Acute Erythroid Leukemia (AEL) Can Be Separated Into Distinct Prognostic Subsets Based On Cytogenetic and Molecular Genetic Characteristics

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1394-1394
Author(s):  
Vera Grossmann ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Ulrike Bacher ◽  
Franziska Poetzinger ◽  
...  
Keyword(s):  
Chromosome Banding ◽  
Cox Regression ◽  
Mutation Screening ◽  
Intermediate Risk ◽  
Complex Karyotype ◽  
Cgh Array ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
N 93

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.

Cytogenetic Clonal Evolution in MDS Is Associated with Shifts towards Unfavorable Karyotypes According to IPSS and Shorter Overall Survival: A Study on 988 MDS Patients Studied Sequentially by Chromosome Banding Analysis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 968-968 ◽  
Author(s):  
Claudia Haferlach ◽  
Melanie Zenger ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chromosome Banding ◽  
Cox Regression ◽  
Clonal Evolution ◽  
Employment Equity ◽  
Cox Regression Analysis ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
Acquired Abnormalities

Abstract Abstract 968 Background and Aim: The karyotype is one of the most important prognostic factors in MDS with respect to survival and evolution to AML and may change during the course of the disease. The aim of this study was to evaluate 1. the frequency of acquisition of additional chromosome abnormalities during the course of the disease (clonal evolution), 2. the pattern of acquired genetic abnormalities, 3. the association of karyotype at diagnosis and clonal evolution and 4. the impact of clonal evolution on transformation to AML and overall survival (OS). Patients and Methods: 988 MDS patients were evaluated by chromosome banding analysis (CBA) during the course of their disease. According to IPSS 729 (73.8%) cases showed a favorable karyotype, 146 (14.8%) patients an intermediate karyotype and 113 (11.4%) cases an unfavorable karyotype at first investigation. Progression to AML occurred in 180 of 988 patients during follow-up. Results: 2,454 chromosome banding analyses were performed in 988 cases (mean: 2.48 per case, range: 2–9). The median time between the first and the last evaluation was 12.5 months (range 1–60.6 months). Overall, in 171 of 988 patients (17.3%) clonal evolution was observed. Clonal evolution was detected between 1 and 56 months (median 14.3 months) after first evaluation and occurred later in patients with favorable than in patients with intermediate or unfavorable karyotype (mean 19.8 mo vs 15.5 mo vs 10.5 mo, favorable vs intermediate p=0.07, intermediate vs unfavorable p=0.05 and favorable vs unfavorable p<0.001). The abnormalities most frequently acquired during the course of the disease were +8, 7q−/−7, and gain of 21q detected in 29 cases each, followed by loss of 12p (n=22), 5q (n=14), 17p (n=19), and 20q (n=12). Other recurrently acquired abnormalities were +13 (n=12), +1q (n=12), +3q (n=12), −3q (n=10). Clonal evolution was strongly associated with cytogenetic IPSS category: Clonal evolution occurred in 100/729 cases with upfront favorable cytogenetics (13.7%), in 32/146 patients (21.9%) with upfront intermediate cytogenetics, but in 39/113 cases (34.5%) with upfront unfavorable cytogenetics (p<0.001). In 100 patients with favorable cytogenetics and clonal evolution karyotype was intermediate at second evaluation in 43 cases (43%), unfavorable in 25 cases (25%) and stayed favorable in the remaining 32 patients (32%). In 32 patients with intermediate cytogenetics and clonal evolution karyotype shifted to unfavorable at second evaluation in 11 cases (34.4%) and stayed intermediate in 21 patients (65.6%). Progression to AML was more frequent in patients with clonal evolution as compared to patients without (52/171 (30.4%) vs 128/817 (15.7%); p<0.001). In Cox regression analysis the IPSS karyotype at first evaluation, the IPSS karyotype at second evaluation, clonal evolution and progression to AML were associated with OS (relative risk: 2.12, 2.15, 1.87, and 6.6; p<0.001, p<0.001, p=0.011, p<0.001, respectively). In multivariate Cox regression analysis the IPSS karyotype at second evaluation and progression to AML were independently associated with shorter OS (relative risk: 2.0, and 6.1; p=0.013, p<0.001, respectively). Clonal evolution was associated with shorter OS (median 130.4 months vs not reached, OS at 5 years 72.3%vs 82.9%, p=0.01). Also in the subset of patients without transformation to AML outcome was inferior in patients with clonal evolution as compared to those without clonal evolution (OS at 5 years 78.2% vs 83.0%, p=0.05). Conclusions: 1. Clonal evolution was observed in 17.3% of patients with MDS. 2. The pattern of acquired abnormalities resembles the pattern observed in MDS at primary evaluation. 3. A higher frequency of clonal evolution and a shorter time to clonal evolution is observed in higher cytogenetic IPSS scores determined at first evaluation. 4. Clonal evolution is significantly associated with transformation to AML and shorter OS. 5. Sequential cytogenetic analyses allow the identification of subsets of MDS patients with a higher risk for transformation to AML and thus might guide treatment decisions in future. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

17p Deletion Is the Most Frequent Abnormality Acquired During Clonal Evolution in Chronic Lymphocytic Leukemia (CLL): 429 Cases Analyzed by Interphase FISH and Chromosome Banding Analysis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3870-3870
Author(s):  
Claudia Haferlach ◽  
Melanie Zenger ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Median Time ◽  
Chromosome Banding ◽  
Clonal Evolution ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Mutation Status ◽  
Time Points ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Abstract 3870 Background and Aim: CLL is a chronic disease with heterogeneous clinical course. While a subset of patients requires early treatment others are followed without treatment for many years. Cytogenetic aberrations have major impact on the prognosis. The aim of this study was to evaluate 1) the frequency of gain of additional chromosome aberrations during the course of the disease (clonal evolution,CE) 2) the pattern of genetic abnormalities acquired during the CE 3) the association between genetic parameters at diagnosis and CE and 4) the impact of CE on clinical outcome. An additional aim was to compare monitoring by interphase FISH (IP-FISH) or chromosome banding analysis (CBA). Patients and Methods: Two different cohorts were evaluated: A) 363 CLL patients who were analyzed during the course of their disease at least at 2 time points by IP-FISH. In this cohort only patients were enrolled who were analyzed at each time point with the complete FISH panel using probes for 13q14 (D13S25, D13S319), 11q22 (ATM), 17p (TP53), 6q21/6q23, chromosome 12 centromer and IGH -CCND1. B) 245 CLL patients who were evaluated by CBA at least at 2 time points. 179 cases were included in both cohorts. Results: In cohort A 954 FISH analyses were performed in 363 cases (mean: 2.6, range: 2–14). The median time between the first and the last evaluation was 21.1 months (range 1.0–68.9 months). Overall, in 42 of 363 patients (11.6%) clonal evolution was observed, 9.3% of untreated and 16.8% of treated patients showed clonal evolution (p=0.05). The most frequently acquired abnormality was a 17p deletion detected in 12/42 (28.6%) cases, followed by deletion of 13q14 and 11q22 (9 cases each, 21.5%). In 6/131 (4.6%) cases with heterozygous 13q14 deletion at first analysis a homozygous 13q14 deletion was observed during follow up. In 290 of 363 the IGHV mutation status was available. An unmutated IGHV status tended to be associated with clonal evolution, 26/35 (74.3%) cases with and 147/255 (57.6%) patients without clonal evolution showed an unmutated IGHV status (p=0.067). No association between any specific abnormality detected by FISH and clonal evolution was observed. The median time between first FISH analysis and the first detection of clonal evolution was 25 months (range 2–65 months). In cohort B 618 CBA were performed in 245 cases (mean: 2.5, range: 2–8). The median time between the first and the last evaluation was 18.8 months (range 1.0–68.9 months). In 73 patients (30.0%) clonal evolution was observed. The most frequently acquired abnormality was loss of 17p detected in 26 cases, followed by deletion of 13q (n=21), and 11q (n=8). Other recurrent aberrations occurring during CE were gains of 8q (n=14), 13q (n=11), 17q (n=8), 1q (n=7), 3q (n=6), 16q (n=6), 4q (n=5), 1p (n=5), 9q (n=4), 15q (n=4), losses of 8p (n=10), 9q (n=8), 8q (n=7), 9p (n=7), 6q (n=7), 1q (n=6), 6p (n=5), 1p (n=5), 10q (n=4), 7q (n=3) and 14q32-rearrangement (n=6) with different partners (2p11, 4p16, 10p11, 2x 8q24, 19q13). In 202 of 245 patients the IGHV mutation status was available. An unmutated IGHV status was significantly more frequent in cases with as compared to patients without CE (44/62 (71.0%) vs 75/140 (53.6%), p=0.021). The median time between first CBA and the first detection of clonal evolution was 21 months (range 1–65 months). Clonal evolution was observed in 7/48 (14.6%) patients with normal karyotype, in 48/159 (30.2%) cases with non-complex aberrant karyotype and in 18/38 (47.4%) patients with complex karyotype (≥ 3 abnormalities) (p=0.04 for normal vs non-complex aberrant and p=0.056 for non-complex aberrant vs complex). For 135 of 245 cases clinical data with respect to treatment was available (45 cases with and 90 without CE). 33/45 (73%) patients with and 52/90 (57.8%) without clonal evolution had received treatment. A tendency towards a shorter overall survival was observed in patients with as compared to patients without CE detected by CBA (alive at 10 yrs 75.4% vs 93.5%). Conclusions: 1. Chromosome banding analysis detects clonal evolution more frequently than IP-FISH (30.0% vs 11.6%). 2. Clonal evolution occurs more frequently in patients with an unmutated IGHV status and an aberrant karyotype with the highest frequency in patients with complex karyotype. 3. Sequential analyses by FISH and CBA seem reasonable as especially 17p abnormalities occur frequently during the course of the disease, which impacts on treatment decisions. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

In Depth Characterization of CLL with Normal Karyotype By Array CGH and Mutation Screening

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1707-1707 ◽  
Author(s):  
Claudia Haferlach ◽  
Sabine Jeromin ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Chromosome Banding ◽  
Array Cgh ◽  
Mutation Screening ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Cytogenetic Abnormalities ◽  
Interphase Fish ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Background: CLL is characterized by a distinct pattern of cytogenetic abnormalities. The most frequent aberrations are deletions of 13q, 11q, 6q and 17p and trisomy 12. However, based on chromosome banding analysis complemented by interphase FISH no abnormalities are identified in approximately 15-20% of cases. In these cases either no cytogenetic aberrations are present or these may be missed by chromosome banding analysis (CBA) due to insufficient cell division in vitro or to too low resolution of chromosome banding analysis (10 MB). On the other hand by FISH a respective abnormality can only be detected if it is covered by the applied probe panel. Aims: 1. Apply array CGH and molecular mutation screening to characterize CLL cases in which CBA and FISH both did not reveal any cytogenetic abnormalities. 2. Determine prognostic factors in this CLL subset. Patients and Methods: Diagnosis of CLL was based on cytomorphology and immunophenotyping. All cases showed at least 15% of CLL cells. The median age was 67 years (range: 40-84, mean 64 years). Overall survival (OS) at 10 years was 81% and median time to treatment (TTT) was 8.9 years. 136 CLL patients were selected based on a normal karyotype in CBA and no abnormalities in interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), the centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH -CCND1). For all 136 patients the IGHV mutation status was determined and array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) was performed. Further, mutation analysis by DNA sequencing was performed in the following genes: TP53 (n=106), SF3B1 (n=106), MYD88 (n=83), XPO1 (n=83), NOTCH1 (n=83), FBXW7 (n=83), BIRC3 (n=45) and ATM (n=44). Results: In total 55 abnormalities were detected in 26/136 (19%) patients by array CGH. Of these 25 were deletions (size of 17 deletions was <10MB and 8 were >10MB), 23 were gains (17 <10MB; 6 >10MB) and 7 were CN-LOH (2 <10MB; 5 >10MB). The following recurrent abnormalities were identified: deletions of 13q14 (n=3); 1q42.12 (n=4), 4p16.3 (n=2), 7p14 (n=3); gains of Xp22.31 (n=2), 3q26-28 (n=2); and CN-LOH 17q (n=2). A mutated IGHV status was present in 68% of cases. Mutations were observed in SF3B1 (19%), NOTCH1 (7%), ATM (5%), XPO1 (4%), TP53 (3%), MYD88 (2%), FBXW7 (1%) and no mutation in BIRC3. Compared to a cohort of 1,115 CLL with aberrant karyotype by CBA/FISH, in the present CLL cohort with normal karyotype SF3B1 mutations were significantly more frequent (19% vs 8%, p=0.001), while TP53 mutations tended to be less frequent (3% vs 8%, p=0.07). In the 26 patients with normal karyotype by CBA/FISH but aberrant karyotype by array CGH (CGHpos) SF3B1 mutations were even more frequent than in cases with normal karyotype by both CBA/FISH and array CGH (CGHneg) (33% vs 14%, p=0.043). A mutated IGHV status was found in 71% of CGHneg patients compared to 58% of CGHpos cases (n.s.). Only age (relative risk (RR): 1.16 per decade, p=0.006) and percentage of CLL cells as determined by flow cytometry (% CLL cells) (RR: 1.36 per 10% increase) were significantly associated with OS and the impact of both parameters was independent of each other. TTT was significantly influenced by the following parameters: CGHpos (RR: 2.4, p=0.017), unmutated IGHV (RR: 4.7, p<0.0001), SF3B1 mutation (RR: 2.9, p=0.006), % CLL cells (RR: 1.32 per 10% increase, p<0.0001), and leucocyte count (RR: 1.043 per 10,000 increase, p=0.031). Multivariate Cox regression analysis revealed an independent impact on TTT for an unmutated IGHV status (RR: 4.7, p<0.0001), mutated SF3B1 (RR: 2.9, p=0.006), and % CLL cells (RR: 1.32 per 10%, p<0.0001). The median TTT was significantly shorter in patients with unmutated IGHV status and/or SF3B1 mutation (n=55) as compared to those without (n=57) (5.1 years vs not reached, p<0.0001). Conclusions: 1. CLL with normal karyotype as determined by chromosome banding analysis and FISH is characterized by a high frequency of SF3B1 mutations (19%). 2. Array CGH detects abnormalities in 19% of CLL with normal karyotype by CBA/FISH. 3. In CLL with normal karyotype by CBA/FISH a negative effect on TTT was found for the presence of any abnormalities detected by array CGH, SF3B1 mutations, an unmutated IGHV status, and the percentage of CLL cells. Thus, in younger patients the analysis of these parameters should be discussed to better define prognosis. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin: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.

Sequential Evolution Versus a Single Catastrophic Event (Chromothripsis) in the Pathogenesis of AML with Complex Aberrant Karyotype

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 517-517 ◽  
Author(s):  
Claudia Haferlach ◽  
Melanie Zenger ◽  
Marita Staller ◽  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Tp53 Mutation ◽  
Array Cgh ◽  
De Novo ◽  
Clonal Evolution ◽  
Complex Karyotype ◽  
Catastrophic Event ◽  
Single Chromosome ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

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.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1516-1516
Author(s):  
Claudia Haferlach ◽  
Melanie Zenger ◽  
Marita Staller ◽  
Andreas Roller ◽  
Kathrin Raitner ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Array Cgh ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Interphase Fish ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
Copy Number Changes

Abstract Background 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.

scholarly journals Biology and Treatment of High-Risk CLL: Significance of Complex Karyotype

2021 ◽  
Vol 11 ◽  
Author(s):  
Thomas Chatzikonstantinou ◽  
Christos Demosthenous ◽  
Panagiotis Baliakas
Keyword(s):  
High Risk ◽  
Chromosome Banding ◽  
Current Knowledge ◽  
Immune Therapy ◽  
Clinical Impact ◽  
Lymphocytic Leukemia ◽  
Complex Karyotype ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Open Issues

Several reports highlight the clinical significance of cytogenetic complexity, namely, complex karyotype (CK) identified though the performance of chromosome banding analysis (CBA) in chronic lymphocytic leukemia. Indeed, apart from a number of studies underscoring the prognostic and predictive value of CK in the chemo(immune)therapy era, mounting evidence suggests that CK could serve as an independent prognosticator and predictor even in patients treated with novel agents. In the present review, we provide an overview of the current knowledge regarding the clinical impact of CK in CLL, touching upon open issues related to the incorporation of CK in the clinical setting.

scholarly journals Abnormalities Detected By Array CGH and Fluorescence in Situ Hybridization in AML with Normal Karyotype Lacking Mutations in NPM1, CEBPA, RUNX1 and MLL Partial Tandem Duplications Are Associated with Unfavorable Outcome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1371-1371
Author(s):  
Claudia Haferlach ◽  
Kathleen Zieschang ◽  
Susanne Schnittger ◽  
Tamara Alpermann ◽  
Melanie Zenger ◽  
...  
Keyword(s):  
Chromosome Banding ◽  
Array Cgh ◽  
Normal Karyotype ◽  
Employment Equity ◽  
In Vitro Proliferation ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Impact On Survival ◽  
Equity Ownership

Abstract Background: AML is a group of genetically distinct entities which have been defined by the presence of certain recurrent, mutually exclusive genetic abnormalities such as AML-specific fusion genes (e.g. PML-RARA, RUNX1-RUNX1T1) or recurrent molecular mutations (NPM1 mutations and CEBPA double mutations (dm)). In addition partial tandem duplications within the MLL gene (MLL-PTD) and RUNX1 mutations seem to play an important role in certain AML subtypes. A subset of AML with normal karyotype lacking the above-mentioned mutations is still poorly characterized. Array CGH and fluorescence in situ hybridization are able to detect abnormalities which are undetectable by chromosome banding analysis either due to a higher resolution, ability to detect copy neutral loss of heterozygosity (CN-LOH) and independence of in vitro proliferation. Aims: 1. Search for submicroscopic copy number changes and cryptic rearrangements in AML with normal karyotype lacking NPM1 and RUNX1 mutations, CEBPA dm, and MLL-PTD. 2. Determine whether submicroscopic cytogenetic changes impact on survival. Patients and Methods: For 1473 AML cases with normal karyotype complete data on mutation status of NPM1, CEBPA, RUNX1 and MLL -PTD was available. Of these 303 cases (21%) did not carry one of these mutations. Out of these 159 cases with de novo AML (median age: 68 years (range: 19-93)) were selected on the basis of availability of material for array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and FISH screening with probes for MLL, RUNX1, CBFB, NUP98, MECOM/EVI1, NPM1, ETV6 and DEK-NUP214 (MetaSystems, Altlussheim, Germany; ABBOTT, Wiesbaden Germany). Results: In total in 67 of 159 patients (42%) abnormalities were identified by FISH and/or array CGH. In detail, 12 balanced rearrangements were detected by FISH screening involving NUP98 (n=7, in 6 of these a NUP98-NSD1 rearrangement was identified by PCR), MLL (n=2) and MECOM, RUNX1 and CBFB (one each). In addition, 27 gains, 42 losses and 41 copy neutral losses of heterozygosity (CN-LOH) were observed in 58 (37%) patients. Recurrent gains affected regions 6q23.3q23.3 (135.325.751-135.607.060) (n=2) and 8q24.21q24.21 (130.517.732-130.808.381) (n=2) while recurrent losses were found for the regions 21q22.12q22.12 encompassing the RUNX1 gene (36.228.735-36.303.952) (n=5), 5q31.2q31.2 including i.a. EGR1 and CTNNA1 (137.617.569-138.993.959) (n=3), 2q34q34 including i.a. IKZF2 (213.371.237-214.560.488) (n=2), 7q22.1q22.1 encompassing i.a. CUX1 (100.485.221-101.916.623) (n=2), and Yq11.223q12 (24.980.949-28.804.541) (n=2). Recurrent CN-LOH were observed on chromosomes 11q (n=10), 2p (n=5), 4q (n=4), 21q (n=3), 1p (n=2), 17q (n=2) and 18q (n=2). 20/27 gains and 38/42 losses were < 10 megabases in sizes and thus below the resolution of chromosome banding analysis. Only in 7 (4%) patients abnormalities (n=11) were identified which in principle are detectable by chromosome banding analysis. These were missed by chromosome banding analysis due to insufficient in vitro proliferation of the aberrant clone, small clone size or poor chromosome morphology. Comparing age, white blood cell count and bone marrow blast counts revealed no differences between patients with or without abnormalities detected by FISH and/or array CGH. However, FLT3-ITD and WT1 mutations were more frequent in cases with abnormalities (25% vs 7%, p=0.001; 9% vs 1%, p=0.021). Survival analysis was performed for 90 intensively treated patients. Overall survival (OS), event-free survival (EFS) and OS with censoring at time of allogeneic SCT (OSctx) were significantly shorter in patients with abnormalities detected by FISH and/or array CGH compared to those without (median OS: 19 vs 49 months, p=0.027; Figure 1; median EFS: 9 vs 21 months, p=0.016; median OSctx: 13 vs 26 months, p=0.018). Conclusions: 1. AML with normal karyotype based on chromosome banding analysis lacking a disease defining molecular mutation harbor balanced rearrangements, copy number gains and losses as well as CN-LOH at a high frequency (42%). 2. The presence of these abnormalities has a negative impact on survival demonstrating that FISH and array CGH can add prognostic information in the diagnostic work-up of AML with normal karyotype lacking a disease defining mutation. 3. Further investigations of mutations in genes located in regions of recurrent CN-LOH is necessary. Figure 1. Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zieschang:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Landscape of Secondary Genetic Lesions in Acute Myeloid Leukemia with Inv(16)/CBFB-MYH11

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 801-801
Author(s):  
Annette Fasan ◽  
Claudia Haferlach ◽  
Karolína Perglerová ◽  
Sonja Schindela ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chromosome Banding ◽  
Prognostic Impact ◽  
Rt Pcr ◽  
Employment Equity ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Introduction: Acute myeloid leukemia (AML) with inv(16)(p13q22) or t(16;16)(p13;q22) accounts for 5-7% of adult AML and overall is associated with a favorable outcome. However, secondary genetic lesions have been shown to negatively impact on outcome. Aims: To assess the frequency and clinical impact of additional mutations and chromosomal aberrations in AML with inv(16)/CBFB-MYH11. Patients: We analyzed 138 patients (pts) who were referred to our laboratory for diagnosis of de novo AML between 2005 and 2014 (54 females; 84 males; median age 54 years, range: 20-88 years). All patients were proven to have inv(16)(p13q22) or t(16;16)(p13;q22) /CBFB-MYH11 by a combination of chromosome banding analysis, fluorescence in situ hybridization and RT-PCR. All 138 samples were analyzed by next generation sequencing using a 22-gene panel targeting ASXL1, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, RAD21, RUNX1, SF3B1, SMC1A, SMC3, SRSF2, TET2, TP53, U2AF1, and WT1. Results: In total, 127 pts showed an inv(16)(p13q22), 10 pts a t(16;16)(p13;q22). One pt showed a normal karyotype with a cytogenetically cryptic CBFB-MYH11 rearrangement confirmed by RT-PCR. Using standard chromosome banding analysis, additional cytogenetic aberrations (ACA) were observed in 52 pts (38%). The most frequent secondary chromosome aberrations were +8 (15/52; 29%), +22 (15/52; 29%) and +21 (5/52; 10%). With regard to blood counts, cases with sole inv(16) had significantly elevated white blood cell counts compared to patients with inv(16) and ACA (78x109/L vs 20x109/L; p<0.001). 112/138 (81%) pts had at least one mutation in addition to CBFB-MYH11, 47/112 (42%) had at least two additional mutations (maximum: four). Most common were mutations in NRAS (35%), KIT (32%), FLT3-ITD and FLT3-TKD(20%) and KRAS (17%). Mutations in other genes (ASXL1, CBL, DNMT3A, RUNX1, SRSF2, TET2 and WT1) were found in less than 10% of cases. Comparing AML with CBFB-MYH11 withthe other core binding factor AML entity, i.e. AML with RUNX1-RUNX1T1 (Krauth et al., Leukemia 2014), the formershowed a higher incidence of additional mutations (81% vs 50%), however, the landscape of mutated genes was comparable. Solely, the frequency of ASXL1 mutations was higher in RUNX1-RUNX1T1 positive AML compared to CBFB-MYH11 positive AML (12% vs <1%). We additionally analyzed concomitant mutations in CBFB-MYH11 positive AML according to functional pathways. Mutations resulting in activated signaling (FLT3- ITDand FLT3- TKD, KRAS, NRAS, KIT) were identified in the majority of cases (n=107/138; 78%), while mutations of tumor suppressors (CBL, TP53, WT1) were detected in 18/138 cases only (13%). Mutations of myeloid transcription factors (CEBPA, RUNX1, ETV6), mutations of genes that modify the epigenetic status (ASXL1, EZH2, TET2, DNMT3A, IDH1/2 and MLL mutations), mutations of cohesin complex genes (SMC1A, SMC3 and RAD21) and spliceosome genes (SF3B1, U2AF1, SRSF2 and ZRSR2) were identified in less than 10% of cases. There was no difference in frequency and types of additional mutations between patients with inv(16) sole and those with inv(16) and ACA with the exception of WT1 mutations, which were more frequent in patients with inv(16) and ACA (8/51; 16% vs 2/84; 2%; p=0.006). Data regarding the prognostic impact of the concurrent genetic lesions, trisomy 22 and KIT mutations, in CBFB-MYH11 AML are controversial. In our cohort, survival analysis revealed no impact of trisomy 22 or concomitant KIT mutations on prognosis of CBFB-MYH11 AML. However, within patients with inv(16) sole those with concomitant KRAS mutations had a significantly worse overall survival (OS) compared to KRAS wild-type patients (2 year OS: 43% vs 23%; p<0.001). Conclusions: Secondary genetic lesions are detected in 91% of inv(16)/CBFB-MYH11 positive AML patients. NRAS mutations were the most frequent secondary lesions followed by KIT mutations, FLT3-ITD and FLT3-TKD. inv(16)/CBFB-MYH11 positive AML show high frequency of mutations resulting in activated signaling. Considering controversial studies, trisomy 22 and concomitant KIT mutations had no prognostic impact in our cohort of 132 inv(16)/CBFB-MYH11 AML cases. The only additional genetic marker with a significant adverse prognostic impact on OS was KRAS mutation. Disclosures Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Prognostic Impact of High EVI1 expression In AML Is Due to Its Close Correlation to Rearrangements Involving EVI1 or MLL, which Are Cytogenetically Cryptic In a Subset of Patients: a Study on 332 Cases.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1676-1676
Author(s):  
Claudia Haferlach ◽  
Vera Grossmann ◽  
Melanie Zenger ◽  
Tamara Alpermann ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Chromosome Banding ◽  
Normal Karyotype ◽  
Fish Analysis ◽  
Prognostic Impact ◽  
Employment Equity ◽  
Npm1 Mutation ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
The Impact

Abstract Abstract 1676 Introduction: High EVI1 expression has been proposed as a negative prognostic factor in AML. An association between high EVI1 expression and distinct cytogenetic subgroups, such as 3q26-rearrangements, MLL-rearrangements and -7/7q- have been reported. Both 3q26- and MLL-rearrangements can be difficult to detect by chromosome banding analyses or may even be cytogenetically cryptic in a subset of patients due to limited resolution. Therefore, only studies using FISH for the detection of cryptic EVI1- or MLL-rearrangements can clarify their frequencies in AML with elevated EVI1 expression. Methods/Patients:: The study cohort was composed of 332 AML cases with a) normal karyotype (NK) (n=211), b) -7/7q- (n=77), and for comparison c) 3q26-rearrangements (n=38), and d) MLL-rearrangement (n=6). In all cases EVI1 expression was investigated using quantitative PCR calculating a % EVI1/ABL1 expression. In all cases FISH for EVI1 rearrangement was performed in addition to chromosome banding analysis. Cases with high EVI1 expression were also analyzed for MLL rearrangements by FISH. Results: In the total cohort, EVI1 expression varied between 0 and 1614 (median: 21.1). The highest EVI1 expression was measured in cases with cytogenetically identified 3q26-rearrangements (range: 6.1–566.4; median: 81.9) and in AML with MLL-rearrangements (range: 46.7–831; median: 239). The EVI1 expression was significantly lower in AML with NK (range: 0–1614; median: 0.5, p<0.001) and AML with -7/7q- (range: 0.03–199; mean: 34.5; median: 10.7, p<0.001). In the subgroup of cases with NK 4 MLL-rearrangements (1.9%) were detected by FISH and subsequently verified by fusion gene specific PCR. In addition, 4 cases with cryptic EVI1-rearrangements (1.9%) were identified by FISH analysis. Further genetic analysis revealed that these were due to t(3;8)(q26;q24) (n=2) and t(3;21)(q26;q11) (n=1). In one case, the EVI1-rearrangement could not be further analyzed due to lack of material. In the -7/7q- cohort 14/77 cases (18.2%) with cytogenetically cryptic EVI1 rearrangement including 3 novel recurrent abnormalities were detected: t(3;21)(q26;q11) (n=3), inv(3)(p24q26) (n=4) and t(3;8)(q26;q24) (n=2). In 5 cases FISH analysis revealed that the 7q- was not caused by an interstitial deletion but due to an unbalanced rearrangement between chromosomes 7 and 3: der(7)t(3;7)(q26;q21). In these 5 cases high-resolution SNP microarray were performed and revealed breakpoints in the CDK6 gene and centromeric of the EVI1 gene. Further mutation screening revealed that none of the cases with EVI1- or MLL-rearrangement harboured mutations in NPM1 or CEPBA. In 254 cases clinical follow-up data was available. Different cut-off levels of EVI1 expression were tested, and a cut-off at 30% EVI1/ABL1 expression was the lowest level that had a significant impact on outcome. Separating the cohort at this cut-off into high EVI1 (n=67) and low EVI1 expressors (n=187) showed a shorter EFS in patients with high EVI1-expression (p=0.001; relative risk (RR)=1.87, median EFS 6.2 vs 15.0 months (mo)), while no impact on OS was observed. When the same analyses were performed with respect to EVI1-rearrangements we observed both a significantly shorter EFS in cases with EVI1-rearrangement (n=39) vs all others (n=215) (p=0.001; RR=2.03, median EFS 4.6 vs 15.0 mo) and a significantly shorter OS (p=0.026; RR=1.73, median OS 10.1 vs 26.3 mo). Analyzing the impact of high EVI1 expression separately in the cohort without EVI1 rearrangement revealed no impact of EVI1 expression on EFS. Conclusions: The negative prognostic impact of high EVI1 expression is strongly associated with EVI1- or MLL-rearrangements and is absent in AML without EVI1- and MLL-rearrangement. Applying FISH in addition to chromosome banding analysis we identified cryptic rearrangements in 3.8% of AML with normal karyotype and in 18.2% of AML with -7/7q-, including 3 novel recurrent cytogenetically cryptic EVI1-rearrangements. This data supports the routine performance of FISH screening for EVI1- and MLL-rearrangements in patients with normal karyotype or 7q-/-7 and without NPM1 mutation and CEPBA mutation to assign patients to the correct biologic entity. The postulated independent prognostic impact of EVI1 expression should be tested further including this laboratory workflow as these parameters may have important impact on prognosis and future treatment strategies. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Targeted RNA Sequencing Is Capable of Identifying Thus Far Unknown Partner Genes in Leukemias with Rare Translocations and Provides Important Clinical and Therapeutical Information

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2857-2857
Author(s):  
Claudia Haferlach ◽  
Niroshan Nadarajah ◽  
Manja Meggendorfer ◽  
Nadine Dicht ◽  
Anna Stengel ◽  
...  
Keyword(s):  
Precision Medicine ◽  
Rna Sequencing ◽  
San Diego ◽  
Chromosome Banding ◽  
Gene Fusions ◽  
Rt Pcr ◽  
Employment Equity ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Background: The genomic landscape of hematological malignancies has been resolved mainly based on whole exome and whole genome sequencing, primarily targeting gene mutations. Beside mutations also gene fusions function as therapeutic targets, impressively shown for e.g. BCR-ABL1 and ETV6-PDGFRB. Hence, the need for a comprehensive genetic analysis is increasing, as it is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. Aim: To test the value of targeted RNA sequencing in a routine diagnostic work up. Patients and Methods: 38 cases were selected in which rearrangements involving KMT2A (n=8), RUNX1 (n=19), ETV6 (n=9), RARA (n=1) and JAK2 (n=1) had been identified by chromosome banding analysis (CBA) complemented by FISH analysis. In all cases the partner gene could not be identified using standard methods. Targeted RNA sequencing was performed using the TruSight RNA Fusion panel (Illumina, San Diego, CA) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. Library was prepared according to manufacturer's protocol with ~50ng DNA extracted from fresh/frozen samples. This assay allows the capture of all targeted transcripts. Sequencing was performed on two NextSeq runs (Illumina, San Diego, CA) with 20 multiplexed samples including two samples with known fusions as positive control samples. Analysis was performed with the RNA-Seq Alignment App (BaseSpace Sequence Hub) using Star for Alignment and Manta for gene fusion calling with default parameters (Illumina, San Diego, CA). Results: In 22/38 cases with rearrangements involving KMT2A (n=8), RUNX1 (n=8), ETV6 (n=4), RARA (n=1) or JAK2 (n=1) this approach led to important new information: The following partner genes for KMT2A were identified: MLLT10 (n=2), MLLT1 (n=2), ITPR2, FLNC, ASXL2 and ARHGEF12. MLLT10 and MLLT1 are two of the most frequent partner genes of KMT2A, while KMT2A-ARHGEF12 fusions are rare. Fusion of KMT2A to ITPR2, FLNC, or ASXL2 have not been reported yet. Seven different partner genes were identified in RUNX1 translocated cases. These were PLAG1 (n=2), PRDM16, MECOM, ZFPM2, MAN1A2, N6AMT2, and KIAA1549L. PRDM1, MECOM and ZFPM2 have previously been described in the literature as RUNX1 partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements in CBA. The other identified partner genes have not been described so far. Interestingly, PRDM1, MECOM, ZFPM2 and the newly identified PLAG1 are all members of the C2H2-type zinc finger gene family. Four different partner genes were identified in ETV6 rearranged cases: ABL1, CCDC126, CLPTM1L, and CFLAR-AS1. Most strikingly was the identification of the ETV6-ABL1 fusion, which could not be suspected by cytogenetics as the 5' ETV6 FISH signal was located on chromosome 7. This ETV6-ABL1 fusion was confirmed by conventional RT-PCR. In an ALL patient a JAK2-PPFIBP1 fusion was identified leading to classification as a BCR-ABL1-like ALL. In an APL patient showing an ins(17;11)(q12;q14q23) in chromosome banding analysis a ZBTB16-RARA fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. All these fusions were not detectable by our routine RT-PCR analyses as these assays cover only the most frequently occurring breakpoints in fusions with known partner genes, but might miss very rare variants. For all yet undescribed fusion partners routine assays are not available. Based on the results of targeted RNA sequencing quantitative PCR assays for MRD monitoring can now be established. In 11 cases with a RUNX1 rearrangement and 5 cases with an ETV6 rearrangement no fusion transcript was identified. Further analyses will have to clarify whether in these cases no transcript was derived from the genomic rearrangement. Conclusions: 1) Targeted RNA sequencing was able to identify and characterize rare gene fusions and thus provided the basis for the design of RT-PCR based assays for monitoring MRD. 2) Targetable genetic aberrations were identified, which were not identifiable by chromosome banding analysis but would now lead to more individualized treatment. 3) Thus, targeted RNA sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques, also paving the way to precision medicine in a considerable number of patients. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Dicht:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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