scholarly journals Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

2021 ◽  
Author(s):  
Jing Ma ◽  
Xiaofei Ai ◽  
Jinhuan Wang ◽  
Limin Xing ◽  
Chen Tian ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Chromosomal Abnormalities ◽  
Normal Karyotype ◽  
Prognostic Information ◽  
Significant Difference ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Copy Number Changes ◽  
Dependent Probe

AbstractChromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndrome (MDS) patients. However, more than 50% of low-risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding analysis, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. MLPA identifies copy number changes in 16.7% of 144 MDS patients, and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

scholarly journals Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

2020 ◽  
Author(s):  
Jing Ma ◽  
xiaofei Ai ◽  
Jinhuan Wang ◽  
Limin Xing ◽  
Chen Tian ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Chromosomal Abnormalities ◽  
Normal Karyotype ◽  
Prognostic Information ◽  
Significant Difference ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Copy Number Changes ◽  
Dependent Probe

Abstract Background Chromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndromes (MDS) patients. However, more than 50% low risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. Methods To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. Results MLPA identifies copy number changes in 16.7% of 144 MDS patients and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA, versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Conclusion Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

scholarly journals Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

2020 ◽  
Author(s):  
Jing Ma ◽  
xiaofei Ai ◽  
Jinhuan Wang ◽  
Limin Xing ◽  
Chen Tian ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Normal Karyotype ◽  
Prognostic Information ◽  
Cytogenetic Aberrations ◽  
Significant Difference ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Copy Number Changes ◽  
Dependent Probe

Abstract Background In myelodysplastic syndromes (MDS), cytogenetic aberrations play an important role for classification and prognostication. However, more than 50% low risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients.Methods To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA.Results MLPA identifies copy number changes in 16.7% of 144 MDS patients and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA, versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Conclusion Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

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.

Complex Aberrant Karyotypes Are Associated with Dismal Prognosis in CLL: Chromosome Banding Analysis Provides Important Prognostic Information in CLL in Addition to Interphase-FISH

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3132-3132
Author(s):  
Claudia Haferlach ◽  
Frank Dicker ◽  
Tamara Weiss ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Chromosome Banding ◽  
Normal Karyotype ◽  
Prior Treatment ◽  
Prognostic Impact ◽  
Prognostic Information ◽  
Mutation Status ◽  
Interphase Fish ◽  
Cytotoxic Treatment ◽  
Banding Analysis ◽  
Chromosome Banding Analysis

Abstract CLL is a heterogeneous disease with a variable clinical course. Today, therapeutic decisions are based on clinical stage, prognostic information obtained by FISH analyses on interphase nuclei and determination of the IgVH mutation status. However, additional information might be obtained from chromosome banding analysis (CBA) which provides more details on genetic aberrations. Thus far, in CLL data from CBA have been scarce due to the low proliferative activity in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2 leading to a high success rate of CBA in routine diagnostics. Clinical follow-up was available in 533 CLL patients investigated in parallel with CBA and interphase-FISH with probes for the detection of trisomy 12, IGH-rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). Diagnosis of CLL was established by standard criteria based on immunophenotyping. In 463/533 cases IgVH mutation status was also available. 298 cases were analyzed at diagnosis (cohort 1), 121 during the course of their disease without prior treatment (cohort 2), 85 patients had received cytotoxic treatment prior to analysis (cohort 3) and for 29 cases no data were available with respect to prior treatment. First, we focused on the subset of patients who showed no aberrations as determined by FISH (n=120) and defined based on CBA 2 groups: normal karyotype (n=80), aberrant karyotype (n=40). No significant differences were observed with respect to OS or time to treatment (TTT). We then focused on complex aberrant karyotypes (3 or more clonal abnormalities). These are rarely found based on FISH diagnostics: we detected 22 cases (4.1%) that showed 3 or more aberrations based on FISH only as compared to 109 cases (20.5%) based on CBA. In detail, a complex aberrant karyotype was observed with comparable frequencies in the two cohorts analyzed at diagnosis (56/295, 19%) and during the course of their disease without prior treatment (22/123, 17.9%), while it was significantly more often found in the cohort analyzed after cytotoxic treatment (31/86, 36.0%; p=0.002). In both cohorts analyzed prior to any treatment patients with a complex aberrant karyotype had a significant shorter overall survival (p=0.042, HR=2.7 and p=0.003, HR=6.1). As TP53 deletions are associated with a complex aberrant karyotype and are a strong negative prognostic factor per se we analyzed the prognostic impact of complex aberrant karyotype in relation to TP53 deletions. Therefore, CLL patients analyzed at diagnosis with a complex aberrant karyotype by CBA (n=56) were subdivided into cases with TP53 deletion (n=17) versus without TP53 deletion (n=39) in FISH. TTT did not differ significantly between complex aberrant cases with or without TP53 deletion but was significantly shorter for both groups as compared to cases with 13q deletion or normal karyotype (n=135) (p=0.05 and p=0.02). Next, cases of cohort 1 with loss of 13q14 were divided based on CBA into 3 subgroups: as the sole abnormality (n=91), plus one additional abnormality (n=24), and plus 2 or more additional abnormalities (i.e. complex) (n=32). Also for these entities TTT was significantly shorter for subgroups 2 and 3 as compared to subgroup 1 (p=0.022, HR=2.5; p=0.001, HR=1.8). In conclusion, CBA allows to identify patients within the good prognostic FISH group del(13q) sole, who show a shorter TTT if additional abnormalities are identified by CBA. Even more striking, CBA defines a new subgroup of CLL with complex aberrant karyotype which shows a shorter TTT independent of the TP53 deletion status as detectable by FISH.

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.

A Distinct Pattern of Additional Aberrations and Novel Mechanisms of MLLRearrangements Are Detectable in AML with 11q23 Aberrations Using SNP Arrays

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4143-4143
Author(s):  
Claudia Haferlach ◽  
Alexander Kohlmann ◽  
Sonja Rauhut ◽  
Frank Dicker ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Microarray Data ◽  
Copy Number ◽  
Chromosome Banding ◽  
Physical Map ◽  
Distinct Pattern ◽  
Fish Analysis ◽  
Snp Microarray ◽  
Mll Gene ◽  
Banding Analysis ◽  
Chromosome Banding Analysis

Abstract Chromosomal rearrangements involving the MLL gene occur in 3–5% of adult AML. More than 50 different partner genes have been described in acute leukemia with 11q23-abnormalities. Although MLL-rearrangements per se have a high leukemic potential, additional genetic aberrations occur. This study was intended to decipher MLLrearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP 6.0 microarray analyses were performed in 47 newly diagnosed AML with 11q23 aberrations. First, as a proof of principle, all gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included recurring gains of whole chromosomes; 4 (n=3), 8 (n=7), and 19 (n=2). In addition, the following unbalanced abnormalities were detected: gain of 1q31.3 to 1q43 (n=5) and a gain of 3q (n=2). In 40/47 cases the following partner genes had been identified based on the translocation observed in chromosome banding analysis and RTPCR: AF9 (n=27), AF6 (n=4), AF10 (n=3), ELL (n=2), AF4 (n=1), AF17 (n=1), ENL (n=1), SEPT5 (n=1). In 4/47 cases results from chromosome banding analysis suggested partner genes to be located at 11q13 (n=1), 10p11 (n=1), and 19p13 (n=2). In 3/47 cases the MLL rearrangement was cryptic and only suspected by FISH analysis. Two of those (#1, #2) showed a del(11)(q23q25) in chromosome banding analyses and FISH analyses demonstrated a loss of the 3′ flanking MLL probe. In the remaining case (#3) cytogenetics showed an i(21)(q10). FISH analysis on metaphase spreads identified an additional copy of the 5′ flanking MLL probe which localized on 6q27. SNP analyses were able to resolve all three cases: #1) The deletion was fine-mapped by SNP microarray data and ranged from physical map position 117,859,541 to 11qter including exons 10 to 28 of the MLL gene. In addition, SNP microarray data revealed a gained segment on 6q ranging from physical map position 167,977,103 to 6qter including exons 2 to 28 of AF6. #2) In this case the 11q deletion spans from physical map position 117,859,541 to 121,033,713 including exons 10 to 28 of the MLL gene. SNP microarray data revealed a gained segment on 6q ranging from physical map position 168,036,784 to 168,457,799 including exons 9 to 28 of AF6. #3) Corresponding to FISH analysis SNP microarray data revealed a gained segment on 11q ranging from physical map position 117,760,488 to 117,859,673, including exons 1 to 9 of MLL. Moreover, on chromosome 6 a small deletion of 177 kb was detected, starting at physical map position 167,804,673 towards 167,982,457. This deletion included exon 1 of AF6 and a small adjacent centromeric region. In all 3 cases, subsequent RT-PCR analyses confirmed the predicted MLL-AF6 fusion. Analyzing the MLL gene further in the remaining cases revealed copy number changes in 2 cases showing gains of 11q starting from exon 12 of the MLL gene to 11qter (physical map position 117,863,291 to 11qter and 117,862,916 to 11qter). These were due to an extra copy of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3), respectively. In two additional cases very small deletions within MLL with a size of 4.831 kb including exons 10 and 11 (physical map position 117,859,541 to 117,864,372) and 1.699 kb including exons 10 and 11 (physical map position 117,859,541 to 117,861,240) were observed (MLL-AF6- and MLL-AF4-rearrangement). With respect to the various MLL partner genes, deletions starting in the partner genes were observed in 2 cases with MLL-AF9 rearrangement (size: 8 MB and 6.1 MB, physical map position 20,334,335 to 28,350,412 and 20,342,604 to 26,451,390). The region deleted in both cases spanned 37 genes, including several genes of the interferon alpha family and the tumor suppressor candidate TUSC1. Copy number gains were observed in the region of the partner genes in both cases with a doubling of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3). In conclusion, using high resolution SNP arrays we identified three novel mechanisms leading to MLL-AF6 fusions which are cytogenetically cryptic and associated with atypical FISH signal constellations. Furthermore, a distinct pattern of additional aberrations was observed showing trisomies of chromosomes 4, 8 and 19. SNP microarray data also revealed a small deletion on the short arm of chromosome 9 as a recurrent additional genetic change in AML with MLL-AF9-rearrangements.

About 17% of AML with NPM1 mutations Show a Specific Pattern of Chromosome Aberrations but These Cases Do Not Differ Prognostically from AML with NPM1 Mutations Carrying a Normal Karyotype

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2527-2527
Author(s):  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Tamara Weiss ◽  
Wolfgang Kern ◽  
Brunangelo Falini ◽  
...  
Keyword(s):  
Chromosome Aberrations ◽  
Chromosome Banding ◽  
Who Classification ◽  
De Novo ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Additional Chromosome ◽  
De Novo Aml

Abstract AML with mutated nucleophosmin gene (AML NPM1mut) usually carries a normal karyotype and will be suggested as a provisional entity in the new WHO classification. Thus far, the impact of chromosome aberrations in AML NPM1mut has not been evaluated in detail. Aim of this study was to determine the incidence and prognostic impact of clonal chromosome aberrations in NPM1mut. We further compared this pattern to additional aberrations in AML with recurrent genetic aberrations: t(8;21)(q22;q22), inv(16)(p13q22)/t(16;16)(p13;q22), t(15;17)(q22;q12) and 11q23-abnormalities leading to an MLL-rearrangement. In total 415 AML (de novo AML: 392, s-AML: 11, t-AML: 12) showing an NPM1 mutation were analyzed by chromosome banding analysis. 71 of these showed clonal chromosome aberrations (17.1%; de novo AML: 63 (16.1%), s-AML: 5 (45.5%), t-AML: 3 (25%); de novo AML vs. s-AML: p=0.024). Overall, 111 chromosome aberrations were observed. The most frequent abnormalities were +8 (n=30), −Y (n=10), +4 (n=9), del(9q) (n=5), +21 (n=4), −7 (n=3), +5 (n=2), +10 (n=2), +13 (n=2),+18 (n=2), del(12p) (n=2), del(20q) (n=2), other non-recurrent balanced aberrations (n=6), other non-recurrent unbalanced aberrations (n=32). For comparison 63 AML with t(8;21), 37 cases with inv(16)/t(16;16), 83 patients with t(15;17) and 83 AML showing a 11q23/MLL-rearrangement were evaluated. 44 (69.7%), 13 (35.1%), 39 (47%), and 28 (43.1%) cases showed clonal chromosome aberrations in addition, respectively. Therefore, additional chromosomal aberrations are more frequent in all these subgroups than in the AML NPM1mut. Similar to NPM1mut cases +8 (n=2), −X/Y (n=32), +4 (n=2), and del(9q) (n=10) were observed. The only other recurrent additional aberrations was del(11q) (n=2). In inv(16)/t(16;16) we also found +8 (n=5) and −Y (n=1). The only other recurring additional aberrations were +22 (n=6) and del(7q) (n=2). In AML with t(15;17) recurring additional abnormalities were +8 (n=12), −Y (n=3), del(9q) (n=2), ider(17)(q10) t(15;17) (n=7). AML with 11q23/MLL-rearrangement showed +4 (n=2), +8 (n=8), +13 (n=2), +19 (n=4), +21(n=4), +22 (n=2), −Y (n=1). Thus, chromosome aberration in AML NPM1mut share many overlaps to those in AML with recurrent aberrations. Furthermore, the prognostic impact of chromosome aberrations in AML NPM1mut was evaluated. No difference with respect to overall survival (OS) and event-free survival (EFS) was observed between AML NPM1mut with a normal (n=344) and an aberrant karyotype (n=71) (OS at 2 yrs 78% vs. 81%, p=0.969; EFS at 2 yrs 40% vs. 50%, median EFS 544 days vs. 522 days, p=0.253). The FLT3-ITD status was available in 400 cases. 127 (38%) of 334 cases with a normal karyotype showed a FLT3-ITD, while in only 16 (24%) of 66 cases with an aberrant karyotype a FLT3-ITD was observed (p=0.035). While the negative prognostic impact of additional FLT3-ITD was confirmed in our cohort, the presence of chromosome aberrations did not influence prognosis neither in the FLT3-ITD negative nor in the FLT3-ITD positive subgroup. In addition, 31 patients with AML NPM1mut were analyzed by chromosome banding analysis at diagnosis and at relapse (median time diagnosis to relapse: 301 days (range: 71–986). 22 cases (71%) showed a normal karyotype both at diagnosis and relapse. In 4 cases a normal karyotype was observed at diagnosis and an aberrant karyotype at relapse (del(9q) (n=2), t(2;11) (n=1), inv(12) (n=1)). One case with +8 at diagnosis showed +8 also at relapse. One case with +4 at diagnosis showed +4 and additional aberrations at relapse. In 1 case clonal regression was observed (+21 -> normal). One case with an unbalanced 1;3-translocation at diagnosis showed a der(17;18) (q10;q10) at relapse and one case with −Y at diagnosis showed a del(3p) at relapse. In conclusion: 1. Frequency of additional chromosome aberrations is low in AML NPM1mut as compared to other genetically defined WHO entities. 2. The pattern of additional chromosome aberrations is overlapping between the 5 groups analyzed. 3. Chromosome aberrations observed at diagnosis in AML NPM1mut do not influence prognosis in comparison to AML NPM1mut with normal karyotype. 4. The karyotype is stable in most AML NPM1mut patients at diagnosis and at relapse. These results point to chromosomal aberrations occurring in AML NPM1mut as secondary events and further support inclusion of AML NPM1mut as a provisional entity in the new WHO classification.

Recurrent Additional Genetic Aberrations and a Novel Mechanism of CBFB-MYH11-Rearrangement in AML M4eo Are Detected Using High-Resolution Genome-Wide Single Nucleotide Polymorphisms (SNPs) Microarrays

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3115-3115
Author(s):  
Alexander Kohlmann ◽  
Sonja Rauhut ◽  
Frank Dicker ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Banding ◽  
Fusion Gene ◽  
Physical Map ◽  
Snp Array ◽  
Chromosome 8 ◽  
Chromosome 1 ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
7Q Deletion

Abstract Leukemia specific fusion genes such as CBFB-MYH11 play a major role in the pathogenesis of distinct AML entities. However, additional genetic aberrations seem necessary for the development of full blown leukemia. This study was performed to decipher CBFB-MYH11 rearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP Array 6.0 analyses, featuring >1.8 million markers for genetic variation (>906,600 SNPs and >946,000 probes for the detection of copy number variations), were performed in 35 newly diagnosed AML with inv(16) (p13q22) or t(16;16)(p13;q22) and CBFB-MYH11-rearrangement. First, as a proof of principle, additional gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included gains of whole chromosome 8 (n=7) and 22 (n=8). In addition, a partial trisomy 13 and a partial trisomy 6 resulting from an unbalanced translocation were confirmed. In two cases a 7q deletion was observed by chromosome banding analysis. One of these was missed by SNP array as the 7q deletion occurred in a subclone only (11% of cells with 7q deletion as determined by interphase FISH). However, SNP array analyses detected loss of 7q in two additional cases which was missed by cytogenetics. Based on SNP array data the commonly deleted region was identified to range from 7q36.1 to 7q36.3 (size: 8.5 MB; physical map position 147,549,804–156,038,680). In addition to a gain of the whole chromosome 8, frequently observed as an additional aberration, in one case SNP array analyses revealed only a partial gain on 8q ranging from 8q24.13 to 8q24.3 (size: 25.3 MB; physical map position 120,986,982–146,268,936). Furthermore, a recurrent deletion (n=2) on chromosome 18 was detected by SNP array but not detected by cytogenetics. The commonly deleted region was localized in 18q23 (size: 3.1 MB; physical map position 72,481,657–75,604,994). In two cases the CBFB-MYH11 rearrangement was cryptic and could not be detected by chromosome banding analysis or FISH using two probes flanking the breakpoints within the CBFB gene, however, a CBFB-MYH11 transcript was amplified by RT-PCR. In one of these cases SNP array data revealed a small gain on 16p13 including 3′ part of the MYH11 gene (size: 71 kb; physical map position 15,654,558–15,725,636) suggesting the insertion of additional 3′ MYH11 sequences into the CBFB rearrangement leading to a CBFB-MYH11 fusion gene. Interestingly, four cases showed a deletion on 16p13 (sizes: 176 kb, 461 kb, 464 kb, 468 kb; physical map positions 15,729,932–15,906,308, 15,726,920–16,188,116, 15,725,663–16,189,984, 15,721,133–16,189,807). All included the 5′ part of the MYH11 gene, and in 3 cases, the ABCC1 gene (multidrug resistance-associated protein 1) was included in the deleted region, which could have an impact on prognosis. The patient with the smallest deletion in 16p13 also showed a deletion on 16q22 including the ′ part of CBFB (size: 35 kb, physical map position 65,672,864–65,707,954). This would be in line with findings in chronic myeloid leukemia where comparable small deletions in the breakpoint region of BCR and ABL have been described. Furthermore, large regions of copy-neutral loss of heterozygosity were observed for the whole short arm of chromosome 1 in two cases, for 17q12 to 17qter and 19q in one case each. In conclusion, a novel mechanism leading to a CBFB-MYH11 fusion gene was identified: A cytogenetically cryptic insertion of additional MYH11 sequences into the CBFB locus. A distinct pattern of additional aberrations was confirmed showing gains of whole chromosomes 8 and 22. Small copy number changes not observable in chromosome banding analysis were detected on 7q, 8q and 18q. A recurrent region of loss of heterozygosity without copy number change was found for the whole short arm of chromosome 1 suggesting that candidate genes in this region are mutated and potentially play a pathogenetic role in AML with CBFB-MYH11-rearrangement.

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.

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