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.

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.

Characterization of CMML with Normal Karyotype in Comparison to CMML with Aberrant Karyotype

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1674-1674
Author(s):  
Claudia Haferlach ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Chromosome Banding ◽  
Array Cgh ◽  
Negative Impact ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Monosomy 7 ◽  
Cytogenetic Abnormalities ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Background: CMML is a myelodysplastic/myeloproliferative neoplasm with distinct morphological and genetic features. Based on differences in blast count CMML is divided into CMML-1 (<1% blasts in the peripheral blood (pB) and <10% in the bone marrow (BM)) and CMML-2 (5-19% blasts in pB, 10-19% in BM or presence of Auer rods). Clonal cytogenetic abnormalities are detected in only 20-40% of patients by chromosome banding analysis (CBA) while >90% of patients harbor at least one molecular mutation. The most frequent cytogenetic abnormalities include abnormalities of chromosome 7, trisomy 8 and complex karyotype. The genes most frequently mutated in CMML are TET2, ASXL1 and SRSF2. Aims: 1. Evaluate the frequency of submicroscopic gains and losses of chromosomal material as well as copy neutral loss of heterozygosity (CN-LOH) in CMML with normal karyotype in chromosome banding analysis (CBA). 2. Analyze the association of these lesions with molecular mutations and impact on survival. Patients and Methods: 69 patients with CMML-1 and 31 with CMML-2 and normal karyotype by CBA were evaluated by array CGH (SurePrint G3 ISCA CGH+SNP, Agilent, Waldbronn, Germany). 32 patients were female, 68 male, median age was 75 years (range: 50-89 years). These were compared to 41 cases with aberrant karyotype by CBA. Patients were screened for mutations (mut) in ASXL1, CBL, DNMT3A, EZH2, JAK2 V617F, KITD 816, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, and U2AF1. Results: In 35 cases (35%) with normal karyotype by CBA 46 abnormalities were detected by array CGH (CGHpos). These were 6 gains, 17 losses and 23 CN-LOH. No recurrent gain was observed, while recurrent losses of 4q24 (n=2, including TET2) and of 13q14 (n=2) were identified. CN-LOH was recurrently observed on 4q (n=6, including TET2), 11q (n=5, including CBL), 17q (n=4) and 7q (n=2). Mutations were identified at the following frequencies: TET2: 77% (74/96), SRSF2: 56% (54/97), ASXL1: 48% (46/96), RUNX1: 20% (20/98), CBL: 15% (15/97), KRAS: 12% (12/97), JAK2 V617F: 10% (10/98). The following genes were mutated in <10%: NRAS, SETBP1, EZH2, U2AF1, KIT D816, SF3B1, DNMT3A. 85 patients were analysed for all mutations. In median 3 mutations were identified per patient (range 0-6), while only in 1 patient no mutation was detected. 4/5 (80%) cases with 11q CN-LOH harbored a CBL mut and 7/8 (88%) cases with CN-LOH 4q or 4q24 deletion harbored a TET2 mut, indicating that these two gene mutations might contribute in homozygous manner to pathogenesis. NRAS mut were significantly less frequent in CMML CGHpos compared to CGHneg (0% vs 14.3%, p=0.024). Mutations in ASXL1 and RUNX1 frequently occurred together: 35% of ASXL1 mut cases also carried a RUNX1 mut as compared to 8% of ASXL1 wild-type cases (p=0.002). All 7 SETBP1 mut cases also carried an ASXL1 mut (p=0.04) Patients with CGHneg (n=65) and CGHpos (n=35) were compared to 41 cases with aberrant karyotype by CBA. While TET2 mut were detected at comparable frequencies in CGHneg and CGHpos patients (80% and 71%) they were significantly less frequent in CMML with aberrant karyotype (54%, p=0.021). On the other hand SETBP1 mut were more frequent in CMML with aberrant karyotype as compared to CGHpos and CGHneg (21%, 7%, 9%, p=0.08). A distinct mutation profile was identified in 9 patients with monosomy 7 who showed ASXL1 mut in 78%, SETBP1 mut in 75%, CBL mut in 33% and TET2 mut in only 22%. In CMML-2 RUNX1 mut were more frequent than in CMML-1 (33% vs 12%. p=0.008). No differences in overall survival (OS) were observed between patients with CGHneg, CGHpos and aberrant karyotype. However, Cox regression analyses revealed a negative impact on OS for ASXL1 mut (relative risk (RR): 2.4, p=0.027), RUNX 1mut (RR: 2.5, p=0.025) and CMML-2 (RR: 2.2, p=0.02). Conclusions: 1. 35% of CMML cases with normal karyotype based on chromosome banding analysis harbor abnormalities detectable by array CGH. 2. Prognosis in CMML is determined by the molecular mutation profile, cytogenetic abnormalities play a minor role. 3. Mutations in ASXL1 and RUNX1 are associated with a negative impact on survival. 4. The poor prognosis described for monosomy 7 seems to be due to a high frequency of ASXL1 und SETBP1 mutations. 5. Inferior outcome in CMML-2 might be due to a higher frequency of RUNX1 mutations. 6. In CMML a molecular work up including screening for mutations in ASXL1 and RUNX1 provides more relevant prognostic information than chromosome banding analysis and/or array CGH. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: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.

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.

scholarly journals Array Based Comparative Genomic Hybridization Detects Copy Number Alterations in 80.3% of Adult Acute Lymphoblastic Leukemia (ALL) with Normal Karyotype or Failed Chromosome Banding Analysis and Identifies a Subset with Only Submicroscopic Alterations Associated with Favorable Outcome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2409-2409
Author(s):  
Claudia Haferlach ◽  
Melanie Zenger ◽  
Torsten Haferlach ◽  
Wolfgang Kern ◽  
Susanne Schnittger
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Normal Karyotype ◽  
Chromosome Abnormalities ◽  
Comparative Genomic ◽  
Copy Number Alterations ◽  
Employment Equity ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Background: Based on chromosome banding analyses (CBA) more than 70% of ALL harbor chromosome abnormalities. These include balanced translocations and a large spectrum of deletions and gains. In up to 10% of ALL CBA fails. In approximately 20% of cases no chromosome abnormalities are detected. This might be due to the fact that ALL blasts failed to proliferate in vitro, to the submicroscopic size of alterations or to a truly normal karyotype. Aim: To characterize the subset of ALL with normal karyotype or failed CBA using array based comparative genomic hybridization (aCGH) and to evaluate whether this technique can provide relevant information in the diagnostic work-up and prognostication of ALL. Patients and Methods: Out of a total of 757 adult ALL patients (age 18.0-91.4 yrs, median 52.5 yrs) analyzed at diagnosis between 2005 and 2014 we selected a subset of 190 cases with normal karyotype (n=144; 75.8%) or failure of CBA (less than 11 analyzable metaphases without clonal chromosome abnormalities, n=46; 24.2%). All cases were analyzed by aCGH (12 x 270 K microarray slides, Roche Nimblegen, Madison, WI). In addition, data on FISH or PCR for BCR-ABL1 and MLL rearrangements was available in 170/190 pts. Results: 92 cases were classified as B-lineage ALL, 46 as T-lineage ALL and 14 showed a Burkitt phenotype. For 38 pts no data on ALL immunophenotype was available. Out of 170 pts investigated by FISH or PCR 21 (12.4%) pts were positive for BCR-ABL1 and 3 (1.8%) pts showed an MLL rearrangement. All 14 pts with a Burkitt phenotype showed a MYC-rearrangement by FISH. In 12 cases (6.3%) aCGH analyses failed due to poor DNA quality. By aCGH 143/178 (80.3%) pts harbored an aberrant karyotype while only 35 showed no copy number alteration (19.7%). 785 copy number alterations were observed in 143 pts (mean 5.5 per case; range: 1-47). 292 were whole chromosome gains (n=164) or losses (n=128) while 493 were alterations affecting certain chromosome regions. Losses of chromosomal regions were more common than gains (333 vs 160). 253 gains and 222 losses, i.e. 60.5% of all affected chromosomes/chromosomal regions, were larger than 10 Mbp. This size is above the resolution of CBA and thus we concluded that these aberrations were missed by CBA due to lack of proliferation of ALL blasts in vitro. 239 losses and 71 gains were smaller than 10 Mbp and thus are not detectable by CBA. In 40 pts (22.5%) only submicroscopic alterations were detected. Most frequent alterations observed by aCGH were: Losses of 9p21 (CDKN2A) (n=58; 32.6%), 6q (n=21; 11.8%), 13q14 (RB1) (n=21; 11.8%), 7q34 (TCRB) (n=21; 11.8%), 12p13 (ETV6) (n=14; 7.9%), 7p12 (IKZF1) (n=13; 7.3%), 14q32 (IGH) (n=8; 4.5%), 5q33 (EBF1) (n=7; 3.9%), 10q23 (GRID1, PTEN) (n=6; 3.4%), 3p (n=6; 3.4%) as well as gains of 1q (n=14; 7.9%). Deletions of 5q33, 6q, 7p12, 7q34, 9p21, 10q23, 12p13 and 13q14 were observed in both B- and T-cell precursor ALL, respectively. In contrast, losses and gains of whole chromosomes, gains of 1q and 14q32 deletions were only detected in pts with B-cell precursor ALL. In the subset of 40 pts harboring only submicroscopic abnormalities the most frequently affected regions were loss of 9p21 (CDKN2A) (40.0%), 14q32 (IGH) (10.0%), 7q34 (TRBV) (10.%), 13q14 (RB1; RCBTB2) (7.5%), 21q22 (KCNJ15) (7.5%). No recurrent gain was identified. Clinical follow-up data was available for 95 pts. Based on aCGH 12 cases with a typical pattern of chromosome losses characteristic for the ALL subset with a low hypodiploid karyotype were detected. As has been described previously for this group they showed a dismal outcome with a median OS of only 5.3 months. The relative risk for death compared to all others amounted to 4.5 (p=0.047). There was a tendency for better OS in patients showing only submicroscopic abnormalities by aCGH, OS at 3 years was 83.6% compared to 60.5% in all others (p=0.09). Conclusions: 80.3% of ALL with normal karyotype in chromosome banding analysis or failed cytogenetics habor copy number alterations detectable by array CGH. The pattern of lost and gained chromosomal regions is comparable to the alterations most frequently occurring in ALL. 12 patients (6.7%) with a characteristic low hypodiploid karyotype were detected, who showed the known poor outcome. A novel subset of 22.5% of pts was identified showing submicroscopic copy number changes only and a favorable outcome. Thus, nearly a third of the target population can be further classified by aCGH for better prognostication. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger: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 Next-Generation Sequencing Of 2,761 Genes Detects Copy Number States and Molecular Mutations In a Single Approach

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1371-1371
Author(s):  
Alexander Kohlmann ◽  
Andreas Roller ◽  
Sandra Weissmann ◽  
Sabrina Kuznia ◽  
Melanie Zenger ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Copy Number ◽  
Chromosome Banding ◽  
Array Cgh ◽  
Copy Number Alterations ◽  
Employment Equity ◽  
Targeted Exome Sequencing ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Introduction In acute myeloid leukemia (AML), the karyotype and molecular mutation profile are the strongest determinants for prognosis and biological subclassification. Yet, diagnostic analyses rely on chromosome banding technique and sequencing of a constantly growing number of genes. Aims In an era of novel high-throughput sequencing assays becoming viable options for diagnostic implementation we aimed to evaluate whether the application of targeted exome sequencing can reliably identify copy number states and molecular mutations in a single-step procedure. Patients and Methods The pilot cohort included four AML cases with a complex karyotype with known chromosomal alterations as detected by chromosome banding analysis, 24-color FISH and array CGH (12x270K microarrays, NimbleGen, Madison, WI). The size of the aberrant clone was determined by suitable probes using interphase-FISH on bone marrow smears. For sequencing analysis genomic DNA was extracted from mononuclear cells and 50 ng were processed using the TruSight Rapid Capture kit (Illumina, San Diego, CA). Sequencing was performed on a MiSeq instrument using the 2x150 bp paired-end read chemistry targeting a subset of the human exome (2,761 genes; 37,366 exons). This exome enrichment library contained >50,000 probes (7.75 Mb) focusing on disease-causing variants in specific inherited conditions (Illumina). Data analysis was performed applying default settings of the on-board MiSeq Reporter Software version 2.2.29 using the Burrows-Wheeler Aligner to align the reads against the hg19 reference genome. Further processing to delineate copy number states was performed using the ExomeCNV package. Results Each patient was analyzed in a single MiSeq run and in median 22,022,240 (range 19,233,134 - 23,507,016) reads were generated. The median coverage per target region was in the range of 74-186 reads. Coverage uniformity was assessed according to the manufacturer's recommendations. Over 98% of bases were covered at 0.12X mean coverage for each sample. Next, two data analysis pipelines were triggered, i.e. copy number states and mutation analysis. With respect to copy number alterations (CNA), in total 65 CNA were detected by chromosome banding analysis/array CGH. Of these, 21 were gains, 44 were losses. The size of the deletions ranged between 378,377 and 141,048,720 bp (median 10,731,680 bp), the size of the gains ranged between 281,608 and 46,404,876 bp (median 4,947,125 bp), respectively. In total, 63/65 (96.9%) copy number alterations were correctly identified by targeted exome sequencing. The NGS assay was able to detect copy number alterations that were present in only 23% of cells as determined by interphase-FISH. In detail, one of the deletions was homozygous with a larger deletion on the long arm of chromosome 17 (size: 1,070,162 bp) and a small intragenic deletion within the NF1 gene. This homozygous deletion was detected by array-CGH and by exome sequencing. Interestingly, the higher resolution of the exome sequencing assay in this area enabled the exact localization (exons 37 to 58) and size determination (78,415 bp) of the deletion. Overall, only 2 gains escaped detection. These were two small gained regions on a highly rearranged chr. 19. Secondly, with respect to mutation analysis, the same assay detected 19, 20, 21 and 28 mutations in the four analyzed patients. This pipeline took only putative variants into account that were not present in the control sample, were having a coverage ≥30 reads with a mutation load ≥10%, and had a confirmed COSMIC mutation entry (v66). 12/2,761 (0.4%) genes harbored mutations in at least 2/4 patients. This included genes known to be involved in leukemogenesis. TP53 mutations were detected in all four cases and all were confirmed by Sanger sequencing. Conclusions A targeted exome sequencing assay allowed to robustly assess copy number states in AML at diagnosis at a resolution greater than current conventional array CGH analyses. Moreover, exome sequencing read data also can be used to delineate mutation profiles. Thus, this workflow enabled to call gene mutations and copy number states in a single assay and is a promising option for a routine diagnostics assay in the future. The gene panel has to be further optimized by adding genes known to be mutated in hematological malignancies. More data is necessary to precisely determine the detection limit and to optimize software tools for a routine use. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Zenger: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.

Interphase FISH and Comparative Genomic Hybridization Performed in Addition to Chromosome Banding Analysis in AML with Normal Karyotype Detect Prognostically Relevant Chromosome Abnormalities.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2016-2016 ◽  
Author(s):  
Claudia Schoch ◽  
Mirjam Klaus ◽  
Susanne Schnittger ◽  
Wolfgang Hiddemann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Comparative Genomic Hybridization ◽  
Chromosome Banding ◽  
Normal Karyotype ◽  
Trisomy 13 ◽  
Comparative Genomic ◽  
Genomic Hybridization ◽  
Interphase Fish ◽  
Banding Analysis ◽  
Chromosome Banding Analysis

Abstract In AML karyotype abnormalities are not detected in 40 to 45% of cases using classical chromosome banding analysis. For several reasons false negative results might occur in chromosome banding analysis: 1. no proliferation of the aberrant clone in vitro, 2. low resolution due to technical problems or limitations of the method itself, 3. real cryptic rearrangements. In order to determine the proportion of “false negative” karyotypes by chromosome banding analysis we conducted a study using interphase-FISH and comparative genomic hybridization in addition to chromosome banding analysis. In total, chromosome banding analysis have been performed in 3849 AML at diagnosis. Of these 1748 showed a normal karyotype (45.4%). Out of these in 3 cases cytomorphology revealed an APL and in 2 cases an AML M4eo. Using interphase FISH with a PML-RARA or CBFB probe we detected cryptic PML-RARA or CBFB-rearrangements, respectively, in all 5 cases, which were cytogenetically invisible due to submicroscopic insertions. 480 cases of AML with normal karyotype were analyzed for MLL gene rearrangements using FISH with an MLL-probe. 11 cases with a cryptic MLL-rearrangement were detected (FAB-subtypes: M5a: 7, M2: 2, M0: 2). In 273 patients interphase-FISH screening with probes for ETO, ABL, ETV6, RB, P53, AML1 and BCR was performed. In 6 out of 273 (2.2%) pts an abnormality was detectable. In two cases the aberrant clone did not proliferate in vitro: 1 case each with monosomy and trisomy 13. Due to limitations of resolution in chromosome banding analysis translocations or deletions of very small chromosome fragments were only detected with FISH in n=4 cases (ETV6 rearrangements: t(11;12)(q24;p13), t(12;22)(p13;q12), ETV6 deletions: del(12)(p13), n=2). Like interphase-FISH comparative genomic hybridization (CGH) does not rely on proliferating tumor cells but in contrast to interphase-FISH allows the detection of all genomic imbalances and not only of selected genomic regions. Therefore, we selected 48 cases with normal karyotype and low in vitro proliferation (less than 15 analyzable metaphases in chromosome banding analysis). In 8 of 48 cases (16.7%) an aberrant CGH-pattern was identified which was verified using interphase-FISH with suitable probes. In 3 cases a typical pattern of chromosomal gains and losses observed in complex aberrant karyotypes was detected. In one case each a trisomy 4 and 13 was observed, respectively. In one case trisomy 13 was accompanied by gain of material of the long arm of chromosome 11 (11q11 to 11q23). One case each showed loss of chromosome 19 and gain of the long arm of chromosome 10, respectively. In conclusion, CGH in combination with interphase-FISH using probes for the detection of balanced rearrangements is a powerful technique for identifying prognostically relevant karyotype abnormalities in AML assigned to normal karyotype by chromosome banding analysis. Especially this is true in cases with a low yield of metaphases and in AML with a high probability of carrying a specific, cytogenetically cryptic fusion-gene. Thus, in these cases interphase-FISH and CGH should be performed in a diagnostic setting to classify and stratify patients best.

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.

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.

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