Chromosome Banding Analysis Defines Subclasses of CLL with 13q14 Deletion and Identifies a New Mechanism of Submicroscopic 13q14 Deletions Occurring in the Breakpoint Region of Reciprocal Translocations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2064-2064
Author(s):  
Claudia Schoch ◽  
Frank Dicker ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Chromosome Banding ◽  
Fish Analysis ◽  
Prognostic Impact ◽  
Derivative Chromosome ◽  
Routine Diagnostics ◽  
Chromosome 13 ◽  
Interphase Fish ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
13Q Deletion

Abstract 13q14 deletions are the most frequent abnormality in CLL and are overall associated with a favourable prognosis. However, the clinical course of the disease is heterogeneous within this subgroup of CLL. In order to characterize this subgroup, which is identified in routine diagnostics by interphase FISH, in more detail we performed chromosome banding analysis in addition. By improving the cultivation technique using the immunostimulatory CpG-oligonucleotide, DSP30, and IL-2 we reached a high success rate in routine diagnostics. Since August 2005 416 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. 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). 411/416 (98.8%) cases could be successfully stimulated for metaphase generation. 348/411 (84.7%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 332 of 416 (79.8%) successfully evaluated cases. In 229 cases (55%) a 13q14 deletion was detected by FISH, including 58 patients with a homozygous deletion. CBA was not evaluable in 4/229 cases. A normal karyotype was observed in 9/229, due to a small size of the aberrant clone missed by CBA (20% of interphase nuclei) in 1 case and due to the small size of the deletion not visible in CBA in 8 cases (growth of the aberrant clone was confirmed by FISH on metaphases). In 108 cases a deletion 13q was the only abnormality detected in CBA. 29 cases showed one other abnormality in addition to del(13q) (del(11q) n=13, +12 n=2, der(17p) n=3, other abnormalities not detectable by the used FISH panel n=11). In 51 cases 2 or more abnormalities were observed in addition to the 13q deletion. Interestingly, 28 cases did not show a 13q-deletion but a reciprocal translocation or insertion with a breakpoint in 13q14. In all these cases FISH on metaphases was performed with a whole chromosome painting probe for chromosome 13 and a probe for either D13S25 or D13S319, demonstrating a loss of one D13S25/D13S319 signal from the derivative chromosome 13 and the partner. In 9 cases D13S25/D13S25 was also lost from the homologous chromosome 13 (homozygous 13q14 deletion). The translocation partner was confirmed in a second FISH analysis also confirming the reciprocal nature of the abnormality. The breakpoints of the partner chromosomes were distributed all over the genome (1p13, 1q23, 1q24, 1q42, 1q42, 3q21, 3q21, 4p16, 4q23, 5q13, 5q15, 6q11, 6q23, 7p21, 8p23, 8q21, 8q22, 9p22, 9q21, 9q33, 10p15, 10q24, 11p15, 11q23, 13q34, 15q15, 16q24, 16q24). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities in addition to 13q14 deletion which can not be detected with a standard interphase FISH panel. 2) CBA provides new biological insights into different mechanisms leading to loss of 13q14. Prospective clinical trials have to evaluate the prognostic impact of the different subclasses of CLL with 13q14 deletion that now can be identified by chromosome banding analysis.

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.

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.

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.

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.

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 -&gt; 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.

Immunostimulatory CpG-Oligonucleotide Activated Metaphase Cytogenetics Is Feasible in Routine Diagnostics of Chronic Lymphocytic Leukaemia and Reveals More Abnormalities Than Interphase FISH.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3252-3252
Author(s):  
Claudia Schoch ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Frank Dicker
Keyword(s):  
Chromosomal Aberrations ◽  
Interleukin 2 ◽  
Chromosome 17 ◽  
Fish Analysis ◽  
Cpg Odn ◽  
Interphase Fish ◽  
Cpg Oligonucleotides ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Trisomy 12

Abstract Genetic characterization of chronic lymphocytic leukemia (CLL) cells by fluorescence in situ hybridization (FISH) has identified genetic aberrations in 80% of CLL patients. In contrast, conventional metaphase cytogenetics detected chromosomal aberrations in only 40–50% of all cases. Immunostimulatory CpG-oligonucleotides (CpG-ODN) in combination with interleukin 2 (IL-2) have recently been reported to induce proliferation in CLL B cells. Therefore, we evaluated this stimulus for its efficacy in metaphase generation for the detection of chromosomal aberrations by cytogenetic banding techniques. In addition these results were compared with data obtained by interphase FISH. For metaphase induction 107 cells were cultured in growth medium with the CpG-ODN DSP30 and IL-2. After 2 days colchicine was added for another 24h before preparation. Of the 133 samples that were included in this study, all cases could be successfully analyzed by interphase 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%). By FISH 55% of all samples showed a single aberration, 22% two aberrations and 2% 3 aberrations. In comparison, 126 cases (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations, which involved all different chromosomes. 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. In all but 1 of these cases the aberrant clone represented &lt; 30% of the cell population. In addition to the aberrations that were detected by the FISH probes, a substantial amount of other aberrations was revealed by chromosome banding analysis. Interestingly, 10 cases of a total of 27 cases without abnormalities detected by FISH displayed aberrations in chromosome analysis indicating that the true amount of CLL patients with aberrations exceeds 79%. Overall, 47 samples (37%) showed chromosomal aberrations in chromosome banding analysis in addition to FISH analysis. Compared to FISH analysis, which detected 2 cases with 3 aberrations, metaphase cytogenetics detected 22 cases with 3 or more unbalanced aberrations, which can be considered as a complex aberrant karyotype. Loss of p53 referred to as del(17p13) in FISH has attracted considerable attention, because of the poor clinical outcome of affected patients. In our study, all del(17p13) cases (n=7) displayed either a loss of the short arm of chromosome 17 (n=6) or a complete loss of chromosome 17 (n=1) indicating that chromosomal regions in addition to the p53 locus might be relevant for this phenotype. Furthermore, numerous recurrent aberrations have been identified in this study beyond the aberrations that are detected by FISH. Of note are gains of 2p and 3q, losses in 11q13 and gains in 11q23–25, gains in 13q31–34, gains of 17q21–25 and cases with trisomy 18 and 19, which occurred in parallel to trisomy 12. The results of the present study show that immunostimulatory CpG-oligonucleotides plus interleukin 2 are a simple and cheap stimulus for efficient metaphase induction in CLL. The rate of aberrations detected by conventional banding techniques was even slighty increased compared to FISH analysis, however, the complexity of cytogenetic aberrations is underestimated by FISH analysis in a large portion of CLL cases (37%).

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.

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.

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