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  • 1
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    Array CGH Identifies Copy Number Changes In 10% Of 520 MDS Patients With Normal Karyotype: Deletions Encompass The Genes TET2, DNMT3A, ETV6, NF1, RUNX1, and STAG2 and Are Associated With Shorter Survival
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1516-1516
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1516-1516
    Abstract: In MDS, cytogenetic aberrations play an important role for classification and prognostication. The original IPSS and the revised IPSS classifiers have clearly demonstrated the prognostic impact of distinct cytogenetic abnormalities. The vast majority of chromosome aberrations in MDS are gains or losses of chromosomal material while balanced rearrangements are rare. However, more than 50% of MDS and even more in low risk MDS harbor a normal karyotype. Chromosome banding analysis can only detect gains and losses of more than 10 Mb size due to its limited resolution and is dependent on proliferation of the MDS clone in vitro to obtain metaphases. Array CGH has a considerably higher resolution and does not rely on proliferating cells. Aims In this study we addressed the question whether MDS with normal karyotype harbor cytogenetically cryptic gains and losses. Patients and Methods 520 MDS patients with normal karyotype were analyzed by array CGH (Human CGH 12x270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). For all patients cytomorphology and chromosome banding analysis had been performed in our laboratory. The cohort comprised the following MDS subtypes: RA (n=22), RARS (n=43), RARS-T (n=27), RCMD (n=124), RCMD-RS (n=111), RAEB-1 (n=104), and RAEB-2 (n=89). Median age was 72.2 years (range: 8.9-90.1 years). Subsequently, recurrently deleted regions detected by array CGH were validated using interphase-FISH. Results In 52/520 (10.0%) patients copy number changes were identified by array CGH. Only eight cases (1.5%) harbored large copy number alterations 〉 10 Mb in size, as such generally detectable by chromosome banding analysis. These copy number alterations were confirmed by interphase-FISH. They were missed by chromosome banding analysis due to small clone size (n=2), insufficient in vitro proliferation (n=3) or poor chromosome morphology (n=3). In the other 44 patients with submicroscopic copy number alterations 18 gains and 32 losses were detected. The sizes ranged from 193,879 bp to 1,690,880 bp (median: 960,176 bp) in gained regions and 135,309 bp to 3,468,165 bp (median: 850,803 bp) in lost regions. Recurrently deleted regions as confirmed by interphase-FISH encompassed the genes TET2 (4q24; n=9), DNMT3A (2p23; n=3), ETV6 (12p13; n=2), NF1 (17q11; n=2), RUNX1 (21q22; n=2), and STAG2 (Xq25, deleted in 2 female patients). No recurrent submicroscopic gain was detected. In addition, we performed survival analysis and compared the outcome of patients with normal karyotype also proven by array CGH (n=462) to patients with aberrant karyotype as demonstrated by array CGH (n=52). No differences in overall survival were observed. However, overall survival in 35 patients harboring deletions detected solely by array CGH was significantly shorter compared to all others (median OS: 62.1 vs 42.4 months, p=0.023). Conclusions 1. Array CGH detected copy number changes in 10.0% of MDS patients with cytogenetically normal karyotype as investigated by the gold standard method, i.e. chromosome banding analysis. 2. Most of these alterations were submicroscopic deletions encompassing the genes TET2, ETV6, DNMT3A, NF1, RUNX1, and STAG2. 3. Interphase-FISH for these loci can reliably pick up these alterations and is an option to be easily performed in routine diagnostics in MDS with normal karyotype. 4. Patients harboring deletions detected solely by array-CGH showed worse prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Raitner:MLL Munich Leukemia Laboratory: Employment. Holzwarth:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1516.1516
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 2
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    Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit
    Elsevier BV ; 2014
    In:  Cell Stem Cell Vol. 14, No. 6 ( 2014-06), p. 824-837
    Details
    In: Cell Stem Cell, Elsevier BV, Vol. 14, No. 6 ( 2014-06), p. 824-837
    Type of Medium: Online Resource
    ISSN: 1934-5909
    URL: Article
    DOI: 10.1016/j.stem.2014.02.014
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2014
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  • 3
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    Acute Erythroid Leukemia (AEL) Can Be Separated Into Distinct Prognostic Subsets Based On Cytogenetic and Molecular Genetic Characteristics
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 1394-1394
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 1394-1394
    Abstract: Abstract 1394 Background: Acute erythroid leukemia (AEL) is characterized by a predominant erythroid population and is comprising 〈 5% of adult AML cases. Because of the relative rarity of AEL, few large studies have examined underlying clinical and genetic features. Aims: Molecular and cytogenetic characterization of AEL and identification of genes with prognostic impact. Patients and Methods: We studied an unselected cohort of 94 AEL patients including 32 female and 62 male cases; median age was 69.0 yrs (range: 21.3–88.3 yrs). Survival data was available in 73 cases; median survival was 15.9 months. First, chromosome banding analysis (n=94) was performed. In addition, all cases with normal karyotype (NK) were investigated by CGH arrays (n=32) (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation screening for ASXL1 (n=87), CEBPA (n=94), DNMT3A (n=94), FLT3 (both internal tandem duplication (ITD) (n=93), and tyrosine-kinase domain (TKD) mutations (n=85)), IDH1 (n=93), IDH2 (n=65), NRAS (n=91), KRAS (n=93), MLL-PTD (n=79), NPM1 (n=94), RUNX1 (n=94), TP53 (n=94), and WT1 (n=90) was performed by 454 amplicon deep-sequencing (Roche, Branford, CT), Sanger sequencing or melting curve analyses. CGH array data analysis was performed using Nexus Copy Number 6.0 (BioDiscovery Inc, El Segundo, CA). Results: Cytogenetic data was available for all cases: 48 cases (51.1%) presented an intermediate-risk and 46 (48.9%) cases an unfavorable cytogenetic category according to the MRC Classification. By CGH array analysis 30/32 cases retained a NK, whereas in two cases small aberrations were detected: case 1: deletion of the CEBPA gene, case 2: duplication 11q13.3 to 11q25 including the ATM and MLL gene. Molecular mutations were detected in 85/94 patients (90.4%). 63.5% (54/85) of mutated patients carried one, whereas 36.5% (31/85) of cases harbored two (n=22) or more (n=9) mutations. In detail, TP53 was the most frequently mutated gene (41 cases, 43.6%). Other alterations were detected in NPM1 (15/94; 16.0%); DNMT3A (12/94; 12.8%); ASXL1 (8/87; 9.2%); MLL-PTD (7/79; 8.9%); RUNX1 (8/94; 8.5%); IDH1 (6/93; 6.5%); WT1 (5/90; 5.6%); IDH2 (3/65; 4.6%); NRAS (3/91; 3.3%); KRAS (3/93; 3.2%); FLT3-ITD (3/93, 3.2%), FLT3-TKD (3/85, 3.5%), and CEBPA (1/94). First, we were interested in any correlation with the respective karyotype and observed that NPM1, RUNX1, and WT1 mutations correlated with an intermediate-risk karyotype (NPM1: 15/48 vs 0/46, P 〈 0.001; RUNX1: 8/48 vs 0/46, P=0.006; WT1: 5/46 vs 0/44, P=0.056), whereas TP53mut correlated with the unfavorable karyotype (38/46 vs 3/48, P 〈 0.001). Within the cytogenetically adverse subset TP53mut were associated with complex karyotype (36/38 vs 2/8, P 〈 0.001). In addition, NPM1mut correlated with lower age (56±15 vs 67±13 yrs, P=0.002), whereas mutations in ASXL1, DNMT3A, and TP53 correlated with higher age (73±4 vs 64±15, P=0.001; 71±6 vs 65±14, P=0.015; 71±8 vs 61±15, P 〈 0.001). NPM1mut were associated with longer, and RUNX1mut and TP53mut with shorter OS (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 28.3%, P=0.001; RUNX1mut vs wt: 0% vs 45.2%, P=0.007; TP53mut vs wt: 9.4% vs 61.6%, P=0.001). In the univariable Cox regression analyses mutations in NPM1 (HR 0.12; P=0.004), RUNX1 (HR 3.99; P=0.013), TP53 (HR 3.19; P=0.001), age (HR 4.24, P=0.001) and adverse cytogenetics (HR 2.98, P=0.002) were significantly associated with OS. Independent prognostic factors in multivariable Cox regression analysis were: age (HR 2.6, P=0.047) and RUNX1mut (HR 6.3, P=0.006). Of note, when separating MRC intermediate from MRC adverse cases, we confirmed the longer OS of NPM1 and shorter OS of RUNX1 mutated cases in comparison to NPM1, RUNX1 wt cases (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 46.3%, P=0.027; RUNX1mut vs wt: 0% vs 69.0%, P 〈 0.001). Conclusions: (1) The frequency of cases with complex or other adverse karyotypes within the AEL cohort is very high (48.9%), (2) 93.7% of cases with NK also showed a NK using high-resolution CGH arrays. (3) Overall, a remarkably high mutation frequency of 90.4% was found. (4) NPM1 and RUNX1mut were exclusively detected in the cytogenetically intermediate-risk MRC, TP53 mut predominantly in the MRC adverse group and mainly in cases with complex karyotype. (5) In addition to chromosome banding analysis mutation screening of RUNX1 and NPM1 in cases with intermediate-risk karyotype should be considered for better prognostication. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.1394.1394
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 4
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    Gene Amplifications In 84 Patients With Acute Myeloid Leukemia and 31 Patients With Myelodysplastic Syndrome Investigated By Array CGH
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 3724-3724
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 3724-3724
    Abstract: Gains and losses of chromosomal material are frequent in AML and MDS and usually lead to loss or gain of a single copy of a whole chromosome, a chromosome arm or small stretches of the chromosome that may be microscopically invisible. More rarely, amplifications of chromosomal regions (defined as the presence of more than 6 copies of a region per cell) are observed. These supernumerary copies are located either extrachromosomally as small acentric chromosomal structures - so called double-minutes (dmin) - or intrachromosomally as large contiguous stretches of amplified DNA, so called homogeneously staining regions (HSR). Aims Characterize AML and MDS cases with gene amplifications with respect to size, affected genes and accompanying chromosomal abnormalities as well as TP53 status. Patients and Methods 84 AML and 31 MDS cases with cytogenetically visible amplifications were selected for this study. All cases were analyzed by array CGH, chromosome banding analysis, sequencing for TP53 mutations as well as FISH for TP53 deletions. Results The cohort comprised 55 (47.8%) males and 60 (52.2%) females with a median age of 72.0 years (range 38.0 - 90.3 years). A complex karyotype (≥4 aberrations) was present in 92/115 (80.0%) cases (AML=65/84 (77.4%); MDS=27/31 (87.1%)). In total, 385 amplified regions were identified by array CGH. In more detail: 3q26 (AML: n=6; MDS: n=3), 8q24 (AML: n=15; MDS: n=1), 11q21-25 (AML: n=42; MDS: n=13), 13q12 (AML: n=3; MDS: n=1), 13q31 (AML: n=3; MDS: n=2), 19p13 (AML: n=2; MDS: n=4), and 21q21-q22 (AML: n=24; MDS: n=5). The median number of amplified regions was 3 (range 1-18). In 14/115 (12.2%) cases, the amplification was located in dmins (AML: n=11; MDS: n=3) and in 101/115 (87.8%) patients in HSR (AML: n=73; MDS: n=28). In 40 of the latter 101 cases (39.6%) (AML: n=24; MDS: n=16) the amplification was located on a ring chromosome (rc). In patients with complex karyotypes we detected a significantly higher number of amplified regions as compared to non-complex karyotypes (3.5 vs. 2.8; p=0.015). No association between the complexity of the karyotype and the structural type of the amplification (dmin vs rc) was observed. Cases with non-complex karyotypes frequently harbored a 5q deletion (6/23; 26.1%) or chromosome 8 abnormalities (3/23; 13.0%). Within the subgroup of non-complex karyotypes del(5q) cases showed a tendency to a higher number of amplified regions (3.6 vs. 1.9; p=0.140). Further, amplifications of 11q genes were more frequent in complex karyotypes (54.4% vs. 21.7%; p=0.005), whereas 8q amplifications were more frequent in non-complex karyotypes (43.5% vs. 4.4%; p 〈 0.001). We detected a large region on band 11q24, which was amplified in 41/53 (77.4%) cases. This commonly amplified region contains 1,575 genes including the MLL gene. Cases harboring dmins had shorter amplified regions compared to cases with rc (4,428,112.5 bp vs. 18,265,496.9 bp; p=0.028). Moreover, we detected a positive correlation of patients having a rc and gene amplification on chromosome 11q23-25 (p 〈 0.05). On chromosome 3q, 8/9 (88.9%) cases shared a minimal amplified region covering the EVI1 gene. In comparison to samples obtained from healthy donors (n=47), the EVI1 expression was significantly higher in cases with EVI1 amplification (87.4 vs. 0.5; p=0.048). On chromosome 21q the regions of amplifications were heterogeneous. However, we detected a minimal region containing 11 genes including ERG which was amplified in 26/29 (89.7%) patients. ERG expression data was available in 8 cases and was significantly higher compared to a control cohort of AML with normal karyotype (n=331) (729.2 vs. 229.0; p=0.05). On chromosome 8 an amplified region was identified in 15/16 cases. In 14 of these cases (87.5%) the region included MYC. TP53mut were present in 93/115 (80.9%) patients, accompanied by a TP53del in 28/93 (30.1%) cases. Interestingly, cases harboring a TP53mut had more amplified regions compared to TP53wt (3.4 vs. 1.7; p 〈 0.001). Conclusions 1. MLL is the most frequently amplified gene in AML and MDS. 2. Patients with complex karyotypes or TP53mut harbored more amplified regions compared to patients with non-complex karyotypes and TP53wt. 3. Amplifications on 11q were more frequent in complex karyotype whereas gene amplifications on 8q were predominantly observed in non-complex karyotypes. 4. EVI1 and ERG gene amplifications lead to a higher expression of the respective genes. Disclosures: Roller: MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.3724.3724
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 5
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    Next Generation Sequencing-Based Molecular Dissection Of Lineage-Specific Mutational Hierarchies In Oligoclonal Primary and Xenografted Myelodysplasia
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 519-519
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 519-519
    Abstract: The underlying molecular defects in myelodysplastic syndromes (MDS), which are a heterogeneous group of malignant clonal hematologic disorders, are not well understood. Recently, next generation sequencing (NGS) based whole genome and exome sequencing highlighted the oligoclonal nature of persistent MDS clones that are present already at early disease stages. The reconstruction of mutational hierarchies in MDS clones and distinction of primary founder from subsequently acquired lesions has yet to be thoroughly interrogated and is likely to aid dissecting the molecular pathogenesis of MDS. Methods An amplicon-based NGS assay using the Roche 454 GS Junior system was established within the IRON-II framework study in order to screen for 17 commonly mutated genes in MDS. Genomic DNA from purified mononuclear bone marrow (BM) cells of 23 MDS IPSS low/int1 risk subjects was screened for somatic mutations. Called variants were compared to dbSNP and COSMIC database entries to rule out germline polymorphisms. In addition, copy number variation analysis was performed by Affymetrix SNP 6.0 array profiling. Custom pyrosequencing assays and interphase-FISH were applied for sensitive quantification of lesion burdens in FACS-sorted myeloid, erythroid, lymphoid and stem/progenitor cells. These were isolated from patients’ primary BM as well as their long-term engrafted human xenotransplants using our recently established MDS xenograft model. Results In this work, we identified 12 oligoclonal BM samples with ≥2 molecular lesions. Of note, varying frequencies of individual mutations between different sorted cell subsets from primary or human xenografted BM support the notion that distinct MDS (sub-)clones from these subjects contributed to hematopoiesis simultaneously and lead to differential engraftment between xenografts. Comparison of variable subset-specific mutation burdens allowed deciphering the individual hierarchical architecture of the mutational landscape from 9 individuals. ASXL1, SF3B1 and SRSF2 were detected as a primary lesion for 2 patients each. In contrast, large-scale genomic alterations such as del(5q), del(RUNX1) or trisomy 8 occurred as late-end lesion or even defined distinct clones which coexist with others harboring different mutations as detected for 2 subjects. Surprisingly, CD19+ and CD3+ lymphocytes from primary and/or xenografted BM displayed significant mutational burden of at least 1 mutation in 50% of the MDS cohort (5/10). Moreover, mutations were detected simultaneously in lymphocytes (hCD19+) as well as myeloid (hCD33+) and erythroid (hCD235a+) cells from three xenografted samples indicating a potent multilineage engraftment capability of MDS hematopoietic stem cells. Interestingly, one individual presented with high RUNX1 mutational frequency in the primary early progenitor fraction (CD34+CD38+), which was absent in the stem-cell enriched fraction (CD34+CD38-), whereas TET2, ZRSR2 and ASXL1 mutations were detected in both fractions and their xenografts. Intriguingly, only xenotransplantation of primary CD34+38- BM cells lead to long-term engraftment of RUNX1 wild type human BM cells in mice, while CD34+CD38+ BM cells gave rise to short term engraftment of RUNX1 mutated human BM cells indicating that mutated RUNX1might originate in a more committed progenitor fraction with limited self-renewal potential. Conclusion Molecular characterization of oligoclonal mutation patterns in primary and xenograft BM allowed the establishment of individual mutational hierarchies and indicates a relatively random order in the mutational evolution of MDS clones, although spliceosome mutations appear as rather early events. Furthermore, our analysis revealed engraftment of independent MDS clones in different mice xenografted with the same subject material, which opens the door to the in vivo study of isolated clones with respect to their pathomechanisms and response to treatment. Our data also suggests that the occurrence of large-scale genomic aberrations is frequently preceded by small-scale gene mutations, emphasizing their potential role in disease diagnosis and risk stratification. Finally, detection of MDS specific mutations in the lymphocytic compartment might be involved in facilitating impaired immune functionality and needs to be investigated prospectively. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Staller:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.519.519
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 6
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    Sequential Evolution Versus a Single Catastrophic Event (Chromothripsis) in the Pathogenesis of AML with Complex Aberrant Karyotype
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 517-517
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 517-517
    Abstract: Abstract 517 Background: In AML, the concept of gradual evolution through a sequence of genetic alterations and clonal expansion was favored thus far, but has been recently challenged by a hypothesis that one catastrophic event generates multiple lesions across the genome in a single step. The term “chromothripsis” was introduced for a single catastophic event leading to the shattering of a single chromosome followed by rejoining and thereby resulting in a highly recombined chromosome (Stephens PJ et al., Cell 2011). Aim: We addressed the question whether AML with complex karyotype - defined as 4 or more abnormalities - evolves by sequential gradual acquisition of chromosome abnormalities or by a single catastrophic event. Patients and Methods: We selected 889 AML cases (de novo: n=634, secondary AML: n=164, therapy-related AML: n=91) presenting a complex karyotype at diagnosis. These were analyzed by chromosome banding analysis, 24-color-FISH, interphase-FISH, array CGH (n=78) and TP53 mutation analysis (n=195). Results: In 518/889 (58.3%) cases at least one subclone was observed that showed extra chromosome aberrations, thus demonstrating clonal evolution already at the timepoint of AML diagnosis. Within these, 77/518 (14.9%) cases showed a primary clone with only one cytogenetic abnormality. Two of these were recurrent single abnormalities: del(5q) (n=62), +8 (n=4). Clonal evolution was more frequent in cases with del(5q) as compared to those without (404/666 (60.7%) vs 117/223 (52.5%); p=0.034) while no association with loss of 7q, loss of 17p, TP53 mutation or type of AML (de novo vs secondary vs therapy-related AML) was observed. In 46 cases which evolved from MDS (n=43) or MPN (n=3) chromosome banding analysis had been performed prior to the diagnosis of AML. In 21/46 (45.7%) cases karyotype had not changed while in 25/46 (54.3%) cases clonal evolution had occurred. 57 cases were analyzed at relapse of AML; in 28 (49.1%) cases clonal evolution was detected. Additionally, 78/889 cases were evaluated by array CGH. The occurrence of chromothripsis was analyzed following the definition by Rausch et al. (Cell 2012) with at least 10 segmental copy-number changes involving two or three distinct copy-number states on a single chromosome. Evidence of at least one “shattered” chromosome was found in 24/78 (30.8%) cases. In 21 cases only one chromosome fulfilled these criteria, while in 3 cases chromothripsis affected two or more chromosomes. The chromosome most frequently affected by “shattering” was chromosome 11, observed in 18 (85.7%) cases, followed by chromosomes 2 and 21, which were affected in 2 cases each. Chromosomes 1, 5, 7, 13, 15, 16 and 20 showed signs of chromothripsis in single cases only. In 19/24 (79.2%) cases showing evidence of chromothripsis a high level amplification was observed for the MLL gene (11q23) in 17 cases and for the ERG gene (21q22) in 2 cases. Thus, amplifications were more frequent than in cases without chromothripsis (21/54, 38.89%; p=0.001), while no association was observed between chromothripsis and deletions of 5q, 7q or 17p or TP53 mut, presence of clonal evolution or type of AML (de novo vs secondary vs therapy-related AML). With respect to outcome within the subgroup of AML with complex karyotype only TP53 mutations and the presence of 5q deletions were significantly associated with overall survival (relative risk (RR) for shorter OS in TP53 mut cases: 3.19, p 〈 0.0001, and in del(5q) cases: 1.61, p=0.006; median OS in TP53mut vs TP53wt cases: 4.6 vs 22.0 months, p 〈 0.0001; median OS in del(5q) vs non-del(5q) cases: 5.7 vs 14.4 months, p=0.006), while presence of deletions of 7q, or 17p, chromothripsis and clonal evolution showed no impact on outcome. In multivariable Cox regression analysis only TP53mut had an independent association with shorter OS (RR: 3.12, p=0.001). Conclusions: 1. In AML with complex karyotype harboring a 5q deletion the acquisition of additional abnormalities is more frequent than in cases without del(5q). 2. Chromothripsis does occur in AML with complex karyotype. However, the “shattering” of one chromosome was never observed as the sole abnormality, indicating that chromothripsis and sequential genetic evolution are not alternative but more likely combined mechanisms in AML. 3. Our data demonstrates that stepwise clonal evolution is more frequent than chromothripsis and thus likely to be more important in the pathogenesis of AML with complex karyotype. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.517.517
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 7
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    MDS-Derived Stromal Cells Exhibit Altered Gene Expression and Support The Engraftment Of lin-CD34+CD38- Disease-Initiating Stem Cells In a Xenograft Model Of Lower Risk MDS
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 100-100
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    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 100-100
    Abstract: Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis, dysplasia and increased risk of progression to acute myeloid leukemia. The development of targeted therapies for MDS has been lagging behind and remains a key clinical challenge that has been hampered, at least in part, by difficulties to establish in vivo model systems that recapitulate disease heterogeneity and complexity. Attempts to generate a xenograft model of lower risk MDS have only achieved low and often transient levels of engraftment. Recent evidence from mouse studies suggests that MDS is a disease in which both the hematopoietic system and the bone marrow microenvironment might be involved. Thus, we hypothesized that a specific MDS microenvironment might be required for the successful modeling of low risk MDS in mice, proposing a dependency of the “disease propagating cells“ on their corresponding niche cells in human MDS. Methods Our study is based on xenotransplantation of material from 19 MDS patients classified as follows: IPSS low risk (n=6), intermediate-1 risk (n=13), WHO 2008 classification: MDS 5q- (n=7), MDS RCMD (n=7), MDS RAEB I (n=3), MDS-U (n=1), MDS RARS (n=1). MDS CD34+ cells were co-injected with patient-derived mesenchymal stromal cells (MSCs) directly in the bone marrow cavity (i.f) of NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) or NSGS (NSG mice expressing human SCF, IL3 and GM-CSF) mice. Molecular tracking of MDS cells was carried out by copy number analysis (Affymetrix SNP 6.0 Arrays), metaphase cytogenetics, interphase FISH, Roche 454 deep sequencing and pyrosequencing of known mutations. Mice were analyzed after a minimum of 16 weeks post transplantation. Results We show that co-injection of MDS CD34+ cells with their corresponding MSCs leads to significant and long-term engraftment of over 77% of the MDS patients analyzed, both in NSG (10/13 patients, range hCD45+= 1-18%) and NSGS mice (7/8 patients, range hCD45+=2.2-74%). In contrast, absence of MSCs or co-injection of healthy age-matched MSCs only gave rise to limited engraftment in NSG mice (2/7 patients (hCD45+=1-3.8%) and 1/2 patients (hCD45+=2%), respectively). Transplanted samples exhibited a clear myeloid bias and significant engraftment of cells with progenitor (CD34+CD38+) and stem cell phenotype (CD34+CD38-) that could be serially transplanted. In addition, presence of morphologically dysplastic cells was readily detectable in NSGS mice. Importantly, molecular analysis of the engrafted cells confirmed their “diseased” origin as they carried identical lesions to the ones present in the original MDS patient. Furthermore, we could demonstrate that disease-propagating stem cells in lower risk MDS exclusively reside within the lin-CD34+CD38- stem cell fraction. Finally, RNA sequencing analysis comparing MDS and age-matched healthy control MSCs revealed altered expression of key genes involved in cellular adhesion, extra-cellular matrix (ECM) remodeling and cellular cross-talk in diseased MSCs, strongly supporting the notion of a complex interplay between MDS hematopoietic cells and their corresponding stroma. In addition, patient MSCs exhibited clear molecular features of fibrosis, a clinical feature often associated with MDS. Conclusion In this study we have identified patient-derived MSCs as a critical functional component of lower risk MDS. Together with MDS stem cells, these patient MSCs form a functional stem cell-niche unit, which allows the propagation of the disease in a xenograft recipient. The striking changed expression in diseased MSCs of genes involved in processes like cytokine-cytokine receptor interaction, cellular adhesion, ECM remodeling as well as hypoxia further suggests that diseased MDS cells might alter the function of the normal HSC niche into one that can support the requirement of MDS cells. Studying the interaction of MDS stem cells and MSCs at the cellular and molecular level will provide a platform for unraveling the molecular basis of clonal dominance in MDS as well as allow the design of targeted strategies aimed to disrupt the MDS stem cell-MSC niche interactions. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.100.100
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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