A Comprehensive Panel of Molecular Mutations Notably Improves a Cytogenetic Prognostication System in Routine AML Diagnostics

Abstract Background: Based on sequencing studies the molecular landscape of AML has been unraveled. Novel prognostic scores combining molecular mutations and karyotype have been proposed (Grimwade et al. Blood, 2016, Döhner et al. NEJM 2015). However, these proposed classification systems differ in several aspects and yet no consensus has been established which genetic information is required for prognostication in AML today. Aims: 1) Test the prognostic value of a panel of molecular markers in addition to a cytogenetic score in a large cohort of AML patients. 2) Determine the proportion of patients with a suitable molecular marker for disease monitoring (MRD) applying molecular genetics. Patients and Methods: 867 de novo AML cases younger than 60 years were investigated (median age: 48 years, median follow up of 41 months). All patients were evaluated for karyotype, KMT2A-PTD, FLT3-ITD and in addition for mutation status of ASXL1, CEBPA, DNMT3A, NPM1, RUNX1 and TP53 according to the proposal by Grimwade et al. Blood 2016. Results: First, AML were classified according to the refined MRC cytogenetic classification with AML with t(15;17)/PML-RARA regarded as a separate group (n=89 (10%), 90% overall survival (OS) at 5 years). 89 cases (10%) were assigned to the favourable risk group (t(8;21)/RUNX1-RUNX1T1: n=42; inv(16)/t(16;16)/CBFB-MYH11: n=47), 570 (68%) to the intermediate risk group and 119 (14%) to the adverse risk group. OS at 5 years was 66%, 53% and 28%, respectively, and differed significantly between all four subgroups (for all comparisons p<0.001). Next, the following subgroups were separated: CEBPA double mutated (dm) cases (n=44 (5%); OS at 5 years: 83%), NPM1mut/FLT3-ITD- AML (n=181 (21%); OS at 5 years: 62%), and NPM1mut/FLT3-ITD+ AML (n=137 (16%); OS at 5 years: 47%; for all comparisons between these 3 groups p<0.001). Thus, prognosis of CEBPAdm cases was comparable to PML-RARA+ AML. OS in NPM1mut/FLT3-ITD- AML is comparable to CBF-leukemias. In NPM1mut AML no prognostic impact of DNMT3Amut was found. In all these 3 groups defined on molecular genetics no prognostic impact of additional karyotype information on OS was observed. Next, in the remaining cases of the cytogenetic intermediate risk group (n=209) the prognostic impact of mutations in ASXL1, DNMT3A, RUNX1, TP53, KMT2A-PTD and FLT3-ITD was evaluated. In multivariate Cox regression analysis mutations in TP53 (relative risk (RR): 3.5; p=0.04), ASXL1 (RR: 2.2, p=0.004), and FLT3-ITD (RR: 1.8; p=0.04) were independently associated with shorter OS. OS at 5 years was 25% in cases carrying at least one of these mutations compared to 54% in cases with none of these mutations (p=0.001). Within the adverse cytogenetic risk group cases with either a complex karyotype (n=27) or a KMT2A (n=25) or MECOM rearrangement (n=14) had the worst outcome compared to the remaining cases (OS at 5 yrs: 19% vs 54%, p=0.02). In the remaining cases the presence of at least one mutation in either ASXL1, TP53 or FLT3-ITD was associated with worse outcome (OS at 5 yrs: 33% vs 74%, p=0.04). Thus, AML with complex karyotype, KMT2A or MECOM rearrangements had the worst prognosis, while cases with adverse cytogenetics and at least one mutation in either ASXL1, TP53 or FLT3-ITD have a slightly better outcome which is comparable to AML with intermediate risk cytogenetics harbouring one of these mutations. OS in AML with adverse cytogenetics without mutations in ASXL1, TP53 or FLT3-ITD is not worse than in AML with intermediate cytogenetics also lacking these mutations. A fusion gene or a molecular mutation as target for MRD monitoring was present in 791 patients (91%) when all genes analysed were taken into account. If only markers showing prognostic relevance, i.e. fusion genes, mutations in NPM1, CEBPA, FLT3-ITD, ASXL1 and TP53 were considered a MRD marker was still available in 726 cases (84%). Conclusions: 1) In AML a prognostication system is feasible based on the identification of t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, 11q23/KMT2A rearrangements, 3q26/MECOM rearrangements, complex karyotype, and mutation status of NPM1, CEBPA, ASXL1, TP53, and FLT3-ITD (figure 1). 2) The analysis of these parameters allows to identify an MRD marker in 84% of patients. 3) The analysis of additional genes may be required in a comprehensive AML work-up as soon as novel targeted treatment strategies will become available. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan: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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In Chronic Lymphocytic Leukemia the Gain of the Short Arm of Chromosome 2 Is Highly Associated with an Unmutated IGHV status, 11q/ATM Deletion, SF3B1 Mutation and a Complex Karyotype

Blood ◽

10.1182/blood.v128.22.4379.4379 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4379-4379

Author(s):

Claudia Haferlach ◽

Sabine Jeromin ◽

Anna Stengel ◽

Manja Meggendorfer ◽

Melanie Zenger ◽

...

Keyword(s):

Multivariate Analysis ◽

Cox Regression ◽

Prognostic Impact ◽

Chromosome 2 ◽

Complex Karyotype ◽

Employment Equity ◽

Cox Regression Analysis ◽

Tp53 Mutations ◽

Equity Ownership

Abstract Background: CLL is characterized by a distinct pattern of translocations, genomic gains and losses and molecular mutations. The most frequent abnormalities such as trisomy 12 and deletions of 6q, 11q, 13q and 17p have been intensively studied. However, data on less frequent recurrent abnormalities such as the partial gain of the short arm of chromosome 2 is lacking. Aims: a) Determine the frequency of 2p gain in CLL, b) Characterize the size and the commonly gained region, c) Analyze the spectrum of additional cytogenetic abnormalities and molecular mutations, and d) Evaluate the prognostic impact. Patients and Methods: Chromosome banding analysis (CBA) revealed a gain of 2p in 113 out of 5564 (2%) CLL cases. In 72 cases with sufficient material genomic array analysis (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and determination of the mutation status of TP53, SF3B1 and IGHV were performed. Results:76% of patients with gain of 2p were male. The median WBC count was 33,700/µL (range: 5,900 - 228,000). Median age was 66 years (range: 29 - 87). The gain of 2p always encompassed the 2p telomere (2pter) while the centromeric border of the 2p gain varied between 2p21 and the centromere of chromosome 2 (2p10) (genomic positions 45,859,076 to 92,297,003). The gain of 2p was the sole chromosomal abnormality in only 8/72 cases (11%) and was accompanied by one, two or more than two additional aberrations in 10, 20, and 34 cases. In total 209 chromosome abnormalities were observed in addition to the 2p gain (median per patient: 2, range: 0-16). Of these only 21 were balanced while 188 were unbalanced abnormalities leading to gain or loss of chromosomal material. Gain of 2p was most frequently accompanied by deletions in 13q (total: 74%, homozygous: 11%), 11q (56%), 18p (18%), and 6q (13%) and gains of 8q (11%). 17p deletions were present in 6% of cases. In 49 cases (68%) the gain of 2p was present in the main clone while it was present in a subclone only in 23 cases (32%). The gain of 2p material was due to a duplication in the short arm of chromosome 2 in 10 cases, while a gain of an isochromosome 2p was present in 3 cases. In the remaining cases material of the short arm of chromosome 2p was attached to a variety of different partner chromosomes. The most frequent acceptor chromosome was chromosome 18 (n=13; 18%). In two cases (2%) 2 IGH rearrangements were observed of which one was mutated and the other unmutated. The IGHV status was unmutated (IGHV-U) in 66 (92%) and mutated in only 4 cases (6%). Three of these 4 cases with mutated IGHV showed only a low mutation rate (sequence homology to germline 97-97.9%). Stereotyped B-cell receptors were present in 14 cases (19%). SF3B1 mutations were observed in 21 cases (29%) with a median mutation load (ML) of 39% (range: 10-51%). TP53 mutations were detected in 8 (11%) cases (median ML: 60%, range: 13-100%). In 2 patients with a TP53 mutation a TP53 deletion was present and in 3 cases a copy neutral loss of heterozygosity (CN-LOH) of 17p was detected leading to TP53 wild-type loss in these 5 cases. TP53 mutations were less frequent in cases harboring the gain of 2p as the sole abnormality (3% vs 21%, p=0.02) The prognostic impact of 2p gain was evaluated in an unselected cohort of 1381 CLL cases with available follow up data (median follow up: 5.1 years) including 22 cases with 2p gain. The frequency of IGHV-Ustatus, SF3B1 mutations and 11q/ATM deletions was significantly higher in CLL with 2p gain compared to cases without (for all p<0.05). In univariate Cox regression analysis gain of 2p was significantly associated with shorter overall survival (OS) (relative risk (RR): 2.1; p=0.05). 5 year OS was 69% in CLL with 2p gain compared to 85% in cases without 2p gain (p=0.05). However, in multivariate analysis only IGHV-U, mutations in SF3B1 and TP53 and TP53/17p deletion were independently associated with shorter OS, while gain of 2p and 11q/ATM deletion were not. 2p gain was associated with shorter time to treatment (TTT) (RR: 2.0; p=0.02). In multivariate analysis only IGHV-U, SF3B1 mutation and 11q/ATM and TP53/17p deletion were independently associated with shorter TTT, while gain of 2p and TP53 mutations were not. Conclusions:CLL with gain of 2p is highly associated with an unmutated IGHV status (92%), a high frequency of 11q/ATM deletion (56%), 13q deletion (74%), SF3B1 mutation (29%) and a complex karyotype (47%). Data suggest that gain of 2p is a later event in CLL pathogenesis and might be a marker of progression. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Translocations Involving the IGH@locus Occur In 3.7% of Chronic Lymphocytic Leukemia and Are Associated with Unmutated IGHV Status and a Shorter Time to Treatment: A Study on 2,135 Cases

Blood ◽

10.1182/blood.v116.21.3596.3596 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3596-3596

Author(s):

Claudia Haferlach ◽

Frank Dicker ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Multivariate Analysis ◽

Regression Analysis ◽

Chronic Lymphocytic Leukemia ◽

Cox Regression ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Mutation Status ◽

Cox Regression Analysis ◽

Igh Locus ◽

Equity Ownership

Abstract Abstract 3596 Introduction: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course and a large spectrum of treatment options. Based on FISH data, a prognostic classification system has been established with 13q deletions as sole abnormality associated with a favorable prognosis and 17p and 11q deletions correlating with an unfavorable outcome. Recently, the combined evaluation of FISH data, IGHV mutation status and chromosome banding analysis (CBA) revealed that the impact of distinct genetic parameters differs with respect to overall survival (OS) and time to treatment (TTT). Thus far only few data is available on less frequent genetic abnormalities such as 14q deletions and translocations involving the IGH@ locus (tIGH). Therefore, we analyzed CLL with tIGH in detail with respect to frequency, partner genes and impact on prognosis. Methods/Patients: 78 CLL cases with tIGH were identified from 2,135 CLL sent to our laboratory for diagnostic work-up. All cases had been evaluated by immunphentotyping, FISH and CBA. Result: The most frequent tIGH was t(14;19)(q32;q13) (BCL3, n=21) followed by t(14;18)(q32;q21) (BCL2, n=19), t(8;14)(q24;q32) (CMYC, n=7) and t(11;14)(q13;q32) (CCND1, n=6). In the remaining 25 cases 5 recurrent translocations (t(2;14)(p13;q32), n=3; t(4;14)(p16;q32), FGFR3, n=2; t(11;14)(p15;q32), n=2; t(14;17)(q32;q25), n=2; and t(7;14)(q21;q32), n=2) were observed while the remaining 14 translocations were identified in single cases only. In 9/78 cases (11.5%) the tIGH was the sole abnormality. Recurrent additional chromosome abnormalities were +12 (n=7), del(13q) (n=9), del(11q) (n=3). A 17p deletion was observed in 1 case. In two cases tIGH was present only in a subclone and was a secondary abnormality occurring in addition to an del(11q) and a +12, respectively. CLL with tIGH were compared to 401 CLL without tIGH comprising all other genetic subgroups (subdivided according to Döhner et al.: del(17p) n=26, del(11q) n=42, +12 n=42, “normal” n=88, del(13q) sole n=177 and del(14q) n=26). An unmutated IGHV status was more frequent in CLL with tIGH as compared to all others (26/46 (54.3%) vs 128/353 (36.3%); p=0.023). For 53 cases with tIGH and all cases of the non-tIGH cohort clinical follow-up data was available. Median OS was 143.8 months (mo) in CLL with tIGH and 72.9 mo in patients with del(17p) while it was not reached in all other subgroups. In Cox regression analysis only del(17p) and mutated IGHV status were significantly associated with OS (p<0.0001, relative risk (RR)=7.0; p=0.014, RR=0.38). Median TTT was as follows: total cohort: 60.9 mo; tIGH: 27.8 mo; del(17p): 58.9 mo; del(11q): 19.7 mo; +12: n.r.; “normal” 63.9 mo; del(13q) sole: 83.0 mo and del(14q): 21.0 mo. In univariate Cox regression analysis the following parameters were significantly associated with shorter TTT: tIGH (p=0.004, RR=1.82), del(11q) (p<0.0001, RR=2.55), and del(14q) (p=0.007, RR=2.1), while del(13q) sole and mutated IGHV status were associated with longer TTT (p<0.0001, RR=0.40; p<0.0001, RR=0.23). In multivariate analysis including tIGH, del(11q), del(14q) and del(13q) sole all parameters retained their impact on TTT. However, if IGHV mutation status was included in the model only the mutated IGHV mutation status retained an impact on TTT (p<0.0001, RR=0.26). Next, patients with tIGH were subdivided according to their partner genes. Median OS was not reached in all subgroups, while median TTT was as follows: t(11;14): 101.2 mo, t(14;18): 47.9 mo, t(14;19): 11.0 mo, t(8;14): 18.5 mo and other partner genes: 27.8 mo. In univariate Cox regression analysis only t(14;19) was significantly associated with shorter TTT (p<0.001, RR=3.1). Including t(14;19) into multivariate analysis revealed a significant impact of both mutated IGHV mutation status and t(14;19) on TTT (p<0.0001, RR=0.286; p=0.004, RR=3.60). Conclusion: Translocations involving the IGH@ locus occur at low frequency in CLL. They are associated with unmutated IGHV status and a shorter TTT. TTT is especially short in cases with t(14;19). The prognostic impact of t(14;19) is independent of IGHV mutation status. In contrast CLL with t(11;14) and t(14;18) are neither associated with shorter OS nor shorter TTT. This data supports the application of CBA in CLL in order to identify all clinically relevant chromosomal aberrations, including those not detected by routine FISH analysis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Impact of Expression of BAALC, CDKN1B, ERG, EVI1, and MN1 on Prognosis and Their Association with Karyotype, FLT3-ITD, NPM1 and MLL-PTD Status In Adult AML: A Comprehensive Study on 286 Cases

Blood ◽

10.1182/blood.v116.21.953.953 ◽

2010 ◽

Vol 116 (21) ◽

pp. 953-953

Author(s):

Claudia Haferlach ◽

Alexander Kohlmann ◽

Sonja Schindela ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Genetic ◽

Normal Karyotype ◽

Genetic Alterations ◽

Complex Karyotype ◽

Employment Equity ◽

Aberrant Expression ◽

Cox Regression Analysis ◽

Total Cohort ◽

Equity Ownership ◽

Low Expression

Abstract Abstract 953 Introduction: The WHO classification in 2008 listed for the first time aberrant expression of genes as molecular genetic alterations affecting outcome in AML. High expression of BAALC, ERG and MN1 were shown thus far to be associated with unfavorable outcome in normal karyotype AML (AML-NK). In addition high EVI1 expression was suggested to predict poor outcome. Recently, our group identified low expression of CDKN1B as a favorable prognostic marker. The aim of this study was to evaluate the expression of BAALC, CDKN1B, ERG, EVI1 and MN1 in AML comprising all cytogenetic risk groups with respect to their association with distinct cytogenetic and known molecular genetic subgroups and their impact on prognosis. Patients/Methods:: Expression levels of BAALC, CDKN1B, ERG, EVI1 and MN1 were determined by oligonucleotide microarrays (HG-U133 Plus 2.0, Affymetrix) in 286 AML (t(15;17) n=15; t(8;21) n=16; inv(16) n=7; normal karyotype n=99; 11q23/MLL-rearrangements n=10; complex karyotype n=51; other abnormalities n=88). Patients were further analyzed for mutations in NPM1, FLT3-ITD, CEPBA and MLL-PTD. Results: Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varied significantly between genetic subgroups: While t(15;17), t(8;21) and 11q23/MLL-rearrangements were associated with low CDKN1B expression, AML-NK and NPM+ cases showed a higher CDKN1B expression. Lower BAALC expression was observed in AML with t(15;17), 11q23/MLL-rearrangement and AML-NK as well as in FLT3-ITD+ AML and in NPM1+ AML, while in AML with other abnormalities a higher BAALC expression was observed. ERG expression was lower in AML with 11q23/MLL-rearrangement and normal karyotype, while it was higher in AML with complex karyotype. Low EVI1 expression was observed in AML with t(15;17), t(8;21), inv(16) and AML-NK, while it was higher in AML with 11q23/MLL-rearrangements. Low MN1 expression was associated with t(15;17), t(8;21) and AML-NK, while it was increased in cases with inv(16) or other abnormalities. Next, Cox regression analysis was performed with respect to overall survival (OS) and event free survival (EFS). In the total cohort high BAALC and ERG expression as continuous variables were associated with shorter OS and EFS while CDKN1B, EVI1 and MN1 had no impact. Furthermore the cohort was subdivided into quartiles of expression for each gene. After inspection of the survival curves the cut-off for high vs low expression was set as follows: BAALC: 75th percentile, CDKN1B: 25th percentile, ERG and MN1: 50th percentile. For EVI1 expression pts were separated into expressers (n=44) and non-expressers (n=242). Low CDKN1B expression was associated with longer OS and EFS in the total cohort (p=0.005, not reached (n.r.) vs 14.9 months (mo); p=0.013, 31 vs 9.7 mo). High BAALC expression had no impact on OS, but was associated with shorter EFS in the total cohort as well as in AML with intermediate cytogenetics and AML with other abnormalities (p=0.032, 6.2 vs 13.0 mo; p=0.027, 5.1 vs 11.3 mo; p=0.006, 2.3 vs 14.8 mo). High ERG expression was significantly associated with shorter OS and EFS in the total cohort (p=0.002, 12.5 mo vs n.r.; p=0.001, 8.1 vs 15.7 mo) as well as in AML-NK (p=0.001, 11.3 mo vs n.r.; p=0.010, 7.2 vs 22.1 mo). OS was also shorter in AML with unfavorable karyotype (p=0.048, median OS 9.3 mo vs n. r.). With respect to MN1 high expressers had a significantly shorter OS and EFS in the total cohort (p=0.004, 12.3 mo vs. n.r.; p=0.001, 8.1 vs 16.7 mo) as well as in AML-NK (p=0.001, 9.7 mo vs n.r.; p=0.001, 5.1 vs 22.1 mo). In a multivariate analysis including CDKN1B, ERG and MN1 all parameters retained their impact on OS as well as on EFS, while BAALC lost its impact on EFS. Adding MLL-PTD, NPM1+/FLT3-ITD-, favorable and unfavorable karyotype into the model demonstrated an independent significant adverse impact on OS for MLL-PTD (p=0.027, relative risk (RR): 2.38) and ERG expression (p=0.044, RR: 1.59) only. In the respective analysis for EFS only favorable karyotype showed an independent association (p=0.002, RR: 0.261). Conclusion: 1) Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varies significantly between cytogenetic subgroups. 2) BAALC as a continuous variable and CDKN1B, ERG and MN1 as dichotomized variables are independently predictive for OS and EFS in AML. 3) ERG expression even retains its independent prediction of shorter OS if cytogenetic and other molecular genetic markers are taken into account. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela: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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TP53 Alterations In CLL - Parameters Influencing The Prognostic Impact: A Study On 3,988 Patients

Blood ◽

10.1182/blood.v122.21.865.865 ◽

2013 ◽

Vol 122 (21) ◽

pp. 865-865

Author(s):

Claudia Haferlach ◽

Frank Dicker ◽

Sabine Jeromin ◽

Sandra Weissmann ◽

Andreas Roller ◽

...

Keyword(s):

Cox Regression ◽

Small Subset ◽

Mutation Load ◽

Employment Equity ◽

Mutation Status ◽

Cox Regression Analysis ◽

Tp53 Mutations ◽

Entire Cohort ◽

Impact On Survival ◽

Equity Ownership

Abstract Background In CLL, the TP53 gene may be inactivated by deletion and/or mutations. Most cases with 17p deletion also carry TP53 mutations on the second allele. However, in a subset of cases only one allele seems to be disrupted by either mutation or deletion. It is still a matter of debate whether monoallelic TP53 abnormalities have the same poor prognostic effect as biallelic alterations. Further, a small subset of patients with TP53 deletions harboring mutated IGHV genes were described to exhibit a slowly progressive disease without treatment indication for years. Aims In this study, we addressed the following questions: 1. Frequency of TP53 alterations: mutation and deletion. 2. Characterization of the TP53 altered subsets with respect to IGHV mutation status, other molecular mutations and cytogenetics. 3. Impact on survival. Patients and Methods 3,988 CLL patients were analyzed by DNA sequencing for TP53 mutations and by FISH for TP53 deletion status as well as for del(13q), del(11q) and +12. IGHV mutation status was determined in 3,505 patients. Further, SF3B1 (n=1,245), MYD88 (n=1,026), XPO1 (n=1,025), NOTCH1 (n=973), and FBXW7 (n=962) were analyzed by DNA sequencing. Results 488/3,988 (12.2%) harbored a TP53 mutation (TP53mut) and 308/3,988 (7.7%) patients showed a TP53 deletion (TP53del) by FISH. 268 cases (6.7%) showed both a TP53del and a TP53mut, while 220 cases (5.5%) harbored a TP53mut only and 40 (1.0%) a TP53del only. 20.5% of TP53mut cases harbored more than one TP53mut. The frequency of TP53mut and TP53del increased significantly with age (≤40 yrs: 2.4%/2.4%; 41-50 yrs: 7.5%/4.0%; 51-60 yrs: 12.4%/6.8%; 61-70 yrs: 12.1%/8.1%; 71-80 yrs: 13.4%/9.1%; >80 yrs: 16.0%/9.9%; p=0.006 and p=0.013, respectively). In the entire cohort, 1,428/3,505 (40.7%) cases showed an unmutated and 2,077/3,505 (59.3%) a mutated IGHV status. The lowest frequency of IGHV unmutated was observed in cases without TP53 alteration (1,148/3,094; 37.1%) and the highest in patients with both TP53mut and TP53del (156/201; 77.6%). The frequency was in between in patients with TP53mut sole (106/176; 60.2%) and TP53del sole (18/34; 52.9%). Patients with both TP53mut and TP53del as well as patients with TP53del sole had a significantly shorter overall survival (OS) compared to patients with TP53mut sole or patients without TP53 alteration (OS at 5 yrs: 40.2% vs. 36.4% vs. 68.8% vs 85.4%; p<0.001; TP53mut sole vs TP53wt: p=0.003). Next, we evaluated the impact of the TP53 mutation load on survival. Therefore, we divided patients into 10 subgroups according to their mutation load (increments of 10%). The OS of patients with a mutation load <20% (n=150) did not differ from patients with TP53wt, while a mutation load ≥20% was significantly associated with shorter OS (HR: 4.9, p<0.001). An unmutated IGHV status was associated with shorter OS in the total cohort (HR: 2.3, p<0.001). In the subset of patients with TP53wt an unmutated IGHV status was also an adverse prognostic factor (OS at 5 yrs: IGHV unmutated vs mutated: 80.3% vs 88.6%, p=0.007). This was true also in cases with TP53del sole (median OS: 12 months vs not reached, p=0.001). In contrast, in patients with either TP53mut sole or both TP53mut and TP53del the IGHV status had no impact on OS. In the entire cohort univariate Cox regression analysis revealed the following parameters to be significantly associated with OS: TP53mut (HR: 4.0), TP53mut ≥20% (HR: 4.9), TP53del (HR: 7.1), IGHV unmutated (HR: 2.3), age >60 yrs (HR: 3.3), del(11q) (HR: 2.1), del(13q) sole (HR: 0.6), SF3B1mut (HR: 2.5) (for all p<0.001), and NOTCH1mut (HR: 1.6, p=0.025). Multivariate Cox regression analysis including parameters significantly associated with OS in univariate analyses revealed the following factors to be independently associated with shorter OS: TP53del (HR: 4.2, p<0.001), TP53mut ≥20% (HR: 2.4, p=0.008), age >60 yrs (HR: 2.6, p<0.001), SF3B1mut (HR: 2.4, p<0.001), and del(11q) (HR: 2.2, p=0.002). Conclusions 1. TP53 alterations were observed in 13.2% of CLL patients, 6.7% showed both a deletion and a mutation, while 1% showed a deletion only and 5.5% a mutation only. 2. Both TP53 mutations and TP53 deletions are associated with an unmutated IGHV status. 3. TP53 deletions had the most adverse impact on survival, TP53 mutations had a significant impact on OS only if the mutation load was ≥20%. A small subset of patients with TP53 deletion sole and a mutated IGHV status seems to have a favorable outcome. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Worseg:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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In CLL with Normal Karyotype By Conventional and Genomic Array Karyotyping the Prognostic Impact of an Unmutated IGHV Status Is Stronger Than the Impact of Gene Mutations

Blood ◽

10.1182/blood.v128.22.4357.4357 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4357-4357 ◽

Cited By ~ 1

Author(s):

Calogero Vetro ◽

Torsten Haferlach ◽

Manja Meggendorfer ◽

Sabine Jeromin ◽

Constance Regina Baer ◽

...

Keyword(s):

Gene Mutation ◽

Mutation Rate ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Genomic Array ◽

Equity Ownership ◽

The Impact

Abstract Background: In 15-20% of CLL cases no aberrations are detected by chromosome banding analysis (CBA) and FISH due to limited resolution, lack of evaluable metaphases or presence of aberrations in loci not covered by standard-panel FISH probes. As reported in our previous study (Haferlach C. et al., ASH 2015, abs ID#79545), genomic arrays (GA) detected abnormalities in almost 20% of cases classified as normal by CBA and FISH and these showed an impact on time to first treatment (TTT) (Vetro C. et al., EHA 2016, abs ID# E1069). The CLL subgroup without abnormalities in CBA, FISH, and GA has not been characterised in detail, so far. Aims: 1) to describe CLL without abnormalities by CBA/FISH/GA by evaluating an extended gene panel, the IGHV mutation status and the B-cell receptor (BCR) stereotypy; 2) to determine prognostic impact of these factors. Patients and Methods: CLL diagnosis was based on cytomorphology and immunophenotyping according to standard guidelines. From a cohort of 1190 patients at diagnosis, 133 (11%) were selected based on normal karyotype by CBA, no abnormalities by interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH-CCND1) and no abnormalities by GA (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). IGHV mutation status and BCR stereotypy were determined according to Agathangelidis et al., Blood 2012, and DNA sequencing was performed for the following genes: ATM; SF3B1; TP53; KLHL6; KRAS; MYD88; NOTCH1; NRAS; POT1; FBXW7; HIST1H1E; XPO1; ITPKB; MAPK1; BIRC3; BRAF; DDX3X; EGR2; RIPK1; RPS15; CND2. Results: Median age was 66 years (range: 33-83). Median follow-up was 5.6 years, 33 patients (25%) received treatment since genetic analyses. 10-year overall survival (OS) was 76% and median TTT was 9.2 years. Mutations were observed in 53 patients (40%): SF3B1 (n=17; 13%); NOTCH1 (n=10; 8%); KLHL6 (n=6; 5%); TP53 (n=6; 5%); ATM (n=5; 4%); XPO1 (n=4; 3%); FBXW7 (n=3; 2%); MYD88 (n=3; 2%); DDX3X (n=2; 2%); POT1 (n=2; 1.5%); ITPKB (n=1; 1%); KRAS (n=1; 1%); NRAS (n=1; 1%); and no mutation in RPS15, CCND2, MAPK1, EGR2, BRAF, HIST1H1E, RIPK1, BIRC3. 6 patients had 2 simultaneous gene mutations and 1 patient had 3 (i.e. NOTCH1, ATM and TP53). A mutated IGHV status (IGHV-M) was present in 100 patients (75%) and an unmutated IGHV status (IGHV-U) in 33 patients (25%). IGHV-U was related to both the occurrence of any gene mutation (p<0.001) and the number of gene mutations (p=0.001). NOTCH1 was mutated in 7 out of the 33 IGHV-U patients (21%), but only in 3 out of 99 IGHV-M patients (3%) (p=0.001). XPO1 mutation occurred in 4 IGHV-U patients (12%) and none out of IGHV-M (p<0.001). Two IGHV-U patients showed POT1 mutation (6%), but no IGHV-M case (p=0.014). 9 patients out of 133 (7%) showed BCR-stereotypy. 2 were in cluster CLL#1 (both showing NOTCH1 mutation), 2 in cluster CLL#2 (both of them with SF3B1 mutation), 2 in CLL#4, 1 in CLL#8 (showing NOTCH1 and XPO1 mutations), 1 in CLL#201 (with KLHL6 mutation) and 1 in CLL#202 (with mutations in ATM, TP53 and NOTCH1 genes). In Kaplan-Meier analysis, IGHV-M patients did not reach a median TTT, while IGHV-U had a median of 5.1 years (p<0.001). Stereotypy rate was too low for reliable statistics. At univariate analysis, TTT was only influenced by: IGHV-U (relative risk (RR): 3.9, p<0.001), TP53 mutation (RR: 3.7, p=0.03), % CLL cells (RR: 1.2 per 10% increase, p=0.013), and number of mutations (RR: 1.8 per each mutation, p=0.031). Multivariate Cox regression analysis showed an independent role for IGHV-U status (RR: 3.3, p=0.002) and % CLL cells (RR: 1.2 per 10% increase, p=0.038) Only age showed an impact on OS (RR: 1.2 per decade, p<0.001). Conclusions: 1. The CLL subset without any genomic event by CBA/FISH/genomic array is characterized by very low frequency of IGHV-U status; 2. IGHV-U subgroup showed higher gene mutation rate compared to IGHV-M subgroup, in particular higher NOTCH1, XPO1 and POT1 mutation rate; 3. BCR stereotypy is less frequent than in CLL in general. 4. IGHV-U, as well as the higher disease burden (i.e. % CLL cells), has an independent negative impact on TTT. 5. Requirement for treatment is low and prognosis very favorable in CLL without any genomic event by CBA/FISH/genomic array and a mutated IGHV status. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Prognostic Value of “Monosomal Karyotype” in Comparison to “Complex Aberrant Karyotype” in AML: A Study on 824 Cases with Aberrant Karyotype

Blood ◽

10.1182/blood.v118.21.418.418 ◽

2011 ◽

Vol 118 (21) ◽

pp. 418-418

Author(s):

Claudia Haferlach ◽

Tamara Alpermann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Cox Regression ◽

Complex Karyotype ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Cox Regression Analysis ◽

Poor Risk ◽

Risk Patients ◽

Equity Ownership ◽

The Impact ◽

Monosomal Karyotype

Abstract Abstract 418 Background: Several classifications based on cytogenetics have been proposed in AML. Typically 3 major categories for prognostication are defined: favorable, intermediate and unfavorable. The assignment to the unfavorable group shows minor differences between the different cytogenetic classifications currently used, however certain cytogenetic subgroups are assigned to the unfavorable subgroup concordantly: −5/5q−, 7q−/−7, −17/abn17p, inv(3)(q21q26)/t(3;3)(q21;q26) and complex karyotype (CK). With respect to CK 3 definitions are used: ≥3, ≥4 or ≥5 unrelated abnormalities. Recently, a so-called “monosomal karyotype” (MSK) defined as a karyotype showing “two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in the presence of structural abnormalities” was introduced (Breems et al. JCO 2008). It was suggested that patients with MSK have a poor outcome being even inferior to CK. Aim: We here evaluated the prognostic power of differently defined cytogenetic subsets in order to identify the best definition for the prognostically most unfavorable subgroup. Patients: From our initial cohort of newly diagnosed AML (n=1,959) patients with t(15;17), t(8;21) or inv(16) (n=170) and AML with normal karyotype (n=965) were excluded. Thus, 824 patients with cytogenetic abnormalities remained for further investigation. Results: 428/824 (51.9%) patients showed an intermediate risk karyotype according to revised MRC criteria (MRC-I) (Grimwade et al. Blood 2010), while the remaining 396/824 (48.1%) cases belonged to the unfavorable MRC group (MRC-U). 162/824 cases (19.7%) fulfilled the criteria of MSK. According to MRC, 4 of these 162 cases with MSK were classified MRC-I while 158 were classified MRC-U. The overlap in classification between CK and MRC-U differed depending on the number of aberrations used to define CK. As such, the number of cases with CK was 272 (33.0%; MRC-I: 17, MRC-U: 255) using ≥3 clonal aberrations, and decreased to 222 (26.9%; all MRC-U) patients using ≥4 clonal aberrations or 196 (23.8%; all MRC-U) cases when applying the criterion of ≥5 clonal aberrations, respectively. Univariate Cox regression analysis revealed that unfavorable cytogenetics as defined by MRC-U, MSK, CK defined as ≥3, ≥4 or ≥5 unrelated abnormalities were all significantly associated with inferior OS as compared to the respective remaining intermediate group (for all p<0.001). Hazard ratios were 1.61, 1.93, 1.68, 1.94, and 1.92, respectively. Median OS in the respective categories was 8.5, 5.7, 6.3, 5.7, and 5.7 months, respectively. We then performed further analyses within the unfavorable risk group defined according to MRC and tested the impact of the 4 definitions for unfavorable subsets. In each comparison the median OS was significantly shorter for the subset with MSK, or CK defined as ≥3, '4 or ≥5 unrelated abnormalities as compared to the remaining MRC-U cases (5.7 vs 11.7 mo p=0.005; 6.3 vs 10.6 mo, p=0.031; 5.7 vs 11.0 mo, p=0.003; 5.7 vs 10.9 mo, p=0.006). Furthermore OS of patients within MRC-U excluding cases with MSK, or CK with ≥3, ≥4 or ≥5 unrelated abnormalities did not differ from patients with cytogenetic abnormalities assigned to MRC-I (median OS 11.7, 10.6, 11.0 and 10.9 mo, respectively vs 21.1 mo, p=0.072, p=0.16, p=0.28, and p=0.11, respectively). Within the MRC-U cohort only 124 cases fulfilled both criteria: MSK and CK≥4 (median OS 5.3 mo), 97 were CK≥4 only (median OS 6.3 mo) and 35 MSK only (median OS 6.7 mo). OS did not differ between these 3 subgroups but was significantly shorter for all comparisons to patients included in none of these subgroups (p<0.001, p=0.009, p=0.012, respectively). On the other hand OS of the 33 cases with 3 unrelated abnormalities did not differ from MRC-U cases with 1 or 2 abnormalities (18.9 vs 10.6, p=0.48). Conclusions: All definitions of very poor risk AML patients allow to identify a subset within MRC-U that shows significantly shorter OS than the remaining MRC-U cases. However, “complex karyotype defined as ≥4 unrelated abnormalities” is the best parameter as it identifies the largest proportion of very poor risk patients. Even more important, the application of the monosomal karyotype for prognostication and clinical guidance in AML misses 24.5% of the very poor risk patients identified based on CK ≥4. This may lead to suboptimal treatment decisions in this clinically proven very high risk patients. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Evaluation of the New Genetic Risk Classification of the European LeukemiaNet Recommendations in 1,110 Patients with De Novo AML and Proposal of a Refined Version

Blood ◽

10.1182/blood.v118.21.413.413 ◽

2011 ◽

Vol 118 (21) ◽

pp. 413-413

Author(s):

Tamara Alpermann ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Molecular Markers ◽

De Novo ◽

Prognostic Impact ◽

Employment Equity ◽

Female Ratio ◽

Mutation Status ◽

Equity Ownership ◽

De Novo Aml ◽

Favorable Group ◽

Refined Version

Abstract Abstract 413 Background: The recently published recommendations for prognostication in AML (Döhner et al. for ELN, Blood 2010;115,453–474) were based on a review of the literature and included cytogenetics as well as NPM1, CEBPA and FLT3-ITD mutation status for risk stratification. We here aimed to evaluate the prognostic impact of this approach in an independent cohort. Patients: We started with a cohort of 1,428 adults with newly diagnosed AML, which were investigated by cytomorphology, immunophenotyping, cytogenetics, and molecular genetics. We first excluded patients with t(15;17) (n=59), therapy-associated AML (n=111) and secondary AML (n=148). Thus, 1,110 patients with de novo AML and cytogenetics available in all cases were further evaluated according to ELN criteria. The following molecular markers were investigated: NPM1 (1,064/1,110), FLT3-ITD (1,066/1,110), CEBPA (880/1,110), MLL-PTD (1,064/1,110) and RUNX1 (454/1,110). Results: Male/female ratio was 1.2 (598/512), median age was 66.6 years (range 18.3 – 100.4). According to the ELN proposal 297 (26.8%) pts were assigned to the favorable group (CBF leukemias, NPM1mut/without FLT3-ITD in normal karyotype (NK), or CEBPAmut in NK), 363 (32.7%) pts were classified as intermediate I (NPM1mut/FLT3-ITD+, or NPM1wt/FLT3-ITD+, or NPM1wt without FLT3-ITD; all NK), 249 (22.4%) as intermediate II (t(9;11) or cytogenetic abnormalities not classified as favorable or adverse), and 201 (18.1%) as adverse (inv(3)/t(3;3); t(6;9); t(v;11)(v;q23); −5 or del(5q); −7; abn(17p); complex karyotype, i.e. ≥ 3 chromosome abnormalities)). Evaluation according to these criteria revealed significant differences in overall survival between the favorable subgroup and all other subgroups (inter I p<0.001; inter II 0.008, adv <0.001). Also adverse vs all other subgroups (all p<0.001) differed significantly with respect to OS. However, no significant differences were observed between both large cohorts of inter I and inter II (together 55.1% of all pts). We therefore intended to revise the ELN criteria for better discrimination of the intermediate groups. In addition to ELN recommendations we considered a threshold of 0.5 for the FLT3-ITD ratio (mut/wt) which had been suggested more valid for prognostication than the mutation status per se. For the revised classification molecular markers were mandatory for all cases with intermediate risk cytogenetics. Therefore, 100 cases had to be excluded due to missing data. Thus, 1,010 pts were reclassified into our new subgroups defined as: favorable I: CBF leukemias; favorable II:NPM1mut or biallelic CEBPAmut (without any other molecular marker and no fav or adv cytogenetics); intermediate I:FLT3-ITD ratio <0.5 (without RUNX1 or MLL-PTD and no fav or adv cytogenetics); intermediate II:FLT3-ITD ratio ≥0.5 and/or RUNX1mut and/or MLL-PTD+ (and no fav or adv cytogenetics); adverse: as defined by ELN. Patients were distributed as follows: fav I: 68 (6.7%), fav II: 286 (28.3%), inter I: 157 (15.5%), inter II: 298 (29.5%), adv: 201 (19.9%). Fav I and fav II had no significant differences in OS (median n.r. vs 62.2 mo, n.s.) and therefore were grouped together as “favorable”. This finally leads to four different prognostic subgroups: favorable: CBF leukemias; NPM1mut or biallelic CEBPAmut, intermediate I:FLT3-ITD ratio <0.5, intermediate II:FLT3-ITD ratio ≥0.5 and/or RUNX1mut and/or MLL-PTD+, adverse. Patients were distributed as follows: fav: 354 (35.0%), inter I 157 (15.5%), inter II: 298 (29.5%), adv: 201 (19.9%). Median OS differed between all subgroups: fav 62.2, inter I 24.3, inter II 12.4, adv 8.7 mo. (fav vs inter I p=0.058, vs inter II <0.001, vs adv <0.001; inter I vs inter II 0.004, vs adv <0.001; inter II vs adv 0.039). Conclusion: The new ELN proposal for prognostication in de novo AML is based on cytogenetic and molecular genetic data. Based on this proposal we further improved prognostication in a series of 1,010 pts by integrating the following molecular markers besides cytogenetics: NPM1mut, biallelic CEBPAmut and FLT3-ITD ratio <0.5 for the favorable group and FLT3-ITD ratio ≥0.5, other CEBPAmut, MLL-PTD+, or RUNX1mut for the intermediate group, and adverse based on cytogenetics only. This refined version may contribute to a better risk assessment in de novo AML patients allowing to separate 4 subgroups with striking differences in OS. Disclosures: Alpermann: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v118.21.3870.3870 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3870-3870

Author(s):

Claudia Haferlach ◽

Melanie Zenger ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Median Time ◽

Chromosome Banding ◽

Clonal Evolution ◽

Complex Karyotype ◽

Employment Equity ◽

Mutation Status ◽

Time Points ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Equity Ownership

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

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IGHV Mutation Status Does Not Add Prognostic Information In The Background Of Mutations In TP53 and SF3B1, and Deletions Of 17p and 11q, Which Are Independent Adverse Prognostic Parameters In CLL

Blood ◽

10.1182/blood.v122.21.4110.4110 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4110-4110 ◽

Cited By ~ 1

Author(s):

Claudia Haferlach ◽

Sabine Jeromin ◽

Sandra Weissmann ◽

Frank Dicker ◽

Wolfgang Kern ◽

...

Keyword(s):

Multivariate Analysis ◽

Genetic Markers ◽

Prognostic Markers ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Status Group ◽

Group A ◽

Equity Ownership ◽

Group B

Abstract Background In CLL, the IGHV mutation status is an established prognostic marker. However, associations between the IGHV mutation status and other genetic markers have been reported. Based on genome sequencing data, novel recurrent mutations have been identified in CLL and first data on their prognostic impact is also available. Aims In this study we addressed the following questions in a large cohort of 934 CLL patients: 1. Associations of IGHV mutation status with cytogenetic and molecular genetic markers 2. Age dependency of IGHV mutation status, cytogenetic and molecular mutations 3. Impact on survival. Patients and Methods In total, in 934 CLL patients the IGHV mutation status was determined. Median age was 66.8 years (range: 29.6 - 90.5 years). Further, all patients were analyzed by DNA sequencing for mutations in TP53, SF3B1, MYD88, XPO1, NOTCH1 and FBXW7 and by FISH for TP53 deletion status as well as for del(13q), del(11q) and +12. Results In the entire cohort 550/934 (58.9%) patients showed a mutated and 384 (41.1%) an unmutated IGHV status. Molecular mutations were observed in the following frequencies: NOTCH1: 12.6%, SF3B1: 11.9%, TP53: 9.2%, XPO1: 4.0%, FBXW7: 2.6%, and MYD88: 1.7%. Patients showed deletions 13q in 58.7% (13q as the sole abnormality in 45.5%), trisomy 12 in 15.6%, 11q in 12.1%, and 17p in 5.8%, respectively. The following genetic aberrations were more frequent in CLL with unmutated vs mutated IGHV status: TP53mut (14.8% vs 5.3%), SF3B1mut (19.0% vs 6.9%), XPO1mut (9.1% vs 0.4%), NOTCH1mut (24.2% vs 4.5%), del(17p) (10.7% vs 2.4%), del(11q) (24.2% vs 3.6%), +12 (21.4% vs 11.6%) (for all p<0.001). In contrast, del(13q) (45.8% vs 67.6%), del(13q) sole (26.3% vs 58.9%) (for all p<0.001), and MYD88mut (0.3% vs 2.7%, p=0.004) were less frequent in CLL with unmutated IGHV status. No association between age and IGHV status was observed. With respect to all other analyzed markers only the frequency of TP53mut and del(17p) increased significantly with age. In univariate analysis the following parameters were significantly associated with shorter overall survival (OS): IGHV unmutated (HR: 2.0), age (≤70 vs >70 yrs: HR:3.4), TP53mut (HR: 3.8), SF3B1mut (HR: 2.3), del(17p) (HR: 6.2), del(11q) (HR: 2.3) (for all p<0.001), and NOTCH1mut (HR: 1.5, p=0.05), while del(13q) sole was associated with longer OS (HR: 0.6; p=0.003). In multivariate analysis the following parameters were independently associated with shorter OS: age (≤70 vs >70 yrs: HR: 3.1; p<0.001), TP53mut (HR: 2.0; p=0.018), SF3B1mut (HR: 2.1; p=0.001), del(17p) (HR: 3.6, p<0.001), del(11q) (HR: 2.1; p=0.005), while IGHV mutation status surprisingly did not show an independent impact on OS. Next we separated the cohort according to the number of adverse prognostic markers TP53mut, SF3B1mut, del(11q), and del(17p) (group A: 0 marker (n=658), group B: 1 marker (n=197), group C: 2 or more markers with alterations (n=78)). Kaplan-Meier-analysis revealed a 5 yrs OS of 89.5% in group A, of 71.3% in group B and of 37.4% in group C (for all comparisons p<0.001). Neither within groups A, B nor within group C did the IGHV mutation status have an impact on OS. On the other hand, OS at 5 yrs differed significantly according to the group within patients with either mutated IGHV or unmutated IGHV status (mutated IGHV status: group A (n=467) 90.6%, group B (n=67) 73.7% and group C (n=16) 47.2%; A vs B: p=0.031; B vs C: p=0.022; unmutated IGHV status: group A (n=191) 86.8%, group B (n=130) 69.6% and group C (n=62) 35.8%; A vs B: p=0.006; B vs C: p<0.001). Interestingly, a significant association between a higher number of adverse prognostic markers and age was observed (p=0.019). Further we performed multivariate analysis with the number of adverse prognostic factors (TP53mut, SF3B1mut, del(11q), del(17p): 0 markers to 4 markers positive) and age per decade. Both parameters were independently associated with OS (HR: 2.3 per adverse marker positive and HR: 1.7 per decade, for both p<0.001). Conclusions 1. Several adverse prognostic markers (TP53mut, SF3B1mut, NOTCH1, del(17p), del(11q)) were associated with an unmutated IGHV status. 2. However, in the background of the genetic markers TP53mut, SF3B1mut, del(11q), or del(17p) IGHV status lost its independent prognostic impact. 3. An accumulation of adverse prognostic markers worsens prognosis in CLL. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker: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.

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Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Blood ◽

10.1182/blood.v128.22.4288.4288 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4288-4288 ◽

Cited By ~ 1

Author(s):

Claudia Haferlach ◽

Anna Stengel ◽

Manja Meggendorfer ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Overall Survival ◽

Relative Risk ◽

Molecular Genetic ◽

Normal Karyotype ◽

Prognostic Impact ◽

Complex Karyotype ◽

Wild Type ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p<0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p<0.001), SRSF2 (27% vs 12%, p<0.001), and ZRSR2 (15% vs 3%, p<0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p<0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p<0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was < 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p<0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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