1 1056 160 CLINICAL MANIFESTATIONS AND EPIGENETIC MECHANISMS OF GASTRIC MUCOSA ASSOCIATED LYMPHOID TISSUE LYMPHOMA AND LONG-TERM FOLLOW-UP FOLLOWING HELICOBACTER PYLORI ERADICATION. THE CURRENT STUDY AIMED TO SUMMARIZE THE CLINICAL MANIFESTATIONS AND IDENTIFY THE EPIGENETIC MECHANISMS OF GASTRIC MUCOSA ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMA, AS WELL AS EVALUATE THE LONG-TERM EFFECTS OF HELICOBACTER PYLORI (H. PYLORI) ERADICATION. A TOTAL OF 122 PATIENTS WITH MARGINAL ZONE B-CELL LYMPHOMA OF PRIMARY GASTRIC MALT LYMPHOMA WERE ENROLLED IN THE PRESENT STUDY. THE CLINICAL MANIFESTATIONS OF GASTRIC MALT LYMPHOMA, INCLUDING SYMPTOMS, H. PYLORI STATE AND ENDOSCOPIC TYPE, WERE SUMMARIZED. THE RESPONSE TO THERAPY WAS EVALUATED IN PATIENTS THAT UNDERWENT H. PYLORI ERADICATION. SURVIVAL ANALYSIS WAS ESTIMATED USING THE KAPLAN-MEIER METHOD. THE EXPRESSION OF MICRORNA-383 (MIR-383) IN TUMOR TISSUES AND CELL LINES WAS DETERMINED USING REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION. FURTHERMORE, BIOINFORMATIC ANALYSES, LUCIFERASE REPORTER ASSAYS. AND WESTERN BLOT ANALYSIS IDENTIFIED ZINC FINGER E-BOX BINDING HOMEOBOX 2 (ZEB2) AS A DIRECT TARGET GENE OF MIR-383. AN MTT ASSAY WAS USED TO EXAMINE THE FUNCTION OF MIR-383 AND ZEB2 IN MALT LYMPHOMA. THE CLINICAL SYMPTOMS OF PATIENTS WITH GASTRIC MALT LYMPHOMA WERE NON-SPECIFIC AND INCLUDED EPIGASTRIC PAIN, ABDOMINAL DISCOMFORT AND BLEEDING. THE MAJORITY OF ENDOSCOPIC TYPES WERE CLASSIFIED AS ULCER, EROSION AND MUCOSA EDEMA. THE H. PYLORI INFECTION RATE WAS 79.5% (97/122) AND A TOTAL OF 47 PATIENTS UNDERWENT ERADICATION THERAPY. LYMPHOMA REMISSION WAS ACHIEVED IN 93.6% (44/47) OF PATIENTS AND COMPLETE REMISSION (CR) WAS ACHIEVED IN 74.4% (35/47). THE MEDIAN FOLLOW-UP TIME WAS 38 MONTHS (RANGE, 10-132 MONTHS) AND THE MEDIAN TIME TAKEN TO ACHIEVE CR WAS 4 MONTHS (RANGE, 3-7 MONTHS). THE ESTIMATED 3-YEAR SURVIVAL RATE WAS 90.3% AND THE 5-YEAR SURVIVAL RATE WAS 76.2%. THEREFORE, IT WAS DETERMINED THAT PATIENTS WITH STAGE I OR II GASTRIC MALT LYMPHOMA ARE ABLE TO UNDERGO H. PYLORI ERADICATION AS A FIRST-LINE TREATMENT AND THAT THE SURVIVAL RATE OF PATIENTS UNDERGOING THIS TREATMENT IS HIGH. FURTHERMORE, IT WAS DETERMINED THAT THE MECHANISM BY WHICH MIR-383 AND ZEB2 CONTRIBUTE TO MALT LYMPHOMA PROGRESSION IS BY THE TARGETING OF ZEB2 BY MIR-383, WHICH INHIBITS THE PROLIFERATION OF CANCER CELLS. 2018 2 3756 35 INTEGRATED GENOMIC SEQUENCING IN MYELOID BLAST CRISIS CHRONIC MYELOID LEUKEMIA (MBC-CML), IDENTIFIED POTENTIALLY IMPORTANT FINDINGS IN THE CONTEXT OF LEUKEMOGENESIS MODEL. CHRONIC MYELOID LEUKEMIA (CML) IS A MODEL OF LEUKEMOGENESIS IN WHICH THE EXACT MOLECULAR MECHANISMS UNDERLYING BLAST CRISIS STILL REMAINED UNEXPLORED. THE CURRENT STUDY IDENTIFIED MULTIPLE COMMON AND RARE IMPORTANT FINDINGS IN MYELOID BLAST CRISIS CML (MBC-CML) USING INTEGRATED GENOMIC SEQUENCING, COVERING ALL CLASSES OF GENES IMPLICATED IN THE LEUKEMOGENESIS MODEL. INTEGRATED GENOMIC SEQUENCING VIA WHOLE EXOME SEQUENCING (WES), CHROMOSOME-SEQ AND RNA-SEQUENCING WERE CONDUCTED ON THE PERIPHERAL BLOOD SAMPLES OF THREE CML PATIENTS IN THE MYELOID BLAST CRISIS. AN IN-HOUSE FILTERING PIPELINE WAS APPLIED TO ASSESS IMPORTANT VARIANTS IN CANCER-RELATED GENES. STANDARD VARIANT INTERPRETATION GUIDELINES WERE USED FOR THE INTERPRETATION OF POTENTIALLY IMPORTANT FINDINGS (PIFS) AND POTENTIALLY ACTIONABLE FINDINGS (PAFS). SINGLE NUCLEOTIDE VARIATION (SNV) AND SMALL INDEL ANALYSIS BY WES DETECTED SIXTEEN PIFS AFFECTING ALL FIVE KNOWN CLASSES OF LEUKEMOGENIC GENES IN MYELOID MALIGNANCIES INCLUDING SIGNALING PATHWAY COMPONENTS (ABL1, PIK3CB, PTPN11), TRANSCRIPTION FACTORS (GATA2, PHF6, IKZF1, WT1), EPIGENETIC REGULATORS (ASXL1), TUMOR SUPPRESSOR AND DNA REPAIR GENES (BRCA2, ATM, CHEK2) AND COMPONENTS OF SPLICEOSOME (PRPF8). THESE VARIANTS AFFECT GENES INVOLVED IN LEUKEMIA STEM CELL PROLIFERATION, SELF-RENEWAL, AND DIFFERENTIATION. BOTH PATIENTS NO.1 AND NO.2 HAD ACTIONABLE KNOWN MISSENSE VARIANTS ON ABL1 (P.Y272H, P.F359V) AND FRAMESHIFT VARIANTS ON ASXL1 (P.A627GFS*8, P.G646WFS*12). THE GATA2-L359S IN PATIENT NO.1, PTPN11-G503V AND IKZF1-R208Q VARIANTS IN THE PATIENT NO.3 WERE ALSO PAFS. RNA-SEQUENCING WAS USED TO CONFIRM ALL OF THE IDENTIFIED VARIANTS. IN THE PATIENT NO. 3, CHROMOSOME SEQUENCING REVEALED MULTIPLE PATHOGENIC DELETIONS IN THE SHORT AND LONG ARMS OF CHROMOSOME 7, AFFECTING AT LEAST THREE CRITICAL LEUKEMOGENIC GENES (IKZF1, EZH2, AND CUX1). THE LARGE DELETION DISCOVERED ON THE SHORT ARM OF CHROMOSOME 17 IN PATIENT NO. 2 RESULTED IN THE DELETION OF TP53 GENE AS WELL. INTEGRATED GENOMIC SEQUENCING COMBINED WITH RNA-SEQUENCING CAN SUCCESSFULLY DISCOVER AND CONFIRM A WIDE RANGE OF VARIANTS, FROM SNVS TO CNVS. THIS STRATEGY MAY BE AN EFFECTIVE METHOD FOR IDENTIFYING ACTIONABLE FINDINGS AND UNDERSTANDING THE PATHOPHYSIOLOGICAL MECHANISMS UNDERLYING MBC-CML, AS WELL AS PROVIDING FURTHER INSIGHTS INTO THE GENETIC BASIS OF MBC-CML AND ITS MANAGEMENT IN THE FUTURE. 2022 3 6383 32 THE ROLE OF PHF6 IN HEMATOPOIESIS AND HEMATOLOGIC MALIGNANCIES. EPIGENETIC REGULATION OF GENE EXPRESSION REPRESENTS AN IMPORTANT MECHANISM IN THE MAINTENANCE OF STEM CELL FUNCTION. ALTERATIONS IN EPIGENETIC REGULATION CONTRIBUTE TO THE PATHOGENESIS OF HEMATOLOGICAL MALIGNANCIES. PLANT HOMEODOMAIN FINGER PROTEIN 6 (PHF6) IS A MEMBER OF THE PLANT HOMEODOMAIN (PHD)-LIKE ZINC FINGER FAMILY OF PROTEINS THAT IS INVOLVED IN TRANSCRIPTIONAL REGULATION THROUGH THE MODIFICATION OF THE CHROMATIN STATE. GERMLINE MUTATION OF PHF6 IS THE CAUSATIVE GENETIC ALTERATION OF THE X-LINKED MENTAL RETARDATION BORJESON-FORSSMAN-LEHMANN SYNDROME (BFLS). SOMATIC MUTATIONS IN PHF6 ARE IDENTIFIED IN HUMAN LEUKEMIA, SUCH AS ADULT T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL, ~ 38%), PEDIATRIC T-ALL (~ 16%), ACUTE MYELOID LEUKEMIA (AML, ~ 3%), CHRONIC MYELOID LEUKEMIA (CML, ~ 2.5%), MIXED PHENOTYPE ACUTE LEUKEMIA (MPAL, ~ 20%), AND HIGH-GRADE B-CELL LYMPHOMA (HGBCL, ~ 3%). MORE RECENT STUDIES IMPLY AN ONCOGENIC EFFECT OF PHF6 IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL) AND SOLID TUMORS. THESE DATA DEMONSTRATE THAT PHF6 COULD ACT AS A DOUBLE-EDGED SWORD, EITHER A TUMOR SUPPRESSOR OR AN ONCOGENE, IN A LINEAGE-DEPENDENT MANNER. HOWEVER, THE UNDERLYING MECHANISMS OF PHF6 IN NORMAL HEMATOPOIESIS AND LEUKEMOGENESIS REMAIN LARGELY UNKNOWN. IN THIS REVIEW, WE SUMMARIZE CURRENT KNOWLEDGE OF PHF6, EMPHASIZING THE ROLE OF PHF6 IN HEMATOLOGICAL MALIGNANCIES. EPIGENETIC REGULATION OF PHF6 IN B-ALL. PHF6 MAINTAINS A CHROMATIN STRUCTURE THAT IS PERMISSIVE TO B-CELL IDENTITY GENES, BUT NOT T-CELL-SPECIFIC GENES (LEFT). LOSS OF PHF6 LEADS TO ABERRANT EXPRESSION OF B-CELL- AND T-CELL-SPECIFIC GENES RESULTING FROM LINEAGE PROMISCUITY AND BINDING OF T-CELL TRANSCRIPTION FACTORS (RIGHT). 2023 4 2326 35 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 5 5061 33 PHF6 FUNCTIONS AS A TUMOR SUPPRESSOR BY RECRUITING METHYLTRANSFERASE SUV39H1 TO NUCLEOLAR REGION AND OFFERS A NOVEL THERAPEUTIC TARGET FOR PHF6-MUNTANT LEUKEMIA. MUTATIONS IN THE PLANT HOMEODOMAIN-LIKE FINGER PROTEIN 6 (PHF6) GENE ARE STRONGLY ASSOCIATED WITH ACUTE MYELOID (AML) AND T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL). IN THIS STUDY, WE DEMONSTRATED THAT PHF6 CAN BIND TO H3K9ME3 AND H3K27ME1 ON THE NUCLEOLAR CHROMATIN AND RECRUIT HISTONE METHYLTRANSFERASE SUV39H1 TO THE RDNA LOCUS. THE DELETION OF PHF6 CAUSED A DECREASE IN THE RECRUITMENT OF SUV39H1 TO RDNA GENE LOCI, RESULTING IN A REDUCTION IN THE LEVEL OF H3K9ME3 AND THE PROMOTION OF RDNA TRANSCRIPTION. THE KNOCKDOWN OF EITHER SUV39H1 OR PHF6 SIGNIFICANTLY ATTENUATED THE EFFECTS OF INCREASE IN H3K9ME3 AND SUPPRESSED THE TRANSCRIPTION OF RDNA INDUCED BY THE OVEREXPRESSION OF THE OTHER INTERACTING PARTNER, THEREBY ESTABLISHING AN INTERDEPENDENT RELATIONSHIP BETWEEN PHF6 AND SUV39H1 IN THEIR CONTROL OF RRNA TRANSCRIPTION. THE PHF6 CLINICAL MUTANTS SIGNIFICANTLY IMPAIRED THE ABILITY TO BIND AND RECRUIT SUV39H1 TO THE RDNA LOCI, RESULTING IN AN INCREASE IN RDNA TRANSCRIPTION ACTIVITY, THE PROLIFERATION OF IN VITRO LEUKEMIA CELLS, AND THE GROWTH OF IN VIVO MOUSE XENOGRAFTS. IMPORTANTLY, SIGNIFICANTLY ELEVATED LEVELS OF PRE-RRNA WERE OBSERVED IN CLINICAL AML PATIENTS WHO POSSESSED A MUTATED VERSION OF PHF6. THE SPECIFIC RDNA TRANSCRIPTION INHIBITOR CX5461 SIGNIFICANTLY REDUCED THE RESISTANCE OF U937 AML CELLS DEFICIENT IN PHF6 TO CYTARABINE, THE DRUG THAT IS MOST COMMONLY USED TO TREAT AML. COLLECTIVELY, WE REVEALED A NOVEL MOLECULAR MECHANISM BY WHICH PHF6 RECRUITS METHYLTRANSFERASE SUV39H1 TO THE NUCLEOLAR REGION IN LEUKEMIA AND PROVIDED A POTENTIAL THERAPEUTIC TARGET FOR PHF6-MUTANT LEUKEMIA. 2022 6 4231 43 METHYLATION OF PROTOCADHERIN 10, A NOVEL TUMOR SUPPRESSOR, IS ASSOCIATED WITH POOR PROGNOSIS IN PATIENTS WITH GASTRIC CANCER. BACKGROUND & AIMS: BY USING METHYLATION-SENSITIVE REPRESENTATIONAL DIFFERENCE ANALYSIS, WE IDENTIFIED PROTOCADHERIN 10 (PCDH10), A GENE THAT ENCODES A PROTOCADHERIN AND IS SILENCED IN A TUMOR-SPECIFIC MANNER. WE ANALYZED ITS EPIGENETIC INACTIVATION, BIOLOGICAL EFFECTS, AND PROGNOSTIC SIGNIFICANCE IN GASTRIC CANCER. METHODS: METHYLATION STATUS WAS EVALUATED BY COMBINED BISULFITE RESTRICTION ANALYSIS AND BISULFITE SEQUENCING. THE EFFECTS OF PCDH10 RE-EXPRESSION WERE DETERMINED IN GROWTH, APOPTOSIS, PROLIFERATION, AND INVASION ASSAYS. PCDH10 TARGET GENES WERE IDENTIFIED BY COMPLEMENTARY DNA MICROARRAY ANALYSIS. RESULTS: PCDH10 WAS SILENCED OR DOWN-REGULATED IN 94% (16 OF 17) OF GASTRIC CANCER CELL LINES; EXPRESSION LEVELS WERE RESTORED BY EXPOSURE TO DEMETHYLATING AGENTS. RE-EXPRESSION OF PCDH10 IN MKN45 GASTRIC CANCER CELLS REDUCED COLONY FORMATION IN VITRO AND TUMOR GROWTH IN MICE; IT ALSO INHIBITED CELL PROLIFERATION (P < .01), INDUCED CELL APOPTOSIS (P < .001), AND REPRESSED CELL INVASION (P < .05), UP-REGULATING THE PRO-APOPTOSIS GENES FAS, CASPASE 8, JUN, AND CDKN1A; THE ANTIPROLIFERATION GENE FGFR; AND THE ANTI-INVASION GENE HTATIP2. PCDH10 METHYLATION WAS DETECTED IN 82% (85 OF 104) OF GASTRIC TUMORS COMPARED WITH 37% (38 OF 104) OF PAIRED NONTUMOR TISSUES (P < .0001). IN THE LATTER, PCDH10 METHYLATION WAS HIGHER IN PRECANCEROUS LESIONS (27 OF 45; 60%) THAN IN CHRONIC GASTRITIS SAMPLES (11 OF 59; 19%) (P < .0001). AFTER A MEDIAN FOLLOW-UP PERIOD OF 16.8 MONTHS, MULTIVARIATE ANALYSIS REVEALED THAT PATIENTS WITH PCDH10 METHYLATION IN ADJACENT NONTUMOR AREAS HAD A SIGNIFICANT DECREASE IN OVERALL SURVIVAL. KAPLAN-MEIER SURVIVAL CURVES SHOWED THAT PCDH10 METHYLATION WAS ASSOCIATED SIGNIFICANTLY WITH SHORTENED SURVIVAL IN STAGE I-III GASTRIC CANCER PATIENTS. CONCLUSIONS: PCDH10 IS A GASTRIC TUMOR SUPPRESSOR; ITS METHYLATION AT EARLY STAGES OF GASTRIC CARCINOGENESIS IS AN INDEPENDENT PROGNOSTIC FACTOR. 2009 7 5478 31 RESULTS OF A RANDOMIZED STUDY OF 3 SCHEDULES OF LOW-DOSE DECITABINE IN HIGHER-RISK MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA. EPIGENETIC THERAPY WITH HYPOMETHYLATING DRUGS IS NOW THE STANDARD OF CARE IN MYELODYSPLASTIC SYNDROME (MDS). RESPONSE RATES REMAIN LOW, AND MECHANISM-BASED DOSE OPTIMIZATION HAS NOT BEEN REPORTED. WE INVESTIGATED THE CLINICAL AND PHARMACODYNAMIC RESULTS OF DIFFERENT DOSE SCHEDULES OF DECITABINE. ADULTS WITH ADVANCED MDS OR CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) WERE RANDOMIZED TO 1 OF 3 DECITABINE SCHEDULES: (1) 20 MG/M2 INTRAVENOUSLY DAILY FOR 5 DAYS; (2) 20 MG/M2 SUBCUTANEOUSLY DAILY FOR 5 DAYS; AND (3) 10 MG/M2 INTRAVENOUSLY DAILY FOR 10 DAYS. RANDOMIZATION FOLLOWED A BAYESIAN ADAPTIVE DESIGN. NINETY-FIVE PATIENTS WERE TREATED (77 WITH MDS, AND 18 WITH CMML). OVERALL, 32 PATIENTS (34%) ACHIEVED A COMPLETE RESPONSE (CR), AND 69 (73%) HAD AN OBJECTIVE RESPONSE BY THE NEW MODIFIED INTERNATIONAL WORKING GROUP CRITERIA. THE 5-DAY INTRAVENOUS SCHEDULE, WHICH HAD THE HIGHEST DOSE-INTENSITY, WAS SELECTED AS OPTIMAL; THE CR RATE IN THAT ARM WAS 39%, COMPARED WITH 21% IN THE 5-DAY SUBCUTANEOUS ARM AND 24% IN THE 10-DAY INTRAVENOUS ARM (P < .05). THE HIGH DOSE-INTENSITY ARM WAS ALSO SUPERIOR AT INDUCING HYPOMETHYLATION AT DAY 5 AND AT ACTIVATING P15 EXPRESSION AT DAYS 12 OR 28 AFTER THERAPY. WE CONCLUDE THAT A LOW-DOSE, DOSE-INTENSITY SCHEDULE OF DECITABINE OPTIMIZES EPIGENETIC MODULATION AND CLINICAL RESPONSES IN MDS. 2007 8 1107 32 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 9 2426 36 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 10 960 28 CHRONIC MYELOMONOCYTIC LEUKEMIA: 2016 UPDATE ON DIAGNOSIS, RISK STRATIFICATION, AND MANAGEMENT. CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) IS A CLONAL HEMATOPOIETIC STEM CELL DISORDER CHARACTERIZED BY OVERLAPPING FEATURES OF MYELODYSPLASTIC SYNDROMES AND MYELOPROLIFERATIVE NEOPLASMS. DIAGNOSIS IS BASED ON THE PRESENCE OF PERSISTENT (>3 MONTHS) PERIPHERAL BLOOD MONOCYTOSIS (>1 X 10(9) /L), ALONG WITH BONE MARROW DYSPLASIA. CLONAL CYTOGENETIC ABNORMALITIES OCCUR IN APPROXIMATELY 20-30% OF PATIENTS, WHILE >90% HAVE GENE MUTATIONS. MUTATIONS INVOLVING TET2 ( APPROXIMATELY 60%), SRSF2 ( APPROXIMATELY 50%), ASXL1 ( APPROXIMATELY 40%), AND RAS ( APPROXIMATELY 30%) ARE FREQUENT; WITH ONLY ASXL1 MUTATIONS NEGATIVELY IMPACTING OVERALL SURVIVAL. TWO MOLECULARLY INTEGRATED, CMML-SPECIFIC PROGNOSTIC MODELS INCLUDE; THE GROUPE FRANCAIS DES MYELODYSPLASIES (GFM) AND THE MOLECULAR MAYO MODEL (MMM). THE GFM MODEL SEGREGATES PATIENTS INTO 3 GROUPS BASED ON: AGE >65 YEARS, WBC >15 X 10(9) /L, ANEMIA, PLATELETS <100 X 10(9) /L, AND ASXL1 MUTATION STATUS, WITH RESPECTIVE MEDIAN SURVIVALS OF 56 (LOW), 27.4 (INTERMEDIATE), AND 9.2 (HIGH) MONTHS. THE MMM IS BASED ON ASXL1 MUTATIONAL STATUS, ABSOLUTE MONOCYTE COUNT >10 X 10(9) /L, HEMOGLOBIN <10 G/DL, PLATELETS <100 X 109/L AND CIRCULATING IMMATURE MYELOID CELLS. THIS MODEL STRATIFIES PATIENTS INTO FOUR GROUPS; HIGH (>/=3 RISK FACTORS), INTERMEDIATE-2 (2 RISK FACTORS), INTERMEDIATE-1 (1 RISK FACTOR) AND LOW (NO RISK FACTORS), WITH MEDIAN SURVIVALS OF 16, 31, 59, AND 97 MONTHS, RESPECTIVELY. HYPOMETHYLATING AGENTS SUCH AS 5-AZACITIDINE AND DECITABINE ARE COMMONLY USED, WITH OVERALL RESPONSE RATES OF APPROXIMATELY 30-40% AND COMPLETE REMISSION RATES OF APPROXIMATELY 7-17%. ALLOGENEIC STEM CELL TRANSPLANT IS THE ONLY POTENTIALLY CURATIVE OPTION, BUT IS ASSOCIATED WITH SIGNIFICANT MORBIDITY AND MORTALITY. INDIVIDUALIZED THERAPY, INCLUDING EPIGENETIC MODIFIERS AND SMALL MOLECULE INHIBITORS, ARE EXCITING PROSPECTS. AM. J. HEMATOL. 91:632-642, 2016. (C) 2016 WILEY PERIODICALS, INC. 2016 11 2678 30 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 12 66 27 A KEY ROLE FOR EZH2 IN EPIGENETIC SILENCING OF HOX GENES IN MANTLE CELL LYMPHOMA. THE CHROMATIN MODIFIER EZH2 IS OVEREXPRESSED AND ASSOCIATED WITH INFERIOR OUTCOME IN MANTLE CELL LYMPHOMA (MCL). RECENTLY, WE DEMONSTRATED PREFERENTIAL DNA METHYLATION OF HOX GENES IN MCL COMPARED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), DESPITE THESE GENES NOT BEING EXPRESSED IN EITHER ENTITY. SINCE EZH2 HAS BEEN SHOWN TO REGULATE HOX GENE EXPRESSION, TO GAIN FURTHER INSIGHT INTO ITS POSSIBLE ROLE IN DIFFERENTIAL SILENCING OF HOX GENES IN MCL VS. CLL, WE PERFORMED DETAILED EPIGENETIC CHARACTERIZATION USING REPRESENTATIVE CELL LINES AND PRIMARY SAMPLES. WE OBSERVED SIGNIFICANT OVEREXPRESSION OF EZH2 IN MCL VS. CLL. CHROMATIN IMMUNE PRECIPITATION (CHIP) ASSAYS REVEALED THAT EZH2 CATALYZED REPRESSIVE H3 LYSINE 27 TRIMETHYLATION (H3K27ME3), WHICH WAS SUFFICIENT TO SILENCE HOX GENES IN CLL, WHEREAS IN MCL H3K27ME3 IS ACCOMPANIED BY DNA METHYLATION FOR A MORE STABLE REPRESSION. MORE IMPORTANTLY, HYPERMETHYLATION OF THE HOX GENES IN MCL RESULTED FROM EZH2 OVEREXPRESSION AND SUBSEQUENT RECRUITMENT OF THE DNA METHYLATION MACHINERY ONTO HOX GENE PROMOTERS. THE IMPORTANCE OF EZH2 UPREGULATION IN THIS PROCESS WAS FURTHER UNDERSCORED BY SIRNA TRANSFECTION AND EZH2 INHIBITOR EXPERIMENTS. ALTOGETHER, THESE OBSERVATIONS IMPLICATE EZH2 IN THE LONG-TERM SILENCING OF HOX GENES IN MCL, AND ALLUDE TO ITS POTENTIAL AS A THERAPEUTIC TARGET WITH CLINICAL IMPACT. 2013 13 765 29 CC-486 MAINTENANCE AFTER STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA OR MYELODYSPLASTIC SYNDROMES. RELAPSE IS THE MAIN CAUSE OF TREATMENT FAILURE AFTER ALLOGENEIC STEM CELL TRANSPLANT (ALLOSCT) IN ACUTE MYELOID LEUKEMIA (AML) AND MYELODYSPLASTIC SYNDROMES (MDS). INJECTABLE AZACITIDINE CAN IMPROVE POST-TRANSPLANT OUTCOMES BUT PRESENTS CHALLENGES WITH EXPOSURE AND COMPLIANCE. ORAL CC-486 ALLOWS EXTENDED DOSING TO PROLONG AZACITIDINE ACTIVITY. WE INVESTIGATED USE OF CC-486 MAINTENANCE THERAPY AFTER ALLOSCT. ADULTS WITH MDS OR AML IN MORPHOLOGIC COMPLETE REMISSION AT CC-486 INITIATION (42 TO 84 DAYS AFTER ALLOSCT) WERE INCLUDED. PATIENTS RECEIVED 1 OF 4 CC-486 DOSING SCHEDULES PER 28-DAY CYCLE FOR UP TO 12 CYCLES. ENDPOINTS INCLUDED SAFETY, PHARMACOKINETICS, GRAFT-VERSUS-HOST DISEASE (GVHD) INCIDENCE, RELAPSE/PROGRESSION RATE, AND SURVIVAL. OF 30 PATIENTS, 7 RECEIVED CC-486 ONCE DAILY FOR 7 DAYS PER CYCLE (200 MG, N = 3; 300 MG, N = 4) AND 23 FOR 14 DAYS PER CYCLE (150 MG, N = 4; 200 MG, N = 19 [EXPANSION COHORT]). GRADES 3 TO 4 ADVERSE EVENTS WERE INFREQUENT AND OCCURRED WITH SIMILAR FREQUENCY ACROSS REGIMENS. STANDARD CONCOMITANT MEDICATIONS DID NOT ALTER CC-486 PHARMACOKINETIC PARAMETERS. THREE PATIENTS (10%) EXPERIENCED GRADE III ACUTE GVHD AND 9 EXPERIENCED CHRONIC GVHD. OF 28 EVALUABLE PATIENTS, 6 (21%) RELAPSED OR HAD PROGRESSIVE DISEASE: 3 OF 7 PATIENTS (43%) WHO HAD RECEIVED 7-DAY DOSING AND 3 OF 23 (13%) WHO HAD RECEIVED 14-DAY DOSING. TRANSPLANT-RELATED MORTALITY WAS 3%. AT 19 MONTHS OF FOLLOW-UP, MEDIAN OVERALL SURVIVAL WAS NOT REACHED. ESTIMATED 1-YEAR SURVIVAL RATES WERE 86% AND 81% IN THE 7-DAY AND 14-DAY DOSING COHORTS, RESPECTIVELY. CC-486 MAINTENANCE WAS GENERALLY WELL TOLERATED, WITH LOW RATES OF RELAPSE, DISEASE PROGRESSION, AND GVHD. CC-486 MAINTENANCE MAY PERMIT EPIGENETIC MANIPULATION OF THE ALLOREACTIVE RESPONSE POSTALLOGRAFT. FINDINGS REQUIRE CONFIRMATION IN RANDOMIZED TRIALS. (CLINICALTRIALS.GOV NCT01835587.). 2018 14 3877 34 KDM6A PROMOTES IMATINIB RESISTANCE THROUGH YY1-MEDIATED TRANSCRIPTIONAL UPREGULATION OF TRKA INDEPENDENTLY OF ITS DEMETHYLASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA. RATIONALE: DESPITE LANDMARK THERAPY OF CHRONIC MYELOGENOUS LEUKEMIA (CML) WITH TYROSINE KINASE INHIBITORS (TKIS), DRUG RESISTANCE REMAINS PROBLEMATIC. CANCER PATHOGENESIS INVOLVES EPIGENETIC DYSREGULATION AND IN PARTICULAR, HISTONE LYSINE DEMETHYLASES (KDMS) HAVE BEEN IMPLICATED IN TKI RESISTANCE. WE SOUGHT TO IDENTIFY KDMS WITH ALTERED EXPRESSION IN CML AND DEFINE THEIR CONTRIBUTION TO IMATINIB RESISTANCE. METHODS: BIOINFORMATICS SCREENING COMPARED KDM EXPRESSION IN CML VERSUS NORMAL BONE MARROW WITH SHRNA KNOCKDOWN AND FLOW CYTOMETRY USED TO MEASURE EFFECTS ON IMATINIB-INDUCED APOPTOSIS IN K562 CELLS. TRANSCRIPTOMIC ANALYSES WERE PERFORMED AGAINST KDM6A CRISPR KNOCKOUT/SHRNA KNOCKDOWN K562 CELLS ALONG WITH GENE RESCUE EXPERIMENTS USING WILDTYPE AND MUTANT DEMETHYLASE-DEAD KDM6A CONSTRUCTS. CO-IMMUNOPRECIPITATION, LUCIFERASE REPORTER AND CHIP WERE EMPLOYED TO ELUCIDATE MECHANISMS OF KDM6A-DEPENDENT RESISTANCE. RESULTS: AMONGST FIVE KDMS UPREGULATED IN CML, ONLY KDM6A DEPLETION SENSITIZED CML CELLS TO IMATINIB-INDUCED APOPTOSIS. RE-INTRODUCTION OF DEMETHYLASE-DEAD KDM6A AS WELL AS WILD-TYPE KDM6A RESTORED IMATINIB RESISTANCE. RNA-SEQ IDENTIFIED NTRK1 GENE DOWNREGULATION AFTER DEPLETION OF KDM6A. MOREOVER, NTRK1 EXPRESSION POSITIVELY CORRELATED WITH KDM6A IN A SUBSET OF CLINICAL CML SAMPLES AND KDM6A KNOCKDOWN IN FRESH CML ISOLATES DECREASED NTRK1 ENCODED PROTEIN (TRKA) EXPRESSION. MECHANISTICALLY, KDM6A WAS RECRUITED TO THE NTRK1 PROMOTER BY THE TRANSCRIPTION FACTOR YY1 WITH SUBSEQUENT TRKA UPREGULATION ACTIVATING DOWN-STREAM SURVIVAL PATHWAYS TO INVOKE IMATINIB RESISTANCE. CONCLUSION: CONTRARY TO ITS REPORTED ROLE AS A TUMOR SUPPRESSOR AND INDEPENDENT OF ITS DEMETHYLASE FUNCTION, KDM6A PROMOTES IMATINIB-RESISTANCE IN CML CELLS. THE IDENTIFICATION OF THE KDM6A/YY1/TRKA AXIS AS A NOVEL IMATINIB-RESISTANCE MECHANISM REPRESENTS AN UNEXPLORED AVENUE TO OVERCOME TKI RESISTANCE IN CML. 2021 15 6373 26 THE ROLE OF MIRNAS AND EPIGENETIC MECHANISMS IN PRIMARY GASTRIC MUCOSA-ASSOCIATED LYMPHOID TISSUE LYMPHOMA. GASTRIC MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMA IS A RARE LOW-GRADE B-CELL NON-HODGKIN LYMPHOMA ASSOCIATED WITH HELICOBACTER PYLORI INFECTION AND THE SUBSEQUENT CHRONIC INFLAMMATION. SIGNIFICANT PROGRESS IN UNDERSTANDING THE PATHOGENESIS OF THE DISEASE HAS ALREADY BEEN MADE. HOWEVER, THE EXACT MOLECULAR PATHWAYS OF LYMPHOMAGENESIS REMAIN UNCLEAR. FURTHERMORE, DIFFICULTIES REGARDING ACCURATE DIAGNOSIS OF GASTRIC MALT LYMPHOMA AND ITS DISCRIMINATION FROM GASTRITIS OR OTHER LYMPHOMA SUBTYPES ARISE. RECENT STUDIES EVALUATE THE ROLE OF MIRNAS AND EPIGENETIC ALTERATIONS ON MALT LYMPHOMA PATHOGENESIS AND PROGNOSIS. THIS REVIEW CRITICALLY SUMMARIZES THE MOST IMPORTANT DATA ON THE ROLE OF MIRNAS AND EPIGENETICS IN MALT LYMPHOMAS PATHOGENESIS, PROGNOSIS AND TREATMENT. 2016 16 6366 28 THE ROLE OF METHYLATION IN CML. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 ONCOGENE IS COMMON EPIGENETIC ALTERATION ASSOCIATED WITH CLINICAL PROGRESSION OF CHRONIC MYELOID LEUKEMIA (CML). IN PRESENTED STUDY WE QUERIED WHETHER BOTH THE PH'-ASSOCIATED AND NORMAL ABL1 ALLELES UNDERGO METHYLATION; WHAT MAY BE THE PROPORTION OF HEMATOPOIETIC PROGENITORS BEARING METHYLATED ABL1 PROMOTERS IN CHRONIC VERSUS ACUTE PHASE DISEASE; WHETHER METHYLATION AFFECTS THE PROMOTER UNIFORMLY OR IN PATCHES WITH DISCRETE CLINICAL RELEVANCE; AND, FINALLY WHETHER METHYLATION OF ABL1 REFLECTS A GENERALIZED PROCESS OR IS GENE-SPECIFIC. TO ADDRESS THESE ISSUES, THE TECHNIQUE OF METHYLATION-SPECIFIC PCR AND BISULFITE-SEQUENCING WAS ADAPTED TO STUDY THE REGULATORY REGIONS OF ABL1 AND OTHER GENES. IN CELL LINES ESTABLISHED FROM CML BLAST CRISIS, WHICH ONLY CARRY A SINGLE ABL1 ALLELE NESTED WITHIN THE BCR-ABL FUSION GENE, ABL1 PROMOTERS WERE UNIVERSALLY METHYLATED. IN CLINICAL SAMPLES FROM PATIENTS AT ADVANCED STAGES OF THE DISEASE, BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE DETECTABLE. IN COLONIES DERIVED FROM SINGLE HEMATOPOIETIC PROGENITORS METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE REVEALED AS WELL. ABL1 METHYLATION WAS WAS NOTED IN THE VAST MAJORITY OF COLONIES FROM BLAST CRISIS, BUT NOT CHRONIC-PHASE CML. IT WAS SHOWN FINALLY THAT ABL1 METHYLATION DOES NOT REFLECT A GENERALIZED PROCESS AND MAY BE UNIQUE AMONG DNA REPAIR/GENOTOXIC STRESS RESPONSE GENES. THESE DATA SUGGEST THAT SPECIFIC METHYLATION OF THE PH'-ASSOCIATED ABL1 ALLELE ACCOMPANIES CLONAL EVOLUTION IN CML. 2000 17 163 28 ABL1 METHYLATION IS A DISTINCT MOLECULAR EVENT ASSOCIATED WITH CLONAL EVOLUTION OF CHRONIC MYELOID LEUKEMIA. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 ONCOGENE IS A COMMON EPIGENETIC ALTERATION ASSOCIATED WITH CLINICAL PROGRESSION OF CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY WE QUERIED WHETHER BOTH THE PH'-ASSOCIATED AND NORMAL ABL1 ALLELES UNDERGO METHYLATION; WHAT MAY BE THE PROPORTION OF HEMATOPOIETIC PROGENITORS BEARING METHYLATED ABL1 PROMOTERS IN CHRONIC VERSUS ACUTE PHASE DISEASE; WHETHER METHYLATION AFFECTS THE PROMOTER UNIFORMLY OR IN PATCHES WITH DISCRETE CLINICAL RELEVANCE; AND, FINALLY, WHETHER METHYLATION OF ABL1 REFLECTS A GENERALIZED PROCESS OR IS GENE-SPECIFIC. TO ADDRESS THESE ISSUES, WE ADAPTED THE TECHNIQUES OF METHYLATION-SPECIFIC PCR AND BISULFITE-SEQUENCING TO STUDY THE REGULATORY REGIONS OF ABL1 AND OTHER GENES WITH A ROLE IN DNA REPAIR OR GENOTOXIC STRESS RESPONSE. IN CELL LINES ESTABLISHED FROM CML BLAST CRISIS, WHICH ONLY CARRY A SINGLE ABL1 ALLELE NESTED WITHIN THE BCR-ABL FUSION GENE, ABL1 PROMOTERS WERE UNIVERSALLY METHYLATED. BY CONTRAST, IN CLINICAL SAMPLES FROM PATIENTS AT ADVANCED STAGES OF DISEASE, BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE DETECTABLE. TO DISTINGUISH BETWEEN ALLELE-SPECIFIC METHYLATION AND A MIXED CELL POPULATION PATTERN, WE STUDIED THE METHYLATION STATUS OF ABL1 IN COLONIES DERIVED FROM SINGLE HEMATOPOIETIC PROGENITORS. OUR RESULTS SHOWED THAT BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES COEXISTED IN THE SAME COLONY. FURTHERMORE, ABL1 METHYLATION WAS NOTED IN THE VAST MAJORITY OF COLONIES FROM BLAST CRISIS, BUT NOT CHRONIC-PHASE CML. BOTH CELL LINES AND CLINICAL SAMPLES FROM ACUTE-PHASE CML SHOWED NEARLY UNIFORM HYPERMETHYLATION ALONG THE PROMOTER REGION. FINALLY, WE SHOWED THAT ABL1 METHYLATION DOES NOT REFLECT A GENERALIZED PROCESS AND MAY BE UNIQUE AMONG DNA REPAIR/GENOTOXIC STRESS RESPONSE GENES. OUR DATA SUGGEST THAT SPECIFIC METHYLATION OF THE PH'-ASSOCIATED ABL1 ALLELE ACCOMPANIES CLONAL EVOLUTION IN CML. 1999 18 32 38 A CASE OF TYROSINE KINASE INHIBITOR-RESISTANT CHRONIC MYELOID LEUKEMIA, CHRONIC PHASE WITH ASXL1 MUTATION. HEMATOLOGICAL MALIGNANCIES, INCLUDING CHRONIC MYELOID LEUKEMIA (CML), EXHIBIT ASXL1 MUTATIONS; HOWEVER, THE FUNCTION AND MOLECULAR MECHANISM OF THESE MUTATIONS REMAIN UNCLEAR. ASXL1 WAS ORIGINALLY IDENTIFIED AS TUMOR SUPPRESSOR GENE, IN WHICH LOSS OF FUNCTION CAUSES MYELODYSPLASTIC SYNDROME (MDS). ASXL1 MUTATIONS ARE COMMON AND ASSOCIATED WITH DISEASE PROGRESSION IN MYELOID MALIGNANCIES INCLUDING MDS, ACUTE MYELOID LEUKEMIA, AND SIMILARLY IN CML. IN MDS, ASXL1 MUTATIONS HAVE BEEN ASSOCIATED WITH POOR PROGNOSIS; HOWEVER, THE IMPACT OF ASXL1 MUTATIONS IN CML HAS NOT BEEN WELL DESCRIBED. A 31-YEAR-OLD MALE WAS DIAGNOSED AS CML-CHRONIC PHASE (CP). LABORATORY FINDINGS SHOWED A WHITE BLOOD CELL COUNT OF 187,200/MICROL, WITH ASYMPTOMATIC SPLENOMEGALY. BLAST COUNT WAS 5.0% IN PERIPHERAL BLOOD AND 7.3% IN BONE MARROW. THERE WAS NO ADDITIONAL CHROMOSOMAL ABNORMALITY EXCEPT FOR T(9;22)(Q34;Q11.2) BY CHROMOSOMAL ANALYSIS. AT ONSET, THE SOKAL SCORE WAS 1.4, INDICATING HIGH RISK. THE PATIENT RECEIVED TYROSINE KINASE INHIBITOR (TKI) THERAPY, COMPRISING NILOTINIB APPROXIMATELY 600 MG/DAY, BOSUTINIB APPROXIMATELY 600 MG/DAY, PONATINIB APPROXIMATELY 45 MG/DAY, AND DASATINIB APPROXIMATELY 100 MG/DAY. NEVERTHELESS, AFTER 1.5 YEARS OF CONTINUOUS TKI THERAPY, THE BEST OUTCOME WAS A HEMATOLOGICAL RESPONSE. ALTHOUGH ADDITIONAL CHROMOSOMAL ABERRATIONS AND ABL1 KINASE MUTATIONS WERE ANALYZED REPEATEDLY BEFORE AND DURING TKI THERAPY, KNOWN GENETIC ABNORMALITIES WERE NOT DETECTED. THEREAFTER, THE PATIENT UNDERWENT BONE MARROW TRANSPLANTATION FROM AN HLA 7/8 MATCHED UNRELATED DONOR (HLA-CW 1 LOCUS MISMATCH, GRAFT-VERSUS-HOST DIRECTION). THE PATIENT ACHIEVED NEUTROPHIL ENGRAFTMENT, 18 DAYS AFTER TRANSPLANTATION, LEADING TO COMPLETE REMISSION WITH AN UNDETECTABLE LEVEL OF BCR-ABL1 MRNA. THE PATIENT, HOWEVER, DIED FROM GRAFT-VERSUS-HOST DISEASE AND THROMBOTIC MICROANGIOPATHY AFTER 121 DAYS. GENE SEQUENCE ANALYSIS OF HIS CML CELL BEFORE STEM CELL TRANSPLANTATION REVEALED ASXL1 MUTATIONS. PHYSIOLOGICALLY, ASXL1 CONTRIBUTES TO EPIGENETIC REGULATION. IN THE CML-CP PATIENT IN THIS CASE REPORT, ASXL1 MUTATION CONFERRED RESISTANCE TO TKI THROUGH OBSCURE RESISTANCE MECHANISMS. EVEN THOUGH A MOLECULAR MECHANISM FOR TKI RESISTANCE IN ASXL1 MUTATION IN CML HAS REMAINED OBSCURE, EPIGENETIC MODULATION IS A PLAUSIBLE MODE OF CML DISEASE PROGRESSION. THE CLINICAL IMPACT INCLUDING PROGNOSIS OF ASXL1 FOR CML IS UNDERSCORED. AND THE TREATMENT STRATEGY OF CML WITH ASXL1 MUTATION HAS NOT BEEN ESTABLISHED. A DISCUSSION OF THIS CASE WAS EXPECTED TO FACILITATE TREATMENT OPTIONS. 2020 19 5144 27 POTENTIAL ROLE OF LNCRNA-TSIX, MIR-548-A-3P, AND SOGA1 MRNA IN THE DIAGNOSIS OF HEPATOCELLULAR CARCINOMA. RECENT TRENDS ARE MOVING TOWARDS THE USE OF THE CIRCULATING TRANSCRIPTOME AS A POTENTIAL DIAGNOSTIC AND THERAPEUTIC TOOL FOR HEPATOCELLULAR CARCINOMA (HCC). THE AIM OF THIS STUDY IS TO IDENTIFY CIRCULATORY RNA BASED BIOMARKER PANEL, IN ADDITION TO THEIR RELATIONSHIP TO THE OUTCOME IN HCC. FIRST, UTILIZING BIOINFORMATICS TOOLS, WE SELECTED AN HCC-SPECIFIC RNA-BASED BIOMARKER PANEL THAT DEPENDED ON THE INTEGRATION OF SUPPRESSOR OF GLUCOSE AUTOPHAGY-ASSOCIATED (SOGA1) GENE EXPRESSION WITH THE CHOSEN PANEL OF EPIGENETIC REGULATORS OF THIS GENE [LONG NON-CODING RNA ANTISENSE FOR X-INACTIVE-SPECIFIC TRANSCRIPT (LNCRNA-TSIX) AND MICRORNA-548-A-3P (MIR-548-A-3P)]. SECOND, WE ATTEMPTED TO VALIDATE THESE BIOMARKERS USING THE SERA OF 65 PATIENTS WITH HCC, 34 PATIENTS WITH CHRONIC HEPATITIS C VIRUS (CHC) INFECTION AND 32 HEALTHY VOLUNTEERS. FINALLY, THE EXPRESSION LEVELS OF THE CHOSEN RNA-BASED BIOMARKER PANEL WERE ASSESSED IN THE SERUM SAMPLES USING QRT-PCR ASSAYS. THE PANEL OF 3 RNA-BASED BIOMARKERS (LNCRNA-TSIX, MIR-548-A-3P, AND SOGA1) EXHIBITED HIGH SENSITIVITY AND SPECIFICITY IN DIFFERENTIATING HCC PATIENTS FROM CHC PATIENTS AND HEALTHY CONTROLS. AMONG THESE 3 RNAS, SERUM LNCRNA-TSIX AND SOGA1 WERE INDEPENDENT PROGNOSTIC FACTOR. THE CHOSEN CIRCULATORY RNA-BASED BIOMARKER PANEL MAY SERVE AS A DIAGNOSTIC AND PROGNOSTIC BIOMARKER FOR HCC. 2019 20 5243 24 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