SNP-Array-Based Karyotyping Has Impact on Cytogenetic Diagnosis and Prognosis of Non-Core Binding Factor Primary and Secondary AML.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 597-597
Author(s):  
Ramon V. Tiu ◽  
Lukasz P. Gondek ◽  
Andrew J. Dunbar ◽  
Mikkael A. Sekeres ◽  
Matt E. Kalaycio ◽  
...  
Keyword(s):  
Copy Number Variants ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Point Mutations ◽  
Clinical Value ◽  
Core Binding Factor ◽  
Secondary Aml ◽  
Mutational Status ◽  
Binding Factor ◽  
The Impact

Abstract Non-core binding factor AML has heterogeneous clinical phenotypes, likely due to various modifying genetic lesions (i.e. point mutations such as Flt3, c-kit, or and nucleophosmin). Using metaphase cytogenetics (MC), chromosomal (chr.) abnormalities are found in only 50% of newly diagnosed patients with primary AML (pAML) and secondary AML (sAML) arising from MDS or MPD. Previously, we have demonstrated that SNP arrays (SNP-A) can detect previously cryptic lesions (including uniparental disomy, UPD) and enhance the clinical value of MC in patients with MDS. Here, we hypothesize that SNP-A will improve cytogenetic analysis in AML as well. Our study included 79 healthy control marrows and 103 AML cases; 36 pAML (FAB M0=3, M1=10, M2=10, M4=6, M5=6, M7=1; mean age 53y) and 67 sAML (from MDS, N=40 and MPD/MDS, N=27; mean age 63y). Normal MC was present in 69% and 45% of pAML and sAML, respectively. First, we investigated technical aspects of SNP-A karyotyping. Dilution studies showed that SNP-A can detect clones spanning 25–50% as well as LOH calls >99% of the time as shown X chr. analysis in males. Repetitive/serial testing demonstrated congruent results and somatic derivation of randomly selected lesions was confirmed by microsatellite and SNP-A of non-clonal cells. Copy number variants (CNV) encountered in controls or described in public databases were excluded. Using SNP-A, new cytogenetic abnormalities were found in 52% (28% UPD) and 59% (33% UPD) of pAML and sAML with normal MC, respectively. Moreover, 80% and 88% of pAML and sAML with previously abnormal MC harbored lesions detected by SNP-A. Examples of microdeletions/duplications include regions harboring known leukemia susceptibility genes, such as AML1. Segmental UPD involved regions often affected by deletion, including 5q, 7q, and 11q among others. Results of SNP-A can help characterize recurrent or minimally shared lesions, map their location, or identify causative genes. However, clinical utility of this technology is best demonstrated by the impact of the new defects on survival and other clinical parameters. In both pAML and sAML patients, we found that those with both normal MC and normal SNP-A had a better overall survival (OS) and event-free survival (EFS) as compared to those showing normal MC but abnormal SNP-A. (pAML: OS: p=.04, 21 vs. 5mo; EFS: p=.03, 19 vs. 6mo; sAML: OS: p=.04, 15 vs. 4mo; EFS: p=.04, 10 vs. 4mo). A subset analysis of those sAML patients derived from MDS showed similar results (OS: p=.02, 20 vs. 4mo; EFS: p=.03, 16 vs. 6mo). Most significantly, new lesions detected by SNP-A in AML patients with previously abnormal MC corresponded to a worse prognosis (OS: p=.0004, 10 vs. 3mo). For frequently encountered lesions, we performed survival analysis. For example, the presence of UPD7q negatively affected clinical outcomes (5 patients with UPD7 had equally poor survival to patients with del7/7q, N=10). Subset analyses (e.g., AML with normal MC) also indicated that chr. lesions detected by SNP-A impact stratification schemes independent of known risk factors such as Flt3 mutational status. In summary, SNP-A karyotyping allows for detection of previously cryptic cytogenetic lesions that together with routine MC may aid not only in diagnosis but prognosis in patients with both pAML and sAML.

Enhancement of Cytogenetic Diagnosis of Myeloid Disorders through Application of SNP-Array-Based Karyotyping.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 108-108 ◽  
Author(s):  
Lukasz P. Gondek ◽  
Ramon Tiu ◽  
Marcin Wlodarski ◽  
Christine O’Keefe ◽  
Michael McDevitt ◽  
...  
Keyword(s):  
Clinical Relevance ◽  
Germ Line ◽  
Copy Number Variants ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Disease Process ◽  
Normal Karyotype ◽  
Diagnostic Value ◽  
Differential Analysis ◽  
The Impact

Abstract Cytogenetic testing improves diagnosis in myeloid disorders; chromosomal (chr) aberrations have important clinical implications. SNP arrays (SNP-A) can be applied for karyotyping with a superb resolution of unbalanced defects and detection of uniparental disomy (UPD). We stipulated that SNP-A will enhance diagnostic value of metaphase cytogenetics (MC) and uncover new random/recurrent lesions. We applied 250K SNP-A to analysis of 76 controls and 318 patients, including 95 MDS, 64 AA, 20 PNH, 48 MDS/MPD, and AML both as primary (N=32) and secondary (N=59). Multiple samples were obtained in 13 patients. Minimal clonal size detectable by SNP-A was 25–50% by dilution studies. Repetitive testing resulted in congruent results; analysis of chr X in males showed >99% fidelity. To obtain reference, deletions and duplications seen in controls were analyzed. These abnormalities correspond to germ line encoded copy number variants (CNV). In patients such CNV were not deemed pathogenic. SNP-A confirmed 82% of unbalanced chr lesions detected by MC; discordant cases included defects involving smaller clones (<8/20 metaphases) and aberrations of Y. SNP-A allowed for detection of defects in 63% vs. 37% by MC, including 77% vs. 58% in MDS, 75% vs. 37% in MDS/MPD, 33% vs. 0% in AA, 30% vs. 0% in PNH, 59% vs. 31% in AML and 76% vs. 53% in sAML. New lesions were confirmed by paired SNP-A and microsatellite analysis. Concurrent analysis of blood and marrow showed concordant results suggesting utility of SNP-A performed on blood. Serially followed patients N=6, showed occurrence of new lesions (del(4)(q) and del(7)(q)) and earlier detection of the chr aberrations. In sAML, differential analysis of blasts and granulatocytes revealed occurrence of new lesions e.g., UPD6 or 7. In both MDS and AML, UPD of various chrs was present in 20% of patients and found in up to 35% of MDS/MPD (in addition to 9p involving also chrs 6,7,11 & 14). Other newly detected lesions included isolated/recurrent microdeletions and duplications involving genes such as AML1 or Ftl3 among others. Clinical utility of SNP-A depends on whether SNP-A karyotypig will have impact on disease parameters. In all groups tested the newly detected lesions showed impact on overall survival. While the detailed results will be a subject of our presentation, survival analysis in AML can illustrate our point; cases with a normal karyotype showed superior OS to those with newly detected defects (21 vs. 6 mo, p=.05). Similarly, new additional lesions worsen the survival as compared to those with confirmed MC (3 vs. 10 mo, p=.004). The impact on OS was also established for some of the new recurrent lesions such as UPD7q (3 vs. 39 mo, p=.002). Clinical relevance of SNP-A karyotyping is also demonstrated in AA; it may help to distinguish AA from hypocellular MDS (clonal chr. defects, including UPD, occur in 33% of AA patients), AA with normal SNP-A testing showed superior response to immunosuppression as compared to patients with a totally normal karyotype. Aside of the clinical relevance, new overlapping/recurrent lesions point towards genes involved in the disease process. We conclude that SNP-A karyotyping may enhance MC in diagnosis of chr. defects and allow for a better clinical correlations of the defects with the phnenotypic and clinical features.

Genome-Wide Analysis of MDS/MPD Disclosed Frequent Homozygous C-Cbl mutations Tightly Associated with 11q-UPD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 855-855 ◽  
Author(s):  
Sanada Masashi ◽  
Shih Lee Yung ◽  
Takahiro Suzuki ◽  
Motohiro Kato ◽  
Mamiko Yanagimoto Sakata ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Point Mutations ◽  
Loss Of Function ◽  
Hematopoietic Progenitors ◽  
Gain Of Function ◽  
Gene Target ◽  
Genome Wide ◽  
Homozygous Mutations

Abstract Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by ineffective hematopoiesis and high propensity to leukemias. Although a number of gene targets have been identified, in many MDS cases, particular genetic targets are unknown. In this study, we performed genome-wide profiling of copy number (CN) abnormalities and allelic imbalances in MDS genomes in order to clarify the distribution of LOH (loss of heterozygosity) and to identify their gene targets. We analyzed a total of 171MDS and MDS/MPD specimens, including 7 RA/RARS, 23 RCMD/RCMD-RS, 6 5q-syndrome, 30 RAEB-1, 40 RAEB-2, 4 therapy related-MDS/AML, 5 MDSu, 17 CMML-1, 16 CMML-2, 24 overt AML, using high-density SNP arrays. The data were analyzed by CNAG/AsCNAR software, which enabled allele-specific CN analysis and sensitive LOH detection. MDS showed characteristic CN profiles in SNP array analysis. Of particular interest is the finding of high frequency of CN-neutral LOH (Uniparental disomy,UPD) observed in 51 of 171 (30%) MDS cases. They preferentially involved 1p, 1q, 4q, 7q, 11q, 17p and other chromosomal segments, which were associated with homozygous mutations of both loss-of-function mutations and gain-of function mutations of tumor suppressor genes and cellular oncogenes, including TP53 (17p UPD), AML1/RUNX1 (21q UPD), Nras and cMPL (1p UPD), JAK-2 (9p UPD), and FLT3 (13q UPD). Next we tried to identify a new gene target in 11q UPD, which was most common UPD region in this study and many of these cases were CMML with a normal karyotype. The minimum 11q UPD segment is about 2Mb which existed in 11q23. We sequenced coding exons of c-cbl and detected homozygous mutations in 8 of 9 MDS cases with 11q UPD (CMML=5, RAEB=3, overt leukemia=1), but very rare in cases without 11q UPD (1/162), demonstrating that the mutation is tightly linked to 11q UPD. These mutations were 8 point mutations and 1 micro-deletion, they were accumulated in the linker or RING domain. These c-cbl mutants transformed NIH3T3 in a dominant fashion, in which they were phosphorylated and activate PI3K-Akt pathway. To investigate the functions of these mutants in hematopoietic cells, we introduced these mutants into c-kit(+)Sca1(+)Lin(−) murine bone marrow cells, it prolonged replating capacity of these hematopoietic progenitors, suggesting involvement of aberrant c-cbl functions in the myeloproliferative phenotypes frequently found in 11q-UPD positive cases. In conclusion, UPD is an important mechanism of development of MDS, in which both gain-of-function and loss-of-function mutations are duplicated with exclusion of wild-type allele. Analysis of 11q UPD disclosed novel gain-of-function mutations. Identification of the targets of UPDs in 1q, 4q and 7q should also be important to gain a novel insight into the pathogenesis of MDS.

Detection of Recurrent Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MDS/MPD-Derived Secondary AML

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1542-1542
Author(s):  
Lukasz P. Gondek ◽  
Andrew J. Dunbar ◽  
Michael A. McDevitt ◽  
Hadrian Szpurka ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
Copy Number Variants ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Secondary Aml ◽  
Molecular Abnormalities ◽  
Significant Difference ◽  
Leukemic Clone ◽  
V617f Mutation

Abstract The WHO classification distinguishes MDS/MPD as a distinct entity. The JAK2 V617F mutation is present in a minority of these patients (pts). UPD9p characterizes pts homozygous for the V617F mutation. Other chromosomal abnormalities can also be detected in pts with typical MPD and MDS/MPD, and it is likely that cytogenetic methods with higher resolution could detect additional defects. We applied 250K SNP-arrays to examine genomic composition and identify previously cryptic defects and molecular abnormalities in pts with MDS/MPD-U and secondary AML (sAML) arising from MDS/MPD-U both in pts wild-type for V617F and those with the mutation. Any deletions, duplications, and/or UPD found by SNP-A in 76 controls or on available internet databases were considered copy number variants (CNV) and non-pathogenic. First, we used pts with typical MPD to assess the ability of SNP-A to detect UPD. All pts homozygous for V617F showed UPD9p by SNP-A. In pts with MDS/MPD, several additional cryptic lesions were detected, including segmental micro-deletions on chr 1, 5, 9, and 12. UPD was common, occurring on chromosomes other than 9 in 9/28 patients (32%, i.e. on chr 1,11,12). Shared/overlapping lesions (in &gt;2 pts) included small segmental lesions on chr 7 (N=3), and a small cytoband (q14.1) of chr 11 (N=3). Overall, clonal lesions including segmental UPD were found in 23/28 (82%) pts by SNP-A in comparison to 17/28 (61%) by metaphase cytogenetics (MC). Pts with a history of MDS/MPD-U with (N=14) and without the JAK2 V617F mutation (N=14) were also analyzed. MC revealed chr aberrations in 10/14 (71%) of V617F+ pts, including common lesions such as +8 and del5q. With SNP-A, 1 additional pt with normal MC was found to have an abnormal karyotype (UPD 7 and 9), and 9/10 pts with abnormal MC had additional lesions previously undetected, including UPD on chrs other than 9 in 3/14 pts (21%). Examples of deleted regions include segmental losses within chrs 2, 7, 8 and 13, and UPD on chrs 1p, 7q, and 11. Likewise, additional lesions were identified in MDS/MPD-U pts negative for V617F. 8/14 pts (57%) showed abnormal MC; however SNP-A showed lesions in 12/14. In addition, 6/8 pts with abnormal MC had lesions in addition to those detected by MC. UPD was also common in V617F- pts, occurring in 6/14 (43%), predominantly on chr 11 (in 3/6 pts). No significant difference was found between the number or type of lesions found in pts with and without V617F mutation. SNP-A can also be used to identify lesions acquired during AML evolution. In 1 MPD pt at diagnosis, SNP-A showed UPD9p as a sole abnormality consistent with a homozygous V617F mutation. Upon transformation, repeated SNP-A showed a V617F- leukemic clone (normal chr 9) possessing microdeletions on chr 4 and 19. Similar evolution of a V617 negative leukemic clone was also observed in an MDS/MPD pt in whom a new UPD6 was detected. However, in 3 other MDS/MPD patients, SNP-A showed the presence of a V617+ leukemic clone (showing UPD9) in AML blasts. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chr aberrations in pts with MDS/MPD, including copy-neutral LOH. UPD is common in both JAK2 V617F+ and V617F- disorders, and is not restricted only to chr 9p, indicating other potential causative genes.

Identification of Proteinase 3 (PR3) Gene Overexpresison and Protein Delocalization in Core Binding Factor Leukemias as a Mechanism Leading to Increased Chemosensitivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 204-204
Author(s):  
Daniela Cilloni ◽  
Ilaria Defilippi ◽  
Sonia Carturan ◽  
Chiara Maffè ◽  
Marisa Pautasso ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Mean Value ◽  
Binding Activity ◽  
Proteinase 3 ◽  
Loss Of Function ◽  
Core Binding Factor ◽  
Secondary Aml ◽  
Dna Binding Activity ◽  
Binding Factor

Abstract Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p&lt;0,0002 for t(8;21), p&lt;0,001 for inv16) and lower C/EPBα levels. EMSA assay demonstrated the absence of C/EBPα DNA binding activity in CBF AML cells but not in NK AML. In addition, PR3 overexpression was detected in 60% of RA patients (mean value: 10, range 3–268), and in all the cases of RAEB (mean value 201: range:128–803) and secondary AML (mean value 589, range 207–7131). WB demonstrated the correlation between the mRNA and protein amount. Interestingly, immunofluorescence demonstrated the de-localization of the protein within the nucleous in CBF AML but it is completely cytoplasmatic in leukemic cells with normal karyotype and in MDS. Transfection experiments with PR3 plasmid demonstrated that PR3 overexpression results into a significantly increased proliferation and reduced apoptosis. By contrast transfection with shRNA triggers apoptosis and cell growth inhibition. In addition, WB demonstrated that nuclear PR3 is able to cleavage the p65subunit of NF-kB into a p56 isoform which lacks any transcriptional activity as confirmed by EMSA. In conclusion, PR3 gene expression and protein are significantly increased in AML and MDS, particularly in CBF leukemias in which the protein is not only increased but also completely delocalized within the nucleous. PR3 overepression My be due to a significant downmodulation of C/EBPα. Ectopic expression of PR3 induces increased proliferation and apoptosis arrest. The abnormal nuclear localization of PR3 in CBF leukemias results into the loss of function of NF-kB thus representing one mechanism of chemo sensitivity in this group of patients.

Molecular Diversity Detected by Whole Exome Sequencing in Chronic Myelomonocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 310-310
Author(s):  
Basel Rouphail ◽  
Kenichi Yoshida ◽  
Holleh D Husseinzadeh ◽  
Edward P Evans ◽  
Satoru Miyano ◽  
...  
Keyword(s):  
Research Funding ◽  
Germ Line ◽  
Target Therapy ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Gene Mutations ◽  
Molecular Pathogenesis ◽  
Common Mutation ◽  
Mutational Status ◽  
Recurrent Mutations

Abstract Abstract 310 CMML is characterized by monocytic proliferation, cytomorphologic dysplasia, and frequent progression to AML. Heterogeneity in or subtlety of presentation can make diagnosis challenging. Recent advances in molecular technology set the stage for systematic study of genetic and genomic lesions associated with CMML. Initially, RAS and RUNX1 mutations were identified in CMML; subsequently, mutations in TET2, CBL, ASXL1 and EZH2 have been discovered. Most recently, recurrent mutations in various genes of the spliceosomal machinery have been added, with SRSF2 likely the most common mutation in this condition. We hypothesized that more precise analysis of molecular lesions in CMML may allow for better categorization of this condition according to molecular pathogenesis and may provide clues to target therapy of this rather refractory condition. We identified 136 patients with CMML or secondary AML (sAML) with antecedent CMML: 87 CMML-1, 20 CMML-2 and 29 post-CMML sAML. The original cohort has been expanded by an additional 53 patients since first reported. The mean follow up period was 16 months (range, 0–114). Abnormal cytogenetics were found in 50% of the cohort by both metaphase and SNP-array-based karyotyping. In a representative subset of 27 patients, we have applied whole genome sequencing (WES) for which paired tumor/germ line DNA was used. To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalytic filtering approach and results were aligned using Burrows-Wheeler Aligner and variants detected using the GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on somatic defects with a frequency of >5% of the cohort. For the most commonly affected genes, results were validated using an expanded panel of 18 genes in 72 additional patients and, thus, for the most relevant genes a cohort of 95 patients was studied. The most frequently mutated genes were TET2 (48%), SRSF2 (35%), ASXL1 (17%) and RUNX1 (17%), whereas CBL (13%), EZH2 (13%), UTX (8%) and U2AF1 (8%), SETPB1 (10%), and RIT1 (9%) were less frequent. We also found TP53 and RUNX1 mutations in 5% and 16% of patients, respectively. A JAK2 V617F mutation was present in one case of seemingly typical CMML. BCOR and STAG2 mutations were found in 13% and 9% of patients, respectively; KRAS/NRAS mutations were in 10%. Spliceosomal gene mutations seem to be mutually exclusive, but were frequently associated with other non-spliceosomal gene mutations examined. Within the cohort of 28 SRSF2 mutant cases, 15 had coexisting TET2 mutations, 22 had ASXL1 mutations, 7 had RUNX1 and 5 had CBL mutations. Among 10 U2AF1 mutant cases, 3, 5 and 2 had TET2, ASXL1, and RUNX1 mutations, respectively. SETBP1 mutations were present in 34% of CMML-1/2 and frequently associated with RUNX1, SRSF2, CBL (approximately 2% each) and ASXL1 (4%) mutations. Cohesin mutations were less frequent (10%) because RAD21 and SMC mutations were absent. Mutations of PTPN11 and NF1 were less frequent in adult CMML than those reported in JMML. We also identified several less-recurrent gene mutations that likely modify pathogenesis or clinical outcomes of specific cases. Serial studies performed on 6 cases showed insight into the clonal architecture, producing a series of putative ancestral and secondary events, including uniparental disomy and acquisition of KRAS/NRAS or SETPB1 mutations. Association between mutational status and overall survival (OS) was assessed using Kaplan-Meier statistics. While all permutations were tested, we highlight here only significant positive and relevant negative results. In the whole cohort, presence of CBL mutations conferred worse OS (p=.018; HR 2.44, 95%CI 1.18–4.69). Median OS was 16 months for CMML-1, 6 months for CMML-2, and 14-months for sAML. In subgroup analyses, CBL mutations were also significant worse prognostic factor in CMML-1 cohort (p=.037; HR 3.23, 95%CI 1.07–8.04). In sum, WES provides intricate information on the molecular pathogenesis of CMML and the wide mutational spectrum correlates with the clinical diversity. Expert-based analysis of the genomic data may be supplanted by unsupervised and unbiased approaches which would cluster patients based on molecular similarities. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

The Impact of Clonal Architecture of IDH1 and IDH2 Mutant Cases on the Biology of Myeloid Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1897-1897 ◽  
Author(s):  
Swapna Thota ◽  
Hideki Makishima ◽  
Bartlomiej P Przychodzen ◽  
Aaron D. Viny ◽  
Bhumika J Patel ◽  
...  
Keyword(s):  
Clinical Features ◽  
Cross Sectional ◽  
Secondary Aml ◽  
Clonal Architecture ◽  
Mutational Status ◽  
Idh Mutations ◽  
Idh1 Mutations ◽  
Serial Samples ◽  
Founder Events ◽  
The Impact

Abstract The rearrangements of core binding factors (CBF) are classical founder events in primary AML (pAML). In non-CBF AML, many new somatic mutations have been identified, some of which may also serve as initial ancestral events. In myelodysplastic syndromes (MDS) and secondary AML (sAML), the diversity of ancestral lesions may be even greater, with potential overlap with other myeloid neoplasm in initiating molecular events. E.g., endomorphic IDH1/IDH2 mutations have been discovered in pAML, but also can be present in sAML and MDS. Analysis of the clonal architecture of a limited number of AML cases suggested that IDH1/2 mutations are ancestral. Theoretically, any ancestral lesion could also occur at later stages as a subclone. It is not clear if IDH1/2 mutations play the same role in pAML vs. sAML and MDS. This is a critical distinction as drugs targeting founder events may be able to be curative, while targeting secondary events may reverse clonal evolution, but is unlikely to eliminate the founder clone. Moreover, initiating molecular lesions are more likely to be subcategory-defining than the secondary events. We hypothesized that IDH1/2 mutations may occur at different stages in the clonal hierarchy in patients with various myeloid phenotypes and may affect both the nature of genetic landscape and clinical features should IDH1/2 mutations be ancestral. Conversely, should IDH1/2 be found to be subclonal it will be essential to identify the founder clone. To address these issues, we have investigated a cohort of 2033 patients with secondary AML (N=125), pAML (N=294), MDS (N=1404), MDS/MPN or MPN (N=201) for the presence of IDH1/2mutations and other clonal events using various forms of deep NGS. IDH1 mutations were found in 3%, 1.3%, 7.8% and 8% of MDS, MDS/MPN, pAML and sAML, whereas those of IDH2 were found in 3.4%, 3.3%, 6.5% and 9.1%, respectively. While both IDH1/2 mutations occurred most frequently in pAML (P=.0001), IDH2 was commonly associated with higher-risk MDS (P=.013). IDH1 mutations coincided with younger age (P=.01) while IDH2 mutants were likely to have a normal karyotype (P=.01). The average copy number-adjusted clonal size was 58% for IDH2 and 56% for IDH1 (P=.78). DNMT3A and NPM1 mutations most frequently co-occurred with IDH1/2. RUNX1, ASXL1, SRSF2, and STAG2 were significantly associated with IDH2, whereas PHF6 mutations with IDH1. To reconstruct the clonal architecture in IDH1/2 cases, variant allelic frequencies were compared, along with cross-sectional comparisons of mutational frequencies and serial samples. Cross-sectional analysis of the distribution of IDH1/2 mutations in patients with lower-risk and higher-risk MDS and sAML showed increasing proportions of both IDH1 and IDH2 mutant cases with more advanced stages of the disease, suggesting that in some cases IDH mutations evolved during progression. This finding was substantiated when we analyzed serial samples. In 5/6 cases, IDH1 mutations constituted ancestral events with PHF6, NPM1, JAK2, NRAS and SMC3 serving as the most common subsequent subclonal events. The sixth case had U2AF1 as the ancestral event. IDH2 was an ancestral event in 3/4 serial cases with STAG2, ASXL1, NRAS, TET2 and NPM1 as common subclonal events. When clonal hierarchy of concurrently mutated genes was analyzed using the diversity score calculation of VAFs, IDH1 mutation was identified as a dominant clone in 45% of cases; when it was subclonal, DNMT3A, SRSF2 and SF3B1 were the most commonly detected dominant mutations. In contrast, in pAML cases, IDH1/2 constituted dominant mutations is 80%, with NPM1, FLT3 mutations being the most common secondary events to IDH1, and RUNX1 being the most common secondary event to IDH2. When IDH1 mutations were analyzed, they constituted ancestral events in 59% of cases (P=.9, vs. IDH1). When IDH was found to be subclonal, the ancestral events were heterogeneous. OS was analyzed in 786 patients within this cohort. IDH1 mutational status had no effect on survival of pAML or MDS patients, whereas IDH2 mutant MDS patients had an inferior OS compared to WT patients (24 vs. 40 months, P=.05) in multivariate analysis. In sum, our results suggest that in pAML, IDH1/2 mutations are primary events, whereas in sAML, MDS and MPN/MDS, IDH1/2 mutations may also be subclonal. As a primary event, IDH1 or IDH2 may influence clinical features and be used to categorize a proportion of patients in particular with regard to use of targeted therapies. Disclosures Haferlach: MLL: Equity Ownership.

Allogeneic Hematopoietic Stem Cell Transplantation (HCT) for Acute Myeloid Leukemia (AML) Not in First Remission

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4388-4388 ◽  
Author(s):  
Kendra L Sweet ◽  
Jeffrey E Lancet ◽  
Ryan Hillgruber ◽  
Megan Melody ◽  
Amina Llishi ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Cumulative Incidence ◽  
Research Funding ◽  
Conditioning Regimen ◽  
Disease Free Survival ◽  
Disease Status ◽  
Hematopoietic Stem ◽  
Secondary Aml ◽  
Mutational Status ◽  
The Impact

Abstract Background: Approximately 60 - 80% of AML patients achieve a complete remission [CR] with one or two cycles of induction chemotherapy, leaving many patients with refractory AML [PIF]. Unfortunately, the majority of patients in CR1 ultimately relapse. With salvage therapy, only 30-50% achieve CR2. Those with PIF or relapsed AML have shortened survival and few therapeutic options. Risk stratification is primarily based on karyotype, however other factors including age, initial white blood cell count, secondary AML and mutational status are also utilized to determine prognosis. HCT is an effective option for treatment of AML with intermediate/high risk features in CR1. It has also been utilized in refractory or relapsed disease. Advances in HCT over the last decade have improved overall survival (OS) and extended this option to older patients. Our aim is to characterize outcomes after HCT for AML patients who are not in CR1. Methods: We analyzed 136 AML patients who were not in CR1 at the time of HCT from 2004 - 2013. The conditioning regimen was fludarabine and myeloablative doses of PK targeted busulfan. IWG AML response criteria were used to define disease status at the time of transplant. Cytogenetic risk was based on the NCCN AML guidelines. OS is defined as the time from HCT until the time of death from any cause. Disease free survival (DFS) is defined as the time from HCT to the time of relapse or death from any cause. Results: Disease status consisted of 74 (54.4%) in CR2, 6 (4.4%) in CR3 or beyond, 27 (18.9%) were PIF, 21 (15.4%) with relapsed AML (REL) that was treated but still present at time of transplant, and 8 (5.7%) who received either no treatment or a hypomethylating agent (HMA). Median age was 52.0 (21.8 - 72.5) years, and 80 (59%) were male. Time from most recent treatment to HCT was < 1 month in 8 (5.8%), 1-3 months in 75 (55.8%), >3 months in 50 (36.8%) and not applicable in 3. Ninety-six (70.6%) had de novo AML, while 40 (29.4%) had secondary AML. Cytogenetic risk was favorable in 32 (23.5%), intermediate in 57 (42%), poor in 40 (29.4%) and unknown in 7 (5.1%). Graft-versus-host disease prophylaxis was tacrolimus with methotrexate or sirolimus, or mycophenolate mofetil. Donors included 41 (30.2%) matched related, 2 (1.4%) mismatched related, 65 (47.8%) matched unrelated and 28 (20.6%) mismatched unrelated donors. Peripheral blood stem cells were used in 97.2% of cases. Two year OS, DFS, cumulative incidence (CI) of relapse and CI-NRM for all patients was 45.3%, 35.2%, 47.1% and 18.2%, respectively. Two-year DFS stratified by disease status at time of HCT was 41.9%, 33.3%, 25.9%, 33.3% and 12.5% in CR2, CR3 or beyond, PIF, REL and HMA, respectively(p=0.011 for CR2 vs HMA) (Figure 1). Two-year DFS stratified by cytogenetic risk was 43.8%, 31.6%, 37.1% and 14.3% in favorable, intermediate, poor and unknown, respectively (p>0.05) (Figure2). CI-Rel stratified by disease status was 43.2%, 16.7%, 66.7%, 42.9% and 50% in CR2, CR3 or greater, PIF, REL and HMA, respectively (Figure 3). Conclusions: We analyzed 136 AML patients after undergoing HST outside of CR1 and the cumulative incidence of relapse at two years was 47%. Relapse was highest in those with primary induction failure or residual disease after either no or low intensity therapy. These data suggest that patients with active disease at the time of transplant fare worse than those who are transplanted in remission, highlighting the importance of effective upfront therapies in order to obtain the maximum potential benefit from HCT. Cytogenetic risk stratification did not significantly impact outcomes, although those with favorable risk cytogenetics trend towards higher 2-year DFS vs those with intermediate or poor-risk disease. Trials looking at the impact of maintenance therapy post-transplant may be valuable in this patient population. Table 1. Disease Status @ HSCT CR2 CR3 or beyond PIF RES HMA/untreated 2 years 41.9% (30.6 - 52.8) 33.3% (4.6 - 67.6) 25.9% (11.5 - 43.1) 33.3% (14.9 - 53.1) 12.5% (0.7 - 42.3) Table 2. Cytogenetic Risk Group Favorable Intermediate Unfavorable Unknown 2 years 43.8% (26.5 - 59.8) 31.6% (20.1 - 43.7) 37.1% (22.5 - 51.8) 14.3% (0.7 - 46.5) Table 3. Cumulative Incidence of Relapse CR2 (1) CR3 or beyond (2) PIF (3) REL (4) HMA/untreated (5) 2 years 43.2% (32.2 - 54.6) 16.7% (0.0 - 53.5) 66.7% (48.1 - 82.9) 42.9% (23.0 - 64.0) 50.0% (18.1 - 81.9) Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Sweet: Novartis Pharmaceuticals: Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc: Research Funding; Incyte: Research Funding. Lancet:Celgene: Consultancy, Research Funding; Seattle Genetics: Consultancy; Boehringer-Ingelheim: Consultancy; Pfizer: Research Funding; Kalo-Bios: Consultancy; Amgen: Consultancy. Perkins:PDL Biopharma: Research Funding. Field:PDL Biopharma: Research Funding.

Unlike AML1, CBFβ gene is not deregulated by point mutations in acute myeloid leukemia and in myelodysplastic syndromes

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3848-3850 ◽  
Author(s):  
Hugues Leroy ◽  
Christophe Roumier ◽  
Nathalie Grardel-Duflos ◽  
Elizabeth Macintyre ◽  
Pascale Lepelley ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Point Mutation ◽  
Myelodysplastic Syndromes ◽  
Myeloid Leukemia ◽  
Point Mutations ◽  
Core Binding Factor ◽  
The Core ◽  
Binding Factor ◽  
Acute Myeloid

The core-binding factor (CBF) complex is a heterodimeric transcription factor composed of 2 subunits, CBFα and CBFβ, that play a major role in hematopoiesis. Both members of the CBF complex are frequently altered in acute myeloid leukemia (AML) by translocation, most commonly t(8;21), t(12;21), and t(3;21) for CBFα, located in 21q22, and inv16(p13;q22) for CBFβ, located on 16q22. Recently, a new mechanism of alteration of CBFα, by point mutation, has been reported in myeloid malignancies, particularly in M0 AML. In the present study, we found no point mutation of theCBFβ gene in 30 myelodysplastic syndromes and 100 AMLs, suggesting a limited role, if any, of CBFβ point mutations in those disorders.

scholarly journals Impact of Race/Ethnicity on Pediatric Core Binding Factor AML Outcomes and Response to Gemtuzumab Ozogamicin

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Shannon E. Conneely ◽  
Casey L. McAtee ◽  
Rachel E. Rau
Keyword(s):  
Racial Differences ◽  
Black Children ◽  
Gemtuzumab Ozogamicin ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Hispanic Patients ◽  
Patient Variables ◽  
Race Ethnicity ◽  
The Impact ◽  
Target Database

Introduction: Racial disparities in pediatric acute myeloid leukemia (AML) outcomes have consistently demonstrated inferior event-free survival (EFS) and overall survival (OS) in minority populations with the worst outcomes in Black children. However, AML is a heterogenous disease classified by recurrent genetic aberrations, and these disparities have not been thoroughly investigated within specific cytogenetic groups. In addition, race-based response to individual chemotherapy agents in AML has not been explored. Here, we use data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database to describe the effects of race on outcomes and response to gemtuzumab ozogamicin (GO) therapy in core binding factor (CBF) AML, a favorable risk AML subtype. Methods: CBF AML patient data was extracted from the online TARGET database. Race and ethnicity were self-reported. Estimates of 2-year OS and EFS were calculated using the Kaplan-Meier method and analyzed via log-rank statistic. Pearson's Chi-squared test and Fisher's exact test were used to analyze patient variables. Results: The OS in CBF AML differed significantly based on race/ethnicity at 87% (95% CI, 82-93%) for White non-Hispanic (WNH), 77% (95% CI, 67-89%) for Hispanic, and 71% (95% CI, 58-88%) for Black children (p=0.005). EFS was also significantly lower in Black (46%; 95% CI, 32-66%) compared to non-Black children (66%; 95% CI, 60-73%)(p=0.023). When assessed by CBF AML subtype, EFS and OS in t(8;21) AML varied significantly based on ethnicity at 50% (95% CI, 34-75%) and 67% (95% CI, 50-88%) in Black patients, 63% (95% CI, 49-82%) and 69% (95% CI, 55-87%) in Hispanic patients, and 82% (95% CI, 72-92%)(p=0.01) and 90% (95% CI, 93-98%)(p=0.006) in WNH patients. In inv(16) AML, Hispanic patients experienced the highest EFS (71%; 95% CI, 55-92%) followed by WNH (54%; 95% CI, 43-66%) and Black (36%; 95% CI, 17-79%) patients though this did not reach statistical significance (p=0.12). There was no impact of race/ethnicity on OS in inv(16) AML. The use of GO did not alter OS in any subset of CBF AML. EFS appeared to be slightly improved but was not statistically significant. However, Black children with any type of CBF AML had preferential improvement with GO with significantly higher EFS (69% v. 26%, p=0.03) and a trend toward improved OS (81% v. 63%, p=0.19) with GO therapy. These improvements were primarily driven by a significant increase in EFS in inv(16) AML when treated with GO (80% v. 50%, p=0.01) and a similar but non-significant improvement in OS in this group. In Hispanic children there was a trend toward inferior EFS with the use of GO (63% v. 73%, p=0.37) in CBF AML, particularly in t(8;21) patients with a 1-year EFS of 56% (95% CI, 37-87%) compared to 81% (95% CI, 64-100%)(p=0.23). WNH children were more likely to achieve Complete Remission (CR) after course 1 (OR 2.36, 95% CI 1.09-5.10, p=0.03), and Black children were more likely to have persistent Minimal Residual Disease (MRD) after course 2 (OR 9.64, 95% CI 2.48-37.43, p&lt;0.001). No differences were detected in other patient variables. Conclusion: Children with CBF AML represent a favorable cytogenetic risk group with an EFS of 70% and OS of 85%. However, the impact of race/ethnicity on outcome is significant with Black children experiencing the lowest EFS and OS at 46% and 71%, respectively, with this trend persisting across cytogenetic subtypes. Black children were also more likely to remain MRD positive after Induction II and less likely than WNH patients to achieve CR. However, while the addition of GO to standard therapy did not improve outcomes in CBF AML overall, Black children selectively benefited from GO therapy. Black children trended toward improved EFS and OS with GO with the most pronounced effect on EFS in inv(16) which was improved from 50% to 80% at 1 year. These results demonstrate that Black children with CBF AML are disproportionately at risk for poor outcomes compared to other ethnicities, but these risks may be mitigated by the use of GO which increase EFS and OS primarily in this group. Conversely, Hispanic patients trended toward worse EFS with GO, particularly in t(8;21) AML, possibly due to treatment-related toxicity. As CD33 expression and certain ABCB1 SNPs are known to predict response to GO therapy, our ongoing work investigates racial differences in these variables as a potential explanation for the differing responses to GO. Disclosures Rau: Jazz Pharmaceuticals, Inc.: Consultancy, Other: Travel Fees.

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