATIENTS WITH PAIRED SAMPLES, MIR-34A METHYLATION STATUS REMAINED UNCHANGED AT PROGRESSION. AMONGST LYMPHOID MALIGNANCIES, MIR-34A WAS PREFERENTIALLY METHYLATED IN NHL (P = 0.018), IN PARTICULAR NATURAL KILLER (NK)/T-CELL LYMPHOMA. IN CONCLUSION, AMONGST HEMATOLOGICAL MALIGNANCIES, MIR-34A METHYLATION IS PREFERENTIALLY HYPERMETHYLATED IN NHL, IN PARTICULAR NK/T-CELL LYMPHOMA, IN A TUMOR-SPECIFIC MANNER, THEREFORE THE ROLE OF MIR-34A IN LYMPHOMAGENESIS WARRANTS FURTHER STUDY. 2010 13 3071 49 GENOME-WIDE DNA METHYLATION PROFILING INTEGRATED WITH GENE EXPRESSION PROFILING IDENTIFIES PAX9 AS A NOVEL PROGNOSTIC MARKER IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), EPIGENOMIC AND GENOMIC STUDIES HAVE EXPANDED THE EXISTING KNOWLEDGE ABOUT THE DISEASE BIOLOGY AND LED TO THE IDENTIFICATION OF POTENTIAL BIOMARKERS RELEVANT FOR IMPLEMENTATION OF PERSONALIZED MEDICINE. IN THIS STUDY, AN ATTEMPT HAS BEEN MADE TO EXAMINE AND INTEGRATE THE GLOBAL DNA METHYLATION CHANGES WITH GENE EXPRESSION PROFILE AND THEIR IMPACT ON CLINICAL OUTCOME IN EARLY STAGE CLL PATIENTS. RESULTS: THE INTEGRATION OF DNA METHYLATION PROFILE (N = 14) WITH THE GENE EXPRESSION PROFILE (N = 21) REVEALED 142 GENES AS HYPERMETHYLATED-DOWNREGULATED AND; 62 GENES AS HYPOMETHYLATED-UPREGULATED IN EARLY STAGE CLL PATIENTS COMPARED TO CD19+ B-CELLS FROM HEALTHY INDIVIDUALS. THE MRNA EXPRESSION LEVELS OF 17 GENES IDENTIFIED TO BE DIFFERENTIALLY METHYLATED AND/OR DIFFERENTIALLY EXPRESSED WAS FURTHER EXAMINED IN EARLY STAGE CLL PATIENTS (N = 93) BY QUANTITATIVE REAL TIME PCR (RQ-PCR). SIGNIFICANT DIFFERENCES WERE OBSERVED IN THE MRNA EXPRESSION OF MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, AND SPRY2 GENES IN CLL CELLS AS COMPARED TO B-CELLS FROM HEALTHY INDIVIDUALS. THE ANALYSIS IN THE IGHV MUTATION BASED CATEGORIES (UNMUTATED = 39, MUTATED = 54) REVEALED SIGNIFICANTLY HIGHER MRNA EXPRESSION OF CRY1 AND PAX9 GENES IN THE IGHV UNMUTATED SUBGROUP (P < 0.001). THE RELATIVE RISK OF TREATMENT INITIATION WAS SIGNIFICANTLY HIGHER AMONG PATIENTS WITH HIGH EXPRESSION OF CRY1 (RR = 1.91, P = 0.005) OR PAX9 (RR = 1.87, P = 0.001). HIGH EXPRESSION OF CRY1 (HR: 3.53, P < 0.001) OR PAX9 (HR: 3.14, P < 0.001) GENE WAS SIGNIFICANTLY ASSOCIATED WITH SHORTER TIME TO FIRST TREATMENT. THE HIGH EXPRESSION OF PAX9 GENE (HR: 3.29, 95% CI 1.172-9.272, P = 0.016) WAS ALSO PREDICTIVE OF SHORTER OVERALL SURVIVAL IN CLL. CONCLUSIONS: THE DNA METHYLATION CHANGES ASSOCIATED WITH MRNA EXPRESSION OF CRY1 AND PAX9 GENES ALLOW RISK STRATIFICATION OF EARLY STAGE CLL PATIENTS. THIS COMPREHENSIVE ANALYSIS SUPPORTS THE CONCEPT THAT THE EPIGENETIC CHANGES ALONG WITH THE ALTERED EXPRESSION OF GENES HAVE THE POTENTIAL TO PREDICT CLINICAL OUTCOME IN EARLY STAGE CLL PATIENTS. 2017 14 3448 39 HYPERMETHYLATION OF THE N-MYC DOWNSTREAM-REGULATED GENE 2 PROMOTER IN PERIPHERAL BLOOD MONONUCLEAR CELLS IS ASSOCIATED WITH LIVER FIBROSIS IN CHRONIC HEPATITIS B. DNA METHYLATION IS A FUNDAMENTAL EPIGENETIC MODIFICATION TO REGULATE GENE EXPRESSION. N-MYC DOWNSTREAM-REGULATED GENE (NDRG) 2 IS A CYTOPLASMIC PROTEIN AND PARTICIPATES IN THE PATHOGENESIS OF LIVER FIBROSIS. IN THIS STUDY, THE MRNA EXPRESSION AND METHYLATION STATUS OF NDRG2 WAS EVALUATED IN PATIENTS WITH CHRONIC HEPATITIS B (CHB). THE STUDY INCLUDED 143 CHB PATIENTS AND 65 NORMAL CONTROLS (NC). THE MRNA EXPRESSION OF NDRG2 IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) WAS DETECTED BY QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION. THE METHYLATION STATUS OF THE NDRG2 PROMOTER IN PBMCS WAS DETECTED BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. THE NDRG2 MRNA LEVEL WAS LOWER IN THE CHB GROUP THAN IN THE NC GROUP (P < 0.001). METHYLATION FREQUENCY OF THE NDRG2 PROMOTER WAS SIGNIFICANTLY HIGHER IN CHB PATIENTS THAN IN THE NC GROUP (52.44% VS. 26.15%, P < 0.001). IMPORTANTLY, THE RELATIVE EXPRESSION LEVELS OF NDRG2 MRNA WERE SIGNIFICANTLY LOWER IN THE METHYLATED GROUP THAN IN THE UNMETHYLATED GROUP IN BOTH CHB PATIENTS AND NC (P < 0.001). FURTHERMORE, A LOWER MRNA LEVEL AND HYPERMETHYLATION OF NDRG2 WERE ASSOCIATED WITH LIVER FIBROSIS AND INFLAMMATION GRADE IN CHB. THE ASPARTATE AMINOTRANSFERASE-TO-PLATELET RATIO INDEX (APRI) SCORE IS WIDELY USED TO PREDICT LIVER FIBROSIS. THE MRNA EXPRESSION LEVELS AND METHYLATION STATUS OF NDRG2 SHOWED A BETTER SCORE COMPARED TO APRI FOR DISCRIMINATING THE SEVERITY OF LIVER FIBROSIS. IN CONCLUSION, HYPERMETHYLATION OF NDRG2 IN PBMCS WAS CORRELATED WITH DECREASED MRNA EXPRESSION AND WITH LIVER FIBROSIS. THE METHYLATION STATUS OF THE NDRG2 PROMOTER IN PBMCS IS A POTENTIAL NONINVASIVE BIOMARKER TO PREDICT THE SEVERITY OF LIVER FIBROSIS. 2017 15 2440 44 EPIGENETIC SILENCING OF TUMOR SUPPRESSOR LONG NON-CODING RNA BM742401 IN CHRONIC LYMPHOCYTIC LEUKEMIA. BM742401 IS A TUMOR SUPPRESSOR LNCRNA DOWNREGULATED IN GASTRIC CANCER. AS THE PROMOTER REGION AND THE ENTIRE TRANSCRIPT ARE EMBEDDED IN A CPG ISLAND, WE POSTULATED THAT BM742401 IS A TUMOR SUPPRESSOR LNCRNA INACTIVATED BY DNA METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). THE PROMOTER OF BM742401 WAS UNMETHYLATED IN NORMAL CONTROLS INCLUDING THREE EACH OF NORMAL BONE MARROW, PERIPHERAL BLOOD BUFFY COATS, AND CD19-SORTED PERIPHERAL B-CELLS, BUT METHYLATED IN FOUR (57.1%) CLL CELL LINES. METHYLATION OF BM742401 CORRELATED INVERSELY WITH EXPRESSION. IN THE COMPLETELY METHYLATED WAC3CD5+ CLL CELLS, 5-AZA-2'-DEOXYCYTIDINE TREATMENT LED TO PROMOTER DEMETHYLATION AND RE-EXPRESSION OF BM742401 TRANSCRIPT. FUNCTIONALLY, STABLE OVEREXPRESSION OF BM742401 RESULTED IN INHIBITION OF CELLULAR PROLIFERATION AND ENHANCED APOPTOSIS THROUGH CASPASE-9-DEPENDENT INTRINSIC BUT NOT CASPASE-8-DEPENDENT EXTRINSIC APOPTOSIS PATHWAY, SUGGESTING A TUMOR SUPPRESSOR ROLE OF BM742401 IN CLL. IN PRIMARY CLL SAMPLES, METHYLATION OF BM742401 WAS DETECTED IN 43/98 (43.9%) OF PATIENTS. MOREOVER, AMONG CLL PATIENTS WITH STANDARD-RISK CYTOGENETIC ABERRATIONS, METHYLATION OF BM742401 CORRELATED WITH ADVANCED RAI STAGE (>/= STAGE 2)(P = 0.002). FURTHERMORE, BM742401 METHYLATION WAS ASSOCIATED WITH MIR-129-2 METHYLATION (P = 0.05). BM742401 IS A TUMOR SUPPRESSOR LNCRNA FREQUENTLY METHYLATED IN CLL. THE MECHANISM OF BM742401 AS A TUMOR SUPPRESSOR WARRANTS FURTHER STUDIES. 2016 16 2126 50 EPIGENETIC INACTIVATION OF MIR-34B/C IN ADDITION TO MIR-34A AND DAPK1 IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: TP53 MUTATION/DELETION IS UNCOMMON IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). WE POSTULATED THAT COMPONENTS OF TP53-CENTERED TUMOR SUPPRESSOR NETWORK, MIR-34B/C, IN ADDITION TO DAPK1 AND MIR-34A MIGHT BE INACTIVATED BY DNA HYPERMETHYLATION. MOREOVER, WE TESTED IF MIR-34B/C METHYLATION MIGHT CORRELATE WITH MIR-203 OR MIR-124-1 METHYLATION IN CLL. METHODS: MIR-34B/C, MIR-34A AND DAPK1 METHYLATION WAS STUDIED IN 11 NORMAL CONTROLS, 7 CLL CELL LINES, AND 78 DIAGNOSTIC CLL SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. MEC-1 CELLS WERE TREATED WITH 5-AZA-2'-DEOXYCYTIDINE FOR REVERSAL OF METHYLATION-ASSOCIATED MIRNA SILENCING. TUMOR SUPPRESSOR PROPERTIES OF MIR-34B WERE DEMONSTRATED BY OVER-EXPRESSION OF PRECURSOR MIR-34B IN MEC-1 CELLS. RESULTS: MIR-34B/C PROMOTER WAS UNMETHYLATED IN NORMAL CONTROLS, BUT COMPLETELY METHYLATED IN 4 CLL CELL LINES. MIR-34B/C EXPRESSION WAS INVERSELY CORRELATED WITH MIR-34B/C METHYLATION. DIFFERENT MSP STATUSES OF MIR-34B/C, INCLUDING COMPLETE METHYLATION AND COMPLETE UNMETHYLATION, WERE VERIFIED BY QUANTITATIVE BISULFITE PYROSEQUENCING. 5-AZA-2'-DEOXYCYTIDINE TREATMENT RESULTED IN PROMOTER DEMETHYLATION AND MIR-34B RE-EXPRESSION IN MEC1 CELLS. MOREOVER, OVER-EXPRESSION OF MIR-34B RESULTED IN INHIBITION OF CELLULAR PROLIFERATION AND INCREASED CELL DEATH. IN PRIMARY CLL SAMPLES, MIR-34A, MIR-34B/C AND DAPK1 METHYLATION WAS DETECTED IN 2.6%, 17.9% AND 34.6% OF PATIENTS AT DIAGNOSIS RESPECTIVELY. FURTHERMORE, 39.7%, 3.8% AND 2.6% PATIENTS HAD METHYLATION OF ONE, TWO OR ALL THREE GENES RESPECTIVELY. OVERALL, 46.2% PATIENTS HAD METHYLATION OF AT LEAST ONE OF THESE THREE GENES. BESIDES, MIR-34B/C METHYLATION WAS ASSOCIATED WITH METHYLATION OF MIR-34A (P = 0.03) AND MIR-203 (P = 0.012) IN CLL. CONCLUSIONS: TAKEN TOGETHER, MIR-34B/C IS A TUMOR SUPPRESSOR MIRNA FREQUENTLY METHYLATED, AND HENCE SILENCED IN CLL. TOGETHER WITH DAPK1 METHYLATION, MIR-34B/C METHYLATION IS IMPLICATED IN THE DISRUPTION OF THE TP53-CENTERED TUMOR SUPPRESSOR NETWORK. MOREOVER, THE ASSOCIATION OF MIRNA METHYLATION WARRANTS FURTHER STUDY. 2014 17 2131 39 EPIGENETIC INACTIVATION OF THE HSA-MIR-203 IN HAEMATOLOGICAL MALIGNANCIES. MIR-203 IS A TUMOUR SUPPRESSOR MICRORNA (MIRNA). WE STUDIED THE METHYLATION OF HSA-MIR-203 IN 150 SAMPLES INCLUDING ACUTE MYELOID LEUKAEMIA (AML), ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL), CHRONIC MYELOID LEUKAEMIA (CML), CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) AND NON-HODGKIN'S LYMPHOMA (NHL) BY METHYLATION-SPECIFIC PCR, AND MIRNA EXPRESSION BY STEM-LOOP RT-QPCR. HSA-MIR-203 PROMOTER WAS UNMETHYLATED IN NORMAL CONTROLS BUT HOMOZYGOUSLY METHYLATED IN TWO AML AND FOUR LYMPHOMA CELL LINES, IN WHICH 5-AZA-2'-DEOXYCYTIDINE TREATMENT LED TO PROMOTER DEMETHYLATION AND MIR-203 RE-EXPRESSION. RESTORATION OF MIR-203 EXPRESSION IN LYMPHOMA CELLS INHIBITED CELLULAR PROLIFERATION AND INCREASED CELL DEATH, SUGGESTING AN INHERENT TUMOUR SUPPRESSOR ACTIVITY. IN PRIMARY SAMPLES, HSA-MIR-203 METHYLATION WAS ABSENT IN CML BUT DETECTED IN 5.0% ALL, 10.0% AML, 42.0% CLL AND 38.8% OF NHL (INCLUDING SIX [60.0%] NATURAL KILLER-CELL, NINE [40.9%] B-CELL AND FOUR [23.5%] T CELL NHL). MOREOVER, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH HYPERMETHYLATION OF HSA-MIR-34A, -124A AND -196B IN NHL BUT NOT CLL. IN CLL, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH A HIGHER PRESENTING HB LEVEL (P = 0.033). THE PROJECTED 10 YEAR OVERALL SURVIVAL OF THE CLL PATIENTS WAS 58.2%, WHICH WAS IMPACTED BY RAI STAGE AND HIGH-RISK KARYOTYPES BUT NOT HSA-MIR-203 METHYLATION. HSA-MIR-203 WAS MORE FREQUENTLY METHYLATED IN LYMPHOID THAN MYELOID MALIGNANCIES (P = 0.002). IN CONCLUSION, MIR-203, A TUMOUR SUPPRESSOR GENE, WAS HYPERMETHYLATED IN A TUMOUR-SPECIFIC MANNER WITH GENE SILENCING. HSA-MIR-203 WAS MORE FREQUENTLY HYPERMETHYLATED IN LYMPHOID THAN MYELOID MALIGNANCIES. IN NHL, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH CONCOMITANT METHYLATION OF OTHER TUMOUR SUPPRESSOR MIRNAS. THE FREQUENT HSA-MIR-203 METHYLATION IN LYMPHOID MALIGNANCIES SUGGESTED A PATHOGENETIC ROLE OF HSA-MIR-203 METHYLATION. 2011 18 337 30 ALTERATIONS IN DNA METHYLATION/DEMETHYLATION INTERMEDIATES PREDICT CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA. CYTOSINE DERIVATIVE DYSREGULATIONS REPRESENT IMPORTANT EPIGENETIC MODIFICATIONS WHOSE IMPACT ON THE CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS INCOMPLETELY UNDERSTOOD. HENCE, GLOBAL LEVELS OF 5-METHYLCYTOSINE (5-MCYT), 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), 5-CARBOXYLCYTOSINE (5-CACYT) AND 5-HYDROXYMETHYLURACIL WERE TESTED IN PURIFIED B CELLS FROM CLL PATIENTS (N = 55) AND CONTROLS (N = 17). THE DNA METHYLATION 'WRITERS' (DNA METHYLTRANSFERASES [DNMT1/3A/3B]), 'READERS' (METHYL-CPG-BINDING DOMAIN [MBD2/4]), 'EDITORS' (TEN-ELEVEN TRANSLOCATION [TET1/2/3]) AND 'MODULATORS' (SAT1) WERE ALSO EVALUATED. ACCORDINGLY, PATIENTS WERE STRATIFIED INTO THREE SUBGROUPS. FIRST, A SUBGROUP WITH A GLOBAL DEFICIT IN CYTOSINE DERIVATIVES CHARACTERIZED BY HYPERLYMPHOCYTOSIS, REDUCED MEDIAN PROGRESSION FREE SURVIVAL (PFS = 52 MONTHS) AND SHORTER TREATMENT FREE SURVIVAL (TFS = 112 MONTHS) WAS IDENTIFIED. IN THIS SUBGROUP, MAJOR EPIGENETIC MODIFICATIONS WERE HIGHLIGHTED INCLUDING A REDUCTION OF 5-MCYT, 5-HMCYT, 5-CACYT ASSOCIATED WITH DNMT3A, MBD2/4 AND TET1/2 DOWNREGULATION. SECOND, THE CYTOSINE DERIVATIVE ANALYSIS REVEALED A SUBGROUP WITH A PARTIAL DEFICIT (PFS = 84, TFS = 120 MONTHS), MAINLY AFFECTING DNA DEMETHYLATION (5-HMCYT REDUCTION, SAT1 INDUCTION). THIRD, A SUBGROUP EPIGENETICALLY SIMILAR TO CONTROLS WAS IDENTIFIED (PFS AND TFS > 120 MONTHS). THE PROGNOSTIC IMPACT OF STRATIFYING CLL PATIENTS WITHIN THREE EPIGENETIC SUBGROUPS WAS CONFIRMED IN A VALIDATION COHORT. IN CONCLUSION, OUR RESULTS SUGGEST THAT DYSREGULATIONS OF CYTOSINE DERIVATIVE REGULATORS REPRESENT MAJOR EVENTS ACQUIRED DURING CLL PROGRESSION AND ARE INDEPENDENT FROM IGHV MUTATIONAL STATUS. 2017 19 5246 35 PROGNOSTIC SCORE INCLUDING GENE MUTATIONS IN CHRONIC MYELOMONOCYTIC LEUKEMIA. PURPOSE: SEVERAL PROGNOSTIC SCORING SYSTEMS HAVE BEEN PROPOSED FOR CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML), A DISEASE IN WHICH SOME GENE MUTATIONS-INCLUDING ASXL1-HAVE BEEN ASSOCIATED WITH POOR PROGNOSIS IN UNIVARIABLE ANALYSES. WE DEVELOPED AND VALIDATED A PROGNOSTIC SCORE FOR OVERALL SURVIVAL (OS) BASED ON MUTATIONAL STATUS AND STANDARD CLINICAL VARIABLES. PATIENTS AND METHODS: WE GENOTYPED ASXL1 AND UP TO 18 OTHER GENES INCLUDING EPIGENETIC (TET2, EZH2, IDH1, IDH2, DNMT3A), SPLICING (SF3B1, SRSF2, ZRSF2, U2AF1), TRANSCRIPTION (RUNX1, NPM1, TP53), AND SIGNALING (NRAS, KRAS, CBL, JAK2, FLT3) REGULATORS IN 312 PATIENTS WITH CMML. GENOTYPES AND CLINICAL VARIABLES WERE INCLUDED IN A MULTIVARIABLE COX MODEL OF OS VALIDATED BY BOOTSTRAPPING. A SCORING SYSTEM WAS DEVELOPED USING REGRESSION COEFFICIENTS FROM THIS MODEL. RESULTS: ASXL1 MUTATIONS (P < .0001) AND, TO A LESSER EXTENT, SRSF2 (P = .03), CBL (P = .003), AND IDH2 (P = .03) MUTATIONS PREDICTED INFERIOR OS IN UNIVARIABLE ANALYSIS. THE RETAINED INDEPENDENT PROGNOSTIC FACTORS INCLUDED ASXL1 MUTATIONS, AGE OLDER THAN 65 YEARS, WBC COUNT GREATER THAN 15 X10(9)/L, PLATELET COUNT LESS THAN 100 X10(9)/L, AND ANEMIA (HEMOGLOBIN < 10 G/DL IN FEMALE PATIENTS, < 11G/DL IN MALE PATIENTS). THE RESULTING FIVE-PARAMETER PROGNOSTIC SCORE DELINEATED THREE GROUPS OF PATIENTS WITH MEDIAN OS NOT REACHED, 38.5 MONTHS, AND 14.4 MONTHS, RESPECTIVELY (P < .0001), AND WAS VALIDATED IN AN INDEPENDENT COHORT OF 165 PATIENTS (P < .0001). CONCLUSION: A NEW PROGNOSTIC SCORE INCLUDING ASXL1 STATUS, AGE, HEMOGLOBIN, WBC, AND PLATELET COUNTS DEFINES THREE GROUPS OF CMML PATIENTS WITH DISTINCT OUTCOMES. BASED ON CONCORDANCE ANALYSIS, THIS SCORE APPEARS MORE DISCRIMINATIVE THAN THOSE BASED SOLELY ON CLINICAL PARAMETERS. 2013 20 2426 46 EPIGENETIC SILENCING OF LONG NON-CODING RNA BM742401 IN MULTIPLE MYELOMA: IMPACT ON PROGNOSIS AND MYELOMA DISSEMINATION. BACKGROUND: LONG NON-CODING RNA (LNCRNA) BM742401 IS A TUMOR SUPPRESSOR IN GASTRIC CANCER AND CHRONIC LYMPHOCYTIC LEUKEMIA. AS THE PROMOTER AND CODING REGION OF BM742401 ARE FULLY EMBEDDED IN A CPG ISLAND, WE HYPOTHESIZED THAT BM742401 IS A TUMOR SUPPRESSOR LNCRNA EPIGENETICALLY SILENCED BY PROMOTER DNA METHYLATION IN MULTIPLE MYELOMA. METHODS: METHYLATION-SPECIFIC PCR AND QUANTITATIVE BISULFITE PYROSEQUENCING WERE PERFORMED TO DETECT THE METHYLATION OF BM742401 IN NORMAL PLASMA CELLS, MYELOMA CELL LINES AND PRIMARY MYELOMA SAMPLES. THE EXPRESSION OF BM742401 WAS MEASURED BY QRT-PCR. THE FUNCTION OF BM742401 IN MULTIPLE MYELOMA CELLS WAS ANALYZED BY LENTIVIRUS TRANSDUCTION FOLLOWED BY MIGRATION ASSAY. RESULTS: BM742401 METHYLATION WAS DETECTED IN 10 (66.7%) MYELOMA CELL LINES BUT NOT NORMAL PLASMA CELLS, AND INVERSELY CORRELATED WITH EXPRESSION OF BM742401. IN PRIMARY SAMPLES, BM742401 METHYLATION WAS DETECTED IN 3 (12.5%) MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE, 9 (15.8%) MYELOMA AT DIAGNOSIS AND 8 (17.0%) MYELOMA AT RELAPSE/PROGRESSION. MOREOVER, BM742401 METHYLATION AT DIAGNOSIS WAS ASSOCIATED WITH INFERIOR OVERALL SURVIVAL (MEDIAN OS: 25 VS. 39 MONTHS; P = 0.0496). IN MYELOMA CELL LINE JJN-3, STABLE OVEREXPRESSION OF BM742401 BY LENTIVIRUS TRANSDUCTION RESULTED IN REDUCED CELL MIGRATION (P = 0.0001) BUT NOT IMPACTING CELL DEATH OR PROLIFERATION. CONCLUSIONS: THIS IS THE FIRST REPORT OF TUMOR-SPECIFIC METHYLATION-MEDIATED SILENCING OF BM742401 IN MYELOMA, WHICH IS LIKELY AN EARLY EVENT IN MYELOMAGENESIS WITH ADVERSE IMPACT ON OVERALL SURVIVAL. MOREOVER, BM742401 IS A TUMOR SUPPRESSOR LNCRNA BY INHIBITING MYELOMA CELL MIGRATION, HENCE IMPLICATED IN MYELOMA PLASMA CELL HOMING, METASTASIS AND DISEASE PROGRESSION. 2020