ABSTRACT
Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.
Subject(s)
Exome , Leukemia, Myeloid, Acute , Mutation , fms-Like Tyrosine Kinase 3/genetics , Chromatin/genetics , Chromatin/metabolism , DNA Methylation/genetics , Female , Humans , Induction Chemotherapy , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Male , Recurrence , Retrospective StudiesABSTRACT
In acute myeloid leukemia (AML), acquired genomic gains and losses are common and lead to altered expression of genes located within or nearby the affected regions. Increased expression of the ETS-related transcription factor gene ERG has been described in myeloid malignancies with chromosomal rearrangements involving chromosome band 21q22, but also in cytogenetically normal AML, where it is associated with adverse prognosis. In this study, fluorescence in situ hybridization on interphase nuclei disclosed an amplification of the ERG gene (more than six copies) in 33 AML patients with structural rearrangements of 21q22. Array comparative genomic hybridization of these cases disclosed a minimal amplified region at the position 39.6-40.0 Mbp from pter that harbors ERG as the only gene. Analysis by quantitative real-time reverse transcription polymerase chain reaction revealed significantly higher ERG mRNA expression in these patients and in a group of 95 AML patients with complete or partial gain of chromosome 21 (three to six copies) compared with 351 AML patients without gain of chromosome 21. Quantification of ERG DNA copy numbers revealed a strong correlation with ERG mRNA expression. Furthermore, in patients with gain of chromosome 21, higher ERG expression was found to be associated with RUNX1 mutations. Our results suggest that acquired gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism contributing to increased ERG expression in AML.
Subject(s)
Chromosomes, Human, Pair 21/genetics , Gene Amplification , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid, Acute/genetics , Trans-Activators/genetics , Aged , Aged, 80 and over , Chromosome Duplication , Comparative Genomic Hybridization , Core Binding Factor Alpha 2 Subunit/genetics , Female , Humans , Male , Middle Aged , Mutation , Transcriptional Regulator ERGABSTRACT
BACKGROUND: To obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime. METHODS: This study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin-Frankfurt-Münster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory). RESULTS: The frequencies of cytogenetic subgroups were age-dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to < 18 years; 33%) to the oldest groups (<5% for > 70 years; P < .0001). Unfavorable cytogenetics (-7/del(7), -5/del(5q) or 5p, inv(3)/t(3;3), t(6;9), complex karyotype, 12p, 17p, and 11q23/mixed-lineage leukemia aberrations, excluding t(9;11)) were frequent (42%) in infants (<2 years), had a low frequency in children and young adults (<22%), and increased in frequency up to 36% in patients older than 85 years (P = .01). This was even more significant for complex karyotypes (P ≤ .0001), which also showed a strong increase in the absolute age-specific incidence with age. Interestingly, the frequency of 11q23 abnormalities decreased from infants to older patients. The proportion of clinically relevant molecular aberrations of CCAAT/enhancer binding protein α, nucleophosmin (NPM1), and NPM1/fms-related tyrosine kinase 3-internal tandem duplication increased with age. CONCLUSIONS: Altogether, with the exclusion of infants, a significant decrease in the proportion of favorable cytogenetic subtypes and an increase in unfavorable cytogenetics were observed with increasing age. These findings indicate different mechanisms for the pathogenesis of AML; these different mechanisms also suggest directions for etiological research and contribute to the more unfavorable prognosis with increasing age. Cancer 2016;122:3821-3830. © 2016 American Cancer Society.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Chromosome Aberrations , Cytogenetic Analysis/methods , Cytogenetics/methods , Female , Humans , Infant , Infant, Newborn , Karyotyping , Male , Middle Aged , Molecular Biology/methods , Nucleophosmin , Prognosis , Young AdultABSTRACT
High BAALC gene expression has been associated with poor prognosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and has been suggested as a suitable marker for assessing minimal residual disease (MRD). The purpose of this study was to substantiate these findings by the analysis of a large data set of 632 diagnostic and follow-up samples in 142 intensively treated CN-AML patients. Paired diagnostic/relapse samples of 35 patients revealed stable high BAALC expression in 89%, irrespective of a high proportion of clonal evolution found in 49% of these cases. High BAALC expression, both directly after induction chemotherapy and within 3-6 months after induction chemotherapy, correlated significantly with shorter event-free survival and overall survival. Moreover, 8 of 10 patients displaying high BAALC expression levels after completion of induction therapy as well as 5 of 5 patients exhibiting high BAALC expression levels within 3-6 months after induction chemotherapy experienced relapse with a median of 197 and 101 days, respectively, from sampling to relapse. Thus, BAALC expression-based MRD detection during therapy may be considered a strategy to identify patients at high risk of relapse.
Subject(s)
Leukemia, Myeloid, Acute/diagnosis , Neoplasm Proteins/genetics , Neoplasm, Residual/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Feasibility Studies , Female , Gene Expression , Humans , Karyotype , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Recurrence , Remission Induction , Risk Assessment/methods , Survival Rate , Young AdultABSTRACT
Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.
Subject(s)
Membrane Proteins/genetics , Mutation , Myeloproliferative Disorders/genetics , Aged , Aged, 80 and over , Alleles , Animals , BRCA1 Protein/genetics , Chromosome Aberrations , DNA Mutational Analysis , Deubiquitinating Enzymes , Female , Gene Frequency , Gene Knockdown Techniques , Genetic Association Studies , Genotype , Humans , Male , Mice , Middle Aged , Myeloproliferative Disorders/diagnosis , Phenotype , RNA, Small Interfering/geneticsABSTRACT
Germline polymorphisms in genes mutated in acute myeloid leukemia (AML) may have prognostic impact. Therefore, the relevance of the polymorphism IDH1G105 (IDH1105(GGT) minor allele) was evaluated in the context of concomitant molecular mutations in a cohort of 507 AML cases with intermediate-risk cytogenetics. In addition, a cohort of 475 healthy controls was analyzed for this polymorphism. IDH1105(GGT) minor allele was found in 10 % of AML patients and 9 % of healthy controls. While no differences were seen with regard to cytomorphology or cytogenetics, immunophenotyping revealed significantly reduced expression of the progenitor marker CD34 in AML cases harboring IDH1105(GGT) minor allele. Cases with IDH1105(GGT) minor allele as compared to those with the IDH1105(GGC) major allele had significantly longer event-free survival (EFS) (median 16 vs 11 months, p = 0.013) which was most pronounced in the age group >60 years (median 14 vs 9 months, p = 0.007) and in the NPM1 mutated/FLT3-ITD/FLT3wt ratio <0.5 group (median 61 vs 13 months, p = 0.012). However, this association is not independent of other prognostic parameters, and we conclude that IDH1105(GGT) minor allele has to be considered in the context of the genetic background of the individual AML analyzed.
Subject(s)
Alleles , Biomarkers, Tumor/genetics , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute , Polymorphism, Genetic , Adolescent , Adult , Aged , Antigens, CD34 , Disease-Free Survival , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Nuclear Proteins/genetics , Nucleophosmin , Survival Rate , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Multiparameter flow cytometry (MFC) identifies rare cases of biclonal disease in chronic lymphocytic leukaemia (CLL). By MFC, we identified 76 patients with biclonal disease in a cohort of 5523 CLL patients (1·4%). Fluorescence in situ hybridization and chromosome banding analysis revealed five and six cases, respectively, with two different cytogenetic aberrations due to clonal evolution. Two different B-cell receptor rearrangements and IGHV subtypes were more frequent in biclonal than in monoclonal CLL by MFC (37·1% vs. 2·7%; P < 0·001). Patients with biclonal CLL by MFC showed a trend to a shorter time to treatment than monoclonal CLL (P = 0·080).
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Adult , Aged , Aged, 80 and over , Chromosome Banding , Cohort Studies , Female , Flow Cytometry/methods , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Young AdultABSTRACT
Correct action upon re-emergence of minimal residual disease in acute myeloid leukaemia (AML) patients has not yet been established. The applicability of demethylating agents and use of allogeneic stem cell transplantation will be dependent on pre-relapse AML growth rates. We here delineate molecular growth kinetics of AML harbouring MLL partial tandem duplication (MLL-PTD; 37 cases) compared to those harbouring MLL translocations (43 cases). The kinetics of MLL-PTD relapses was both significantly slower than those of MLL translocation positive ones (median doubling time: MLL-PTD: 24 d, MLL-translocations: 12 d, P = 0·015, Wilcoxon rank sum test), and displayed greater variation depending on additional mutations. Thus, MLL-PTD+ cases with additional RUNX1 mutations or FLT3-internal tandem duplication relapsed significantly faster than cases without one of those two mutations (Wilcoxon rank sum test, P = 0·042). As rapid relapses occurred in all MLL subgroups, frequent sampling are necessary to obtain acceptable relapse detection rates and times from molecular relapse to haematological relapse (blood sampling every second month: MLL-PTD: 75%/50 d; MLL translocations: 85%/25 d). In conclusion, in this cohort relapse kinetics is heavily dependent on AML subtype as well as additional genetic aberrations, with possibly great consequences for the rational choice of pre-emptive therapies.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Tandem Repeat Sequences/genetics , Chromosome Aberrations , DNA Mutational Analysis/methods , DNA, Neoplasm/genetics , Gene Duplication , Genes, Neoplasm/genetics , Histone-Lysine N-Methyltransferase , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Mutation , Neoplasm, Residual/genetics , Prognosis , Recurrence , Translocation, GeneticABSTRACT
In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29-90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization (aCGH). By aCGH (n = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had 'early MDS' without blast increase, 95 (31·1%) 'advanced' MDS with blast increase (5-19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs (P = 0·003) and had a higher mean number of ACAs (P = 0·020) and of molecular mutations (P = 0·060). Spliceosome mutations were frequent (U2AF1: n = 31/155; 20·0%; SRSF2: n = 31/159; 19·5%; SF3B1mut: n = 8/159; 5·0%). ASXL1mut (25/153; 16·3%) were associated with advanced MDS (P = 0·001). Presence of ≥3 ACAs (P = 0·003) and ASXL1mut (P = 0·002) were associated with worse 2-year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1mut is overrepresented in MDS with del(20q), and ASXL1mut is prognostically adverse.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 20 , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/genetics , Adult , Aged , Aged, 80 and over , Cytogenetics , Female , Genomics/methods , Humans , Male , Middle Aged , Mutation , Myelodysplastic Syndromes/pathology , PrognosisABSTRACT
In acute myeloid leukemia (AML) the subset with complex karyotype (CK) is traditionally regarded as the worst prognostic group. However, ≥ 3, ≥ 4, or ≥ 5 abnormalities have been variably used for its definition. Recently, monosomal karyotype (MSK) was suggested to indicate an even inferior outcome. We tested which definition fits best to identify the most unfavorable subgroup. After excluding patients with t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv (16)/t(16;16)/CBFB-MYH11, and normal karyotype, 824 patients with AML with cytogenetic abnormalities were analyzed. Patients with MSK or CK defined as ≥ 3, ≥ 4, or ≥ 5 abnormalities showed an inferior overall survival compared with the respective remaining patients not fulfilling these criteria (for all, P < .001). Hazard ratios were 1.93, 1.68, 1.94, and 1.92. CK ≥ 4 as a single parameter identified the largest proportion of patients with very poor risk. However, combining CK ≥ 4 and MSK detected an even larger number of patients with very unfavorable outcome (261 of 824; 31.7%).
Subject(s)
Abnormal Karyotype , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Humans , Leukemia, Myeloid, Acute/mortality , Prognosis , Survival AnalysisABSTRACT
In 2008, the World Health Organization introduced CEBPA (encoding the CCAAT/enhancer binding protein)-mutated acute myeloid leukemia (AML) as a provisional entity. However, the classification of CEBPA-mutated AML with multilineage dysplasia (MLD; ≥ 50% dysplastic cells in 2-3 lineages) remains to be clarified. In the present study, we investigated 108 CEBPA-mutated AML patients for the impact of MLD, karyotype, and additional mutations. MLD(+) patients differed from MLD(-) patients only by lower mean WBC counts, not by biologic characteristics, cytogenetic risk profiles, or additional mutations. Survival was better for female patients, patients < 60 years of age, for intermediate versus adverse karyotypes, and, in the case of FLT3-ITD negativity, biallelic versus monoallelic/homozygous CEBPA mutations. In contrast, 2-year overall survival and event-free survival did not differ significantly between MLD(+) and MLD(-) patients. By univariable Cox regression analysis, sex, age, WBC count, and cytogenetic risk category were related to overall survival, but MLD was not. Therefore, because dysplasia is not relevant for this subtype, CEBPA-mutated AML patients should be characterized only according to mutation status, cytogenetic risk group, or additional mutations.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , Cell Lineage/genetics , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/diagnosis , Terminology as Topic , World Health Organization , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Mutation/physiology , Neoplasm Staging/methods , Prognosis , Research Design , Retrospective Studies , World Health Organization/organization & administration , Young AdultABSTRACT
We analyzed the mutational hotspot region of SRSF2 (Pro95) in 275 cases with chronic myelomonocytic leukemia (CMML). In addition, ASXL1, CBL, EZH2, JAK2V617F, KRAS, NRAS, RUNX1, and TET2 mutations were investigated in subcohorts. Mutations in SRSF2 (SRSF2mut) were detected in 47% (129 of 275) of all cases. In detail, 120 cases had a missense mutation at Pro95, leading to a change to Pro95His, Pro95Leu, Pro95Arg, Pro95Ala, or Pro95Thr. In 9 cases, 3 new in/del mutations were observed: 7 cases with a 24-bp deletion, 1 case with a 3-bp duplication, and 1 case with a 24-bp duplication. In silico analyses predicted a damaging character for the protein structure of SRSF2 for all mutations. SRSF2mut was correlated with higher age, less pronounced anemia, and normal karyotype. SRSF2mut and EZH2mut were mutually exclusive, but SRSF2mut was associated with TET2mut. In the total cohort, no effect of SRSF2mut on survival was observed. However, in the RUNX1mut subcohort, SRSF2 Pro95His had a favorable effect on overall survival. This comprehensive mutation analysis found that 93% of all patients with CMML carried at least 1 somatic mutation in 9 recurrently mutated genes. In conclusion, these data show the importance of SRSF2mut as new diagnostic marker in CMML.
Subject(s)
Biomarkers, Tumor/genetics , Leukemia, Myelomonocytic, Chronic/genetics , Leukemia, Myelomonocytic, Chronic/mortality , Mutation/genetics , Nuclear Proteins/genetics , Ribonucleoproteins/genetics , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Serine-Arginine Splicing Factors , Survival Rate , Young AdultABSTRACT
Analyses of 164 RUNX1 mutations (RUNX1mut) in 147 of 449 patients (32.7%) with normal karyotype or noncomplex chromosomal imbalances were performed. RUNX1mut were most frequent in acute myeloid leukemia French-American-British classification M0 (65.2%) followed by M2 (32.4%) and M1 (30.2%). Considering cytogenetics, RUNX1mut were most frequent in cases with +13 (27 of 30, 90%), whereas frequencies were similar in other cytogenetic groups (26%-36%). The molecular genetic markers most frequently associated with RUNX1mut were partial tandem duplication in the MLL gene (19.7%), internal tandem duplication in the FLT3 gene (FLT3-ITD; 16.3%), and NRAS mutations (9.5%). Patients with RUNX1mut had shorter overall and event-free survival compared with RUNX1 wild-type cases (median, 378 days vs not reached, P = .003; and median, 285 vs 450 days, P = .003, respectively). In addition, it was shown that the adverse effect of RUNX1 was independent of the adverse effect of FLT3-ITD as well as of the high frequency of prognostically favorable NPM1mut and CEBPAmut in the RUNX1wt group. No effect of the type or localization of the individual RUNX1 mutations was observed. Multivariate analysis showed independent prognostic relevance for overall survival for RUNX1mut (P = .029), FLT3-ITD (P = .003), age (P < .001), and white blood cell count (P < .002).
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Leukemia, Myeloid, Acute/genetics , Mutation , fms-Like Tyrosine Kinase 3/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Cytogenetic Analysis , Humans , Inverted Repeat Sequences , Kaplan-Meier Estimate , Karyotyping , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/diagnosis , Leukocyte Count , Middle Aged , Prognosis , Survival Rate , Young AdultABSTRACT
The significance of flow cytometry indicating myelodysplasia without proof of myelodysplasia by cytomorphology remains to be clarified. We evaluated follow-up analyses in 142 patients analyzed in parallel by flow cytometry, cytomorphology and cytogenetics for suspected myelodysplasia without proof of myelodysplasia by cytomorphology. At initial assessment, flow cytometry indicated myelodysplasia in 64 of 142 (45.1%) patients. In 9 of 142 (6.3%) patients, cytogenetics revealed aberrant karyotypes at first evaluation that were found in 5 of 64 (7.8%) patients rated with myelodysplasia by flow cytometry. The remaining 133 patients without proof of myelodysplasia by cytomorphology and with normal karyotype underwent follow-up analyses that confirmed myelodysplasia by cytomorphology, cytogenetics or molecular genetics in 47 (35.3%) after a median interval of nine months (range 1-53 months). As far as initial flow cytometry results are concerned, this applied to 30 of 59 (50.1%) with myelodysplasia, 10 of 42 (23.8%) with "possible myelodysplasia" (minor antigen aberrancies only) and 7 of 32 (21.9%) without myelodysplasia (P=0.004). Notably, in these latter 7 patients, flow cytometry results changed at follow up to "possible myelodysplasia" (n=4) and "myelodysplasia" (n=2). These data argue in favor of including flow cytometry along with cytomorphology, cytogenetics and molecular genetics to diagnose myelodysplasia, and suggest a closer monitoring of patients with myelodysplasia-typical aberrant antigen expression found by flow cytometry.
Subject(s)
Myelodysplastic Syndromes/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Cytogenetic Analysis , Female , Flow Cytometry , Follow-Up Studies , Humans , Immunophenotyping , Karyotyping , Male , Middle Aged , Reproducibility of Results , Young AdultABSTRACT
In acute myeloid leukemia (AML) mutations in the juxtamembrane and tyrosine kinase 1 domain (exons 13-15) of the FLT3 gene (FLT3-ITD/LM) are heterogeneous with respect to mutation load, size, and localization. We characterized length and structure of these mutations by fragment analysis and sequencing in 689 AML which were identified among 3,365 (20.5%) newly diagnosed AML (1,803 males, 1,562 females; 15.8-91.8 years). Mutations were heterogeneous in length (median: 63, range: 3-1,236 nucleotides; nt). Most frequent were sizes of 21 (8.4%) or 24 nt (6.0%). Ninety-one different insertion sites were observed (between nt 1,788 and 1,934, according to accession "FLT3 [Ensembl/Havana merge: ENSG00000122025]" with nt 1,856 (n = 41) and 1,863 (n = 35) being most frequent. In addition, 89 different insertion end points were observed between nt 1,790 and 1,994. FLT3-mutation/wild-type ratio was available in 615 patients (median, 0.80; range 0.03-181.73). 128 Patients (20.8%) had ratios <0.3, 334 (54.3%) had ratio ≥0.3 <1, 118 (19.2%) ≥1, and 35 (5.7%) showed complete loss of the FLT3-wild-type allele. Overall (OS) and event-free (EFS) survival were better for FLT3-negative than FLT3mut normal karyotype patients (P = 0.078 and P = 0.004, respectively) and patients with low level FLT3-mutations had significantly longer OS and EFS compared with high level mutations (FLT3-mutation/wild-type ratio ≥1) (P < 0.001 and P = 0.002, respectively). The length of the mutation had no prognostic impact. Mutations localized more 5' were associated with better outcome than more 3'mutations, but no strict association to certain functional domains was detected. In conclusion, FLT3-mutations are extremely heterogenous with mutation load being the most relevant parameter.
Subject(s)
Amino Acid Sequence , Leukemia, Myeloid, Acute/genetics , Protein Structure, Tertiary/genetics , Sequence Deletion , fms-Like Tyrosine Kinase 3/genetics , Adolescent , Adult , Aged, 80 and over , Alleles , DNA Mutational Analysis , Exons , Female , Gene Frequency , Genetic Variation , Humans , Karyotype , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Molecular Sequence Data , Mutagenesis, Insertional , Mutation Rate , Prognosis , Survival AnalysisABSTRACT
ETV6 (TEL) rearrangements are favorable in pediatric acute lymphoblastic leukemia but are less well characterized in myeloid malignancies. We investigated 9,550 patients with myeloid disorders for ETV6 rearrangements by chromosome banding analysis and interphase fluorescence in situ hybridization. ETV6 rearrangements were identified in 51 of 9,550 (0.5%) patients (range, 19.2-85.3 years). Frequencies were in detail: acute myeloid leukemia (AML): 40 of 3,798, 1.1%; myelodysplastic syndromes (MDS): 6 of 3,375, 0.2%; myeloproliferative neoplasms (MPNs): 5 of 1,720, 0.3%; MDS/MPN: 0 of 210; and chronic myelomonocytic leukemia: 0 of 447. Thirty-three different partner bands of ETV6 were identified, and most were recurrent: 3q26 (n = 10), 5q33 (n = 4), 17q11 (n = 3), 22q12 (n = 3), 5q31 (n = 2), and 2q31 (n = 2). Additional chromosomal abnormalities were identified in 29 of 51 (57%) ETV6 rearranged cases. In AML, ETV6 rearrangements were frequently associated with NPM1 (9/39, 23%) and RUNX1 mutations (6/31, 19%). The FAB M0 subtype was more frequent in ETV6 rearranged de novo AML than other AML (P < 0.001); expression of CD7 and CD34 by immunophenotyping was higher in ETV6 rearranged AML compared with other subgroups. Survival of 29 ETV6 rearranged de novo AML was compared with 818 AML from other cytogenetic subgroups. Median overall and event-free survival of ETV6 rearranged cases was similar to the intermediate-risk cohort (26.3 vs. 62.2 months and 14.0 vs. 15.4 months) defined according to Medical Research Council criteria. Our study confirms the variety of ETV6 rearrangements in AML, MDS, and MPNs often in association with other genetic events. Prognosis of ETV6 rearranged de novo AML seems to be intermediate, which should be independently confirmed.
Subject(s)
Gene Rearrangement , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/genetics , Myeloproliferative Disorders/genetics , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/genetics , Adult , Aged , Aged, 80 and over , Antigens, CD34/biosynthesis , Antigens, CD7/biosynthesis , Chromosome Banding , Core Binding Factor Alpha 2 Subunit/genetics , Disease-Free Survival , Female , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence , Karyotype , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Myelodysplastic Syndromes/mortality , Myelodysplastic Syndromes/pathology , Myeloproliferative Disorders/mortality , Myeloproliferative Disorders/pathology , Nuclear Proteins/genetics , Nucleophosmin , Prognosis , Young Adult , ETS Translocation Variant 6 ProteinABSTRACT
Expression of BAALC, ERG, and MN1 is associated with outcome in normal karyotype acute myeloid leukemia (AML). In this study, the expression of these markers and of EVI1 and CDKN1B was determined using oligonucleotide microarrays in 286 AML comprising all cytogenetic groups. Higher expression of each gene was associated with an inferior outcome: CDKN1B, median overall survival (mOS): 14.9 months vs. not reached (nr), P = 0.005, median event-free survival (mEFS): 9.7 vs. 31.0 months, P = 0.013; BAALC, no impact on OS, mEFS: 6.2 vs. 13.0 months, P = 0.03; ERG: mOS: 12.5 months vs. nr, P = 0.002, mEFS: 8.1 vs. 15.7 months, P = 0.001; MN1: mOS: 12.3 months vs. nr, P = 0.004, mEFS: 8.1 vs. 16.7 months, P = 0.001. A multivariate analysis revealed an independent impact on OS for CDKN1B, ERG, and MN1 expression. A novel score based on BAALC, CDKN1B, ERG, and MN1 expression had an impact on OS and EFS independent of cytogenetics and age. A score taking into account gene expression and karyotype allowed the separation of four prognostic groups with significant differences in OS and EFS (OS at 2 years: 90.4%, 56.4%, 34.0%, 12.6%; mEFS: n.r., 18.1 months, 8.7 months, 2.5 months). The impact on outcome of this score was independent of NPM1mut/FLT3-ITD- status, MLL-PTD, and age.
Subject(s)
Biomarkers, Tumor/genetics , Gene Expression , Karyotyping , Leukemia, Myeloid, Acute/genetics , Adult , Aged , Aged, 80 and over , Cohort Studies , Cyclin-Dependent Kinase Inhibitor p27/genetics , Disease-Free Survival , Gene Expression Profiling , Germany , Humans , Leukemia, Myeloid, Acute/diagnosis , Middle Aged , Multivariate Analysis , Mutation , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis , Trans-Activators/genetics , Transcriptional Regulator ERG , Tumor Suppressor Proteins/geneticsABSTRACT
The World Health Organization classification uses a cut-off point of 5·0 × 10(9)/l cells with a chronic lymphocytic leukaemia (CLL)-phenotype in peripheral blood to discriminate between monoclonal B-lymphocytosis (MBL) and B-CLL. This study analysed 298 MBL patients by multi-parameter flow cytometry, chromosome banding analysis (CBA)/fluorescence in situ hybridization (FISH), and IGHV mutation status and compared them with 356 CLL patients. In MBL, CBA more frequently revealed a normal karyotype and FISH identified less frequently del(6q), del(13q) (as sole alterations), and del(17)(p13). Within the MBL cohort, a shorter time to treatment (TTT) was found for ZAP-70-positivity, 14q32/IGH-translocations (CBA), del(11)(q22·3) (FISH) and unmutated IGHV status. Higher CD38 and ZAP-70 expression, del(11)(q22·3) (FISH), trisomy 12 (FISH), and 14q32/IGH-translocations (CBA) were correlated with a shorter TTT in the combined cohort (MBL + CLL); a sole del(13)(q14) (FISH) correlated with longer TTT. Regarding overall survival, unmutated IGHV status and 'other' alterations (CBA) had an adverse impact. There was no correlation between the concentration of CLL-cells and TTT or overall survival. Multivariate analysis confirmed a negative impact on TTT for del(11)(q22·3)/ATM, trisomy 12 (both by FISH), and 14q32/IGH-translocations by CBA. These data emphasize a close relationship between MBL and CLL regarding clinically relevant parameters and provide no evidence to strictly separate these entities by a distinct threshold of clonal B-cells.
Subject(s)
B-Lymphocytes , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphocytosis , Adult , Aged , Aged, 80 and over , Chromosome Aberrations , Chromosome Banding , Chromosomes, Human/genetics , Disease-Free Survival , Female , Flow Cytometry , Humans , Immunoglobulin Heavy Chains/genetics , In Situ Hybridization, Fluorescence , Leukemia, Lymphocytic, Chronic, B-Cell/classification , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Lymphocytosis/classification , Lymphocytosis/diagnosis , Lymphocytosis/genetics , Lymphocytosis/mortality , Male , Middle Aged , Prospective Studies , Survival Rate , World Health OrganizationABSTRACT
TET2 mutations have been identified in 10-25% of patients with myeloid malignancies, but TET2 gene copy number alterations remain to be evaluated. We performed interphase and metaphase fluorescence in situ hybridization (FISH), using newly designed probes that span TET2, in 893 patients with acute myeloid leukaemia (AML) and chronic myeloid malignancies and validated the new assay by single nucleotide polymorphism arrays. Next-generation sequencing for molecular TET2 mutations was performed in 37/50 del(TET2) patients. We identified TET2 deletions in 50/893 patients (26 males, 24 females; 44-87 years) resulting in a 5.6% frequency [22/425 AML (5.2%), 15/217 chronic myelomonocytic leukaemia (CMML; 6.9%), 9/188 myelodysplastic syndromes (4.8%), 4/63 myeloproliferative neoplasms (6.3%)]. Cytogenetic alterations (mostly complex) were detected in 35/50 (70.0%) of del(TET2) cases. TET2 deletions were cytogenetically cryptic in 25/50 (50.0%). Sequencing detected TET2mut in 19/37 (51.4%) del(TET2) cases investigated, predominantly CMML (10/14; 71.4%). In de novo AML with intermediate cytogenetics, presence of any TET2 alteration was related to worse median overall survival (347 d vs. not reached; P = 0.052) and event-free survival (164 vs. 457 d; P = 0.024). JAK2(V617F) was found in 6/18 (33.3%) del(TET2) cases analysed. Thus, TET2 haploinsufficiency recurrently occurs in myeloid malignancies, frequently combined with TET2 mutations on the remaining allele. The prognostic impact of del(TET2) in myeloid malignancies should be further evaluated.
Subject(s)
Alleles , DNA-Binding Proteins/genetics , Leukemia, Myeloid/genetics , Mutation , Myelodysplastic Syndromes/genetics , Myeloproliferative Disorders/genetics , Proto-Oncogene Proteins/genetics , Adult , Aged , Aged, 80 and over , Dioxygenases , Female , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , Gene Frequency , Haploinsufficiency , Humans , Kaplan-Meier Estimate , Karyotyping , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/mortality , Male , Middle Aged , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/mortality , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/mortality , PrognosisABSTRACT
Mutations in the IDH1 gene at position R132 coding for the enzyme cytosolic isocitrate dehydrogenase are known in glioma and have recently been detected also in acute myeloid leukemia (AML). These mutations result in an accumulation of α-ketoglutarate to R (2)-2-hydroxyglutarate (2HG). To further clarify the role of this mutation in AML, we have analyzed IDH1R132 in 1414 AML patients. We detected IDH1R132 mutations in 93 of 1414 patients (6.6%) with a clear prevalence in intermediate risk karyotype group (10.4%, P < .001). Although IDH1R132 mutations can incidentally occur together with all other molecular markers, there were strong associations with NPM1 mutations (14.2% vs 5.4% in NPM1wt, P < .001) and MLL-PTD (18.2% vs 7.0% in MLLwt, P = .020). IDH1-mutated cases more often had AML without maturation/French-American-British M1 (P < .001), an immature immunophenotype, and female sex (8.7% vs 4.7% in male, P = .003) compared with IDH1wt cases. Prognosis was adversely affected by IDH1 mutations with trend for shorter overall survival (P = .110), a shorter event-free survival (P < .003) and a higher cumulative risk for relapse (P = .001). IDH1 mutations were of independent prognostic relevance for event-free survival (P = .039) especially in the age group < 60 years (P = .028). In conclusion, these data show that IDH1R132 may significantly add information regarding characterization and prognostication in AML.