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.

scholarly journals Characterization of Nonrandom Chromosomal Gains and Losses in Multiple Myeloma by Comparative Genomic Hybridization

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 3007-3010 ◽  
Author(s):  
Juan C. Cigudosa ◽  
Pulivarthi H. Rao ◽  
M. Jose Calasanz ◽  
M. Dolores Odero ◽  
Joseph Michaeli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Comparative Genomic Hybridization ◽  
Plasma Cells ◽  
Comparative Genomic ◽  
Genomic Hybridization ◽  
Banding Analysis ◽  
Chromosomal Changes ◽  
Gains And Losses

Clonal chromosomal changes in multiple myeloma (MM) and related disorders are not well defined, mainly due to the low in vivo and in vitro mitotic index of plasma cells. This difficulty can be overcome by using comparative genomic hybridization (CGH), a DNA-based technique that gives information about chromosomal copy number changes in tumors. We have performed CGH on 25 cases of MM, 4 cases of monoclonal gammopathy of uncertain significance, and 1 case of Waldenstrom's macroglobulinemia. G-banding analysis of the same group of patients demonstrated clonal chromosomal changes in only 13 (43%), whereas by CGH, the number of cases with clonal chromosomal gains and losses increased to 21 (70%). The most common recurrent changes detected by CGH were gain of chromosome 19 or 19p and complete or partial deletions of chromosome 13. +19, an anomaly that has so far not been detected as primary or recurrent change by G-banding analysis of these tumors, was noted in 2 cases as a unique change. Other recurrent changes included gains of 9q, 11q, 12q, 15q, 17q, and 22q and losses of 6q and 16q. We have been able to narrow the commonly deleted regions on 6q and 13q to bands 6q21 and 13q14-21. Gain of 11q and deletion of 13q, which have previously been associated with poor outcome, can thus be detected by CGH, allowing the use of this technique for prognostic evaluation of patients, without relying on the success of conventional cytogenetic analysis.

Array CGH Identifies Copy Number Changes In 10% Of 520 MDS Patients With Normal Karyotype: Deletions Encompass The Genes TET2, DNMT3A, ETV6, NF1, RUNX1, and STAG2 and Are Associated With Shorter Survival

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

Abstract Background In MDS, cytogenetic aberrations play an important role for classification and prognostication. The original IPSS and the revised IPSS classifiers have clearly demonstrated the prognostic impact of distinct cytogenetic abnormalities. The vast majority of chromosome aberrations in MDS are gains or losses of chromosomal material while balanced rearrangements are rare. However, more than 50% of MDS and even more in low risk MDS harbor a normal karyotype. Chromosome banding analysis can only detect gains and losses of more than 10 Mb size due to its limited resolution and is dependent on proliferation of the MDS clone in vitro to obtain metaphases. Array CGH has a considerably higher resolution and does not rely on proliferating cells. Aims In this study we addressed the question whether MDS with normal karyotype harbor cytogenetically cryptic gains and losses. Patients and Methods 520 MDS patients with normal karyotype were analyzed by array CGH (Human CGH 12x270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). For all patients cytomorphology and chromosome banding analysis had been performed in our laboratory. The cohort comprised the following MDS subtypes: RA (n=22), RARS (n=43), RARS-T (n=27), RCMD (n=124), RCMD-RS (n=111), RAEB-1 (n=104), and RAEB-2 (n=89). Median age was 72.2 years (range: 8.9-90.1 years). Subsequently, recurrently deleted regions detected by array CGH were validated using interphase-FISH. Results In 52/520 (10.0%) patients copy number changes were identified by array CGH. Only eight cases (1.5%) harbored large copy number alterations >10 Mb in size, as such generally detectable by chromosome banding analysis. These copy number alterations were confirmed by interphase-FISH. They were missed by chromosome banding analysis due to small clone size (n=2), insufficient in vitro proliferation (n=3) or poor chromosome morphology (n=3). In the other 44 patients with submicroscopic copy number alterations 18 gains and 32 losses were detected. The sizes ranged from 193,879 bp to 1,690,880 bp (median: 960,176 bp) in gained regions and 135,309 bp to 3,468,165 bp (median: 850,803 bp) in lost regions. Recurrently deleted regions as confirmed by interphase-FISH encompassed the genes TET2 (4q24; n=9), DNMT3A (2p23; n=3), ETV6 (12p13; n=2), NF1 (17q11; n=2), RUNX1 (21q22; n=2), and STAG2 (Xq25, deleted in 2 female patients). No recurrent submicroscopic gain was detected. In addition, we performed survival analysis and compared the outcome of patients with normal karyotype also proven by array CGH (n=462) to patients with aberrant karyotype as demonstrated by array CGH (n=52). No differences in overall survival were observed. However, overall survival in 35 patients harboring deletions detected solely by array CGH was significantly shorter compared to all others (median OS: 62.1 vs 42.4 months, p=0.023). Conclusions 1. Array CGH detected copy number changes in 10.0% of MDS patients with cytogenetically normal karyotype as investigated by the gold standard method, i.e. chromosome banding analysis. 2. Most of these alterations were submicroscopic deletions encompassing the genes TET2, ETV6, DNMT3A, NF1, RUNX1, and STAG2. 3. Interphase-FISH for these loci can reliably pick up these alterations and is an option to be easily performed in routine diagnostics in MDS with normal karyotype. 4. Patients harboring deletions detected solely by array-CGH showed worse prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Raitner:MLL Munich Leukemia Laboratory: Employment. Holzwarth:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals 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.

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.

Complex Aberrant Karyotypes and Unbalanced Translocations in CLL Correlate with an Unmutated IgVH Status: A Study on 133 Patients Studied with Chromosome Banding Analysis, Interphase FISH, IgVH Mutation Status, ZAP-70 RNA Expression and Immunophenotyping.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2939-2939 ◽  
Author(s):  
Frank Dicker ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Schoch
Keyword(s):  
Risk Factors ◽  
Mrna Expression ◽  
Chromosome Banding ◽  
Normal Karyotype ◽  
High Risk Group ◽  
Interphase Fish ◽  
Cd38 Expression ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Conventional Cytogenetics

Abstract CLL is a heterogenous disease from a clinical as well as from a genetic point of view. In order to characterize CLL patients in detail we performed chromosome banding analysis and interphase FISH on 133 cases and analyzed the IgVH status, CD38 and ZAP-70 mRNA expression in parallel. Of the 133 samples that were included in this study, 126 (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations. In comparison, all 133 cases could be successfully analyzed by FISH with a rate of 79% aberrations like deletions of chromosomes 13q (57%, in 38% as sole abnormality), 11q (17%), trisomy 12 (13%), 6q (7%) 17p (6%) and translocations involving 14q32 (4%). 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. Additional aberrations not included in the FISH panel of probes, were detected in 47 samples (37%) by conventional cytogenetics. The assessment of additional risk factors in our cohort of CLL patients revealed an unmutated IgVH status in 55%, a positive ZAP-70 expression in 57% and a CD38 expression ≥ 30% in 29% of the samples. ZAP-70 mRNA expression resulted in concordance with the IgVH status in 75% of the cases with the concurrence of ZAP-70 positive/unmutated IgVH or ZAP-70 negative/ mutated IgVH. Genetic poor risk factors like del(11q) and del(17p )correlated well with an unmutated IgVH status with 89% and 75% concordant cases, respectively and also CD38 expression resulted in a significant correlation with the IgVH status (p = 0.028, Fischer’s exact test). When analyzing the group of 47 samples with cytogenetic aberrations beyond the FISH panel in more detail, it was remarkable that a large number of 22 cases (47%) was characterized by a complex aberrant karyotype (≥ 3 unbalanced aberrations). The status of somatic mutations of the IgVH gene was available for 39 cases in that group with 74% having an unmutated IgVH-status. This value was significantly increased (p = 0.013) compared to the 44% of unmutated samples calculated from the panel of samples that were concordant in FISH and chromosome analysis (IgVH status was available in 57 cases). No significant correlations were obtained for ZAP-70 and CD38 expression with the different cytogenetic groups. Remarkable was also the incidence of unbalanced translocations in 22 cases, not picked up by the FISH probes, which were strongly associated with an unmutated IgVH-status (83%). In conclusion, a comprehensive laboratory work-up is necessary in order to obtain more insights into the pathophysiology of CLL and for individualized treatment approaches. Interestingly, CLL cases with a more complex karyotype (≥ 3 unbalanced aberrations) or with unbalanced translocations correlated with an unmutated IgVH status and might therefore represent a high risk group of patients.

Chromosome Banding Analysis Allows a More Precise Classification of CLL Than Interphase FISH: A Study on 446 Cases.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 297-297
Author(s):  
Claudia Schoch ◽  
Frank Dicker ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Clinical Outcome ◽  
Chromosome Banding ◽  
Chromosome Abnormalities ◽  
Prognostic Impact ◽  
Interphase Fish ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Balanced Translocations ◽  
13Q Deletion

Abstract In CLL data from chromosome banding analysis 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 chromosome banding analysis in routine diagnostics. Since August 2005 446 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. Diagnosis of CLL was established by standard criteria based on cytomorphology and immunophenotyping. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). 440/446 (98.7%) cases could be successfully stimulated for metaphase generation and are the basis of this study. 370/440 (84.0%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 353 of 440 (80.2%) successfully evaluated cases. Overall 452 abnormalities were detected by FISH and 788 abnormalities by CBA. Based on FISH results 277 cases showed 1, 67 cases 2, 8 cases 3 and 1 case 4 abnormalities, respectively. In CBA at least 1 aberration was detected in 177, 2 in 98, 3 in 45, 4 in 19, and 5 or more aberrations in 31 patients. In 31 of 87 cases (35.6%) showing no aberrations in FISH abnormalities were detected in CBA. On the other hand 14 of 70 cases (20.0%) with a normal karyotype demonstrated abnormalities using FISH. In 7 of these cases CBA missed the abnormalities due to the small size of the aberrant clone or insufficient proliferation of the aberrant clone in vitro and in another 7 cases due to the small size of the 13q deletion not visible in CBA. Using CBA, in total 97 balanced translocations, 169 unbalanced translocations leading to gain and/or loss of genetic material, 368 deletions, 77 gains of whole chromosomes, 40 losses of whole chromosomes, and 37 other aberrations were observed. Only 17 of 97 balanced translocations involved the IGH gene. In 28 cases balanced translocations involved the breakpoint 13q14. Although these translocations were reciprocal and seemed balanced in CBA FISH demonstrated a 13q14 deletion in the breakpoint region. Therefore, based on CBA cases with 13q deletion could be subdivided into 3 different categories: 1. del(13q) sole, 2. del(13q) with additional abnormalities and 3. del(13q) due to a reciprocal translocation. This genetic heterogeneity might account for differences in clinical outcome. In cases with TP53 deletions the number of chromosome abnormalities was higher compared to cases without TP53 deletion (mean 5.0 vs 1.5, p<0.0001). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities which can not be detected with a standard interphase FISH panel. These additional abnormalities could explain heterogeneous clinical outcome. 2) CBA provides new biological insights into different CLL subclasses based on a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far for CLL. Therefore, prospective clinical trials should evaluate the prognostic impact of these additional abnormalities that now can be identified by chromosome banding analysis.

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.

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.

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