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

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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WT1 Mutations Are Secondary Events In AML and Show Varying Frequencies Within Genetic Subgroups and Different Impact On Prognosis

Blood ◽

10.1182/blood.v122.21.2542.2542 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2542-2542

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Frank Dicker ◽

Madlen Ulke ◽

...

Keyword(s):

Multivariate Analysis ◽

Median Time ◽

Univariate Analysis ◽

Normal Karyotype ◽

Adverse Impact ◽

Prognostic Impact ◽

Employment Equity ◽

Genetic Aberrations ◽

Equity Ownership ◽

The Impact

Abstract Background Mutations (mut) in the WT1 gene belong to the first genetic aberrations described in AML. In contrast to recurrent fusion genes or NPM1mut WT1mut do not seem to be disease defining. Also in contrast to other mutations in AML, for most of which a certain prognostic value has been established, the impact of WT1mut still is discussed controversially. Aim Analyze the frequency and prognostic impact of WT1 mutations in comparison to other genetic aberrations. Patients and Methods 3,157 unselected AML patients (pts) were analyzed (de novo: n=2,699, s-AML: n=234, t-AML: n=224). 1,708 pts were male and 1,449 female. Median age was 67.1 years (y) (range: 17.8-100.4 y) with 1,108 pts <60 y and 2,049 ≥60 y. The mutational hot spot regions of WT1 (exons 7 and 9) were analyzed by direct Sanger sequencing with a sensitivity of ∼10%. Karyotype and WT1 mutation status was available in all cases. Other mutations were assessed in subsets: ASXL1 (n=1,951), CEBPA (n=2,670), DNMT3A (n=1,293), FLT3-ITD (n=3,149), FLT3-TKD (n=3,004), IDH1R132 (n=2,431), IDH2R140 (n=2,380), IDH2R172 (n=2,412), KRAS (n=1,409), NRAS (n=1,780), NPM1 (n=3,003), MLL-PTD (n=2,961), RUNX1 (n=2,390), TET2 (n=1,016) and TP53 (n=1,215). Results A total of 189 WT1 mutations were detected (exon 7: n=151, exon 9: n=38). The total frequency of WT1mut pts was 175/3,157 (5.5%). 11 pts were double to quadruple mutated. The frequency was heterogeneous with respect to AML subtypes. Compared to all others, significantly higher frequencies were detected in biallelic CEBPAmut (15/110; 13.6%; p=0.001), followed by t(15;17)/PML-RARA (18/164; 11.0%, p=0.004), and FLT3-ITD (58/682; 8.5%, p<0.001). Lower frequencies were observed in DNMT3Amut (18/412; 4.3%, p=0.014, ASXL1mut (6/355; 1.7%, p<0.001), IDH2R140 (5/286; 1.7%, p=0.001), and IDH1R132 (2/222; 0.9%, p<0.001). WT1mut were never detected in pts with complex karyotypes (0/175; p=0.047) or those with IDH2R172 (0/68; p=0.020). Further, WT1mut were more frequent in females (95/1,449, 6.6%) than in males (80/1,708, 4.7%) (p=0.014) and in younger pts (<60 y: 102/1,108, 9.2% vs ≥ 60 y: 73/2,049, 3.6%; p<0.001). Median age of pts with WT1mut was 55.5 y compared to 63.6 in WT1wt (p<0.001). Further, WT1mut were associated with lower platelet count (58.4 vs 84.7 x109/L; p<0.001) and lower hemoglobin level (8.8 vs 9.3 g/dL, p=0.001). There was no association to the history of the disease or white blood cell count. Stability of WT1mut was analyzed in 35 paired diagnostic and relapse samples (median time of relapse after diagnoses: 11.1 months (m); range: 2.6-60.6 m). In 23 cases (65.7%) the WT1mut was retained at relapse and in 12 cases (34.3%) it was lost. In 5 cases a sample at 2nd relapse was available (median time from 1st relapse: 8.5 m, range: 6.0-18.0 m). 3 of these cases retained and 2 lost the WT1mut. Analysis of prognostic impact was restricted to intensively treated pts (n=1,936, WT1mut: n=132, 6.8%). In the total cohort, there was no impact of WT1mut on prognosis. In pts ≥60 y there was a trend to shorter event free survival (EFS) for WT1mut (9.3 vs 12.3 m, p=0.052). In the two prognostically favorable groups with high WT1mut incidences (biallelic CEBPAmut and PML-RARA) no effect on outcome was seen. When restricting the analysis to normal karyotype AML (WT1mut: n=85, WT1wt: n=1,093) WT1mut pts had shorter EFS (10.8 vs 17.9 m, p=0.008). This was true for the younger (12.2 vs 29.0 m, p=0.007) as well as for the older pts (9.3 vs 13.9 m, p=0.016). In a multivariate analysis all parameters with significant impact on EFS in univariate analysis were included: age (p<0.001, HR: 1.24), ASXL1mut (p<0.001, HR: 1.36), FLT3-ITD (p<0.001, HR: 1.55), NPM1mut/FLT3-ITD wild-type (p<0.001, HR:1.55), RUNX1 (p=0.019, HR: 1.23, and WT1mut (p=0.009, HR: 1.64). In multivariate analysis WT1mut was found to have independent adverse impact on EFS (p=0.002, HR: 1.64) besides FLT3-ITD status (p<0.001, HR: 1.71) and age (p<0.001, HR: 1.28). Conclusions WT1 mutations are 1) more frequent in females and younger AML, 2) more frequent in t(15;17)/PML-RARA, biallelic CEBPAmut, FLT3-ITD mutated AML, and nearly mutually exclusive of ASXL1, IDH1, IDH2 and complex karyotype. 3) The distribution pattern in different genetic subtypes and the instability during follow-up as shown by paired sample analyses clearly emphasize a secondary character of this mutation. 4) For AML with normal karyotype an independent adverse impact of WT1mut on EFS was shown. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Kern: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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Comprehensive Molecular Analyses of BCR-ABL1 Negative MPN Show That PMF Is Genetically Much More Heterogeneous Than ET and PV

Blood ◽

10.1182/blood.v124.21.3224.3224 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3224-3224

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Normal Karyotype ◽

Great Majority ◽

Primary Myelofibrosis ◽

Gene Panel ◽

Prognostic Impact ◽

Prognostic Information ◽

Employment Equity ◽

Equity Ownership

Abstract Introduction: In the WHO classification (2008) JAK2 and MPL mutations are major criteria for the diagnosis of myeloproliferative neoplasms (MPN): polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET). Cytogenetic aberrations are rare in these entities. Although the prognostic impact of JAK2 mutations beside some other gene mutations has been shown in PMF patients, the driving events for establishing accelerated phase or blast crises are unknown. In recent years, novel molecular markers such as ASXL1, SRSF2, and CALR were identified and PMF was investigated in several studies. However, comprehensive mutational analyses of MPN entities in comparison to each other are still rare. Aim: To identify gene mutations beyond JAK2, CALR, and MPL using a 28 gene panel, and to compare mutational data with clinical data and prognostic information in order to identify a risk profile. Patients and Methods: We in the first step investigated 56 patients (19 ET, 18 PMF, and 19 PV; 21 females, 35 males) diagnosed by cytomorphology following WHO criteria and accompanied by genetic studies. All patients underwent mutation analyses by a 28 gene panel containing: ASXL1, BCOR, BRAF, CALR, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, NRAS, KRAS, MPL, NPM1, PHF6, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). BCR-ABL1 fusion transcripts were shown to be negative in all cases by PCR. Not yet described genetic variants (n=6) were excluded from statistical analyses. Cytogenetics was available in 55/56 cases and grouped in normal karyotype (n=45, 82%) or aberrant karyotype (n=10, 18%). Results: In the total cohort JAK2 (44/56, 79%) was the most frequently mutated gene, followed by TET2 (13/56, 23%), ASXL1 (11/56, 20%), SRSF2 (7/56, 13%), and CALR (6/56, 11%). All other analyzed genes showed mutation frequencies below 10% (10 genes) or even no mutation (13 genes). Analyzing the number of mutations per patient revealed that only 4 patients showed no mutation (4/56, 7%), the great majority had 1 mutation (19/56, 34%) and 2 mutations (23/56, 41%), while 5 patients showed 3 mutations (5/56, 9%), 4 patients had 4 (4/56, 7%) and 1 patient even 5 mutations (1/56, 2%). Accordingly, the mean number of mutations per patient was 1.9. Summing up the mutations in JAK2, CALR, and MPL resulted in 52/56 (93%) patients that had a mutation in at least 1 of these genes, indicating that most of the patients had just 1 or 2 additional gene mutations to one of the 3 known key player MPN genes (mean: 1.3 additional mutations). Cytogenetically there were no significant differences between the 3 entities in frequencies of normal (65-90%) and aberrant karyotypes (11-35%), although in the PMF cohort there were more aberrant karyotypes (6/17, 35%) in comparison to ET and PV (for each 2/19, 11%). Addressing the mutation patterns of these 3 MPN entities revealed similar frequencies of TET2 mutations. In contrast, as expected JAK2 was more often mutated in PV (18/19, 95%) compared to ET (12/19, 63%, p=0.042) and PMF (14/18, 78%) and CALR was more often mutated in ET (5/19, 26%) in comparison to PMF (1/18, 6%) and PV (0/19, 0%, p=0.046). In PMF ASXL1 (8/18, 44%) and SRSF2 (6/18, 33%) were more often mutated compared to ET (1/19, 5%, p=0.008; 1/19, 5%, p=0.042) and PV (2/19, 11%; p=0.029; 0/19, 0%; p=0.008), respectively. Investigating the numbers of mutated genes per patient resulted in a significantly different distribution within MPN entities: in the ET and PV cohorts patients carried mostly 1 or 2 mutations (36/38, 95%; mean: 1.5), while in PMF 9/18 (50%) patients carried >2 mutations (mean: 2.5; p=0.045). Looking at the affected genes besides JAK2 and CALR showed that in ET and PV 4 more genes were affected, while in PMF 11 different additional genes showed mutations, indicating that PMF is genetically much more heterogeneous than ET or PV. This nicely matches to the finding that PMF is also marked by the highest cytogenetic aberration rate of these 3 BCR-ABL1 negative MPN (24-42%). Conclusions: 1)JAK2 is the most and TET2 the second most frequently mutated gene in BCR-ABL1 negative MPN. 2) Most patients carry only 1 or 2 gene mutations. 3) However, PMF patients are genetically much more heterogeneous than ET and PV patients regarding both cytogenetic and molecular alterations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment; Novartis: Research Funding. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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IDH Mutations Can Be Detected In 28.7% of All Normal Karyotype AML and Have Unfavourable Impact on the NPM1+/FLT3-ITD- Genotype

Blood ◽

10.1182/blood.v116.21.102.102 ◽

2010 ◽

Vol 116 (21) ◽

pp. 102-102

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Hot Spot ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Therapeutic Decisions ◽

Idh Mutations ◽

Equity Ownership ◽

De Novo Aml ◽

The Impact

Abstract Abstract 102 Introduction: Mutations in IDH1 and IHD2 have recently been shown to play an important role in AML. As they code for enzymes from the citric acid cycle mutations within these genes from the mechanistical point of view are a totally new kind of mutation associated with AML. In IDH1 one mutational hot spot (amino acid R132) and in IDH2 two hotspots (R140 and R172) have been reported. We aimed at further delineating the impact of IDH1 and IDH2 mutations in AML and analyzed the interaction with other mutations in normal karyotype (NK) AML. Methods: 526 AML patients were selected according to normal karyotype and availability of mutational status for FLT3-ITD, NPM1 and MLL-PTD. Further mutation analyses were available in subgroups of the cohort (FLT3-TKD: n=318, CEBPA: n=369, RUNX1: n=174, NRAS: n=220). Female/male ratio was 283/243 and age ranged from 20.0–90.1 years (median, 66.9 years). 435 had de novo AML (82.6%), 71 AML following MDS (s-AML,13.5%) and 20 AML after previous treatment of other malignancies (t-AML, 3.8%). The respective base exchanges in R132, R140, and R172 were analysed by a melting curve assay with subsequent sequencing of the positive samples. Results: Overall, in 151 pts (28.7%) IDH mutations (IDHmut) were detected. In detail, 68 mutations (12.9% of all cases) were detected in IDH1 (R131C: n=35, R131L: n=17, R131H: n=7, R131G: n=6, R131S: n=3) and 83 mutations (15.8%) in IDH2 (R140Q: n=72, R140L: n=2, R140W: n=1, N141G: n=1, R174K: n=7). IDH1mut and IDH2mut were mutually exclusive in this cohort. IDH1mut were more frequent in females (18.2% vs 8.6 % in males, p=0.001), whereas there was no sex difference for IDH2. According to history IDH1 was equally distributed in de novo AML, s-AML and t-AML whereas IDH2 was more frequent in de novo compared to s- and t-AML (19.6% vs. 7.6 vs 11.8%, p=0.048). According to FAB the most prevalent subtype was FAB M1 with IDHmut in 23.2% compared to 9.8% in all other FAB (in detail: IDH1: 44.8% vs. 23.9%, IDH2: 27.0% vs. 15.1%; p<0.001, for both). IDH1 was underrepresented in M4 (4.9% vs. 15.0 % in all other subtypes, p=0.004), whereas the distribution of IHD2 was not different in M4 vs. all others. The immunophenotype (n= 297) of IDHmut cases tended to be more immature and featured a lower expression of monocytic markers. The analyzed 78 IDHmut cases, as compared to 219 IDHwt cases, showed a significantly higher expression of MPO and CD117 while CD116, CD11b, CD14, CD15, CD36. CD56, CD64, CD65 and CD7 were lower expressed. Age, WBC count, and platelet count were not different between IDH1, IDH2 and IDHwt cases. IDH mutations are not mutually exclusive of other mutations. However, the frequency of CEBPAmut in IDHmut compared to IDHwt was decreased (7.7% vs. 13.7, p=0.001) (IDH1: 0% vs 11.7%, p=0.022 and IDH2: 7.7% vs 13.4%, p=0.053). MLL-PTD was more frequent in IDHmut vs. IDHwt (44.7 vs. 5.8%, p=0.039), however, this is restricted to IDH1mut vs. IDH1wt (26.3 vs. 6.3%, p=0.018). RUNX1mut are distributed equally in IDH2mut and IDH2wt (20.0% vs 27.3%) but are underrepresented in IDH1mut compared to IDH1wt (2.2% vs. 28.7%, p=0.068). FLT3-ITDs are equally distributed between IDHmut and IDHwt, however, those IDH1mut with FLT3-ITD have lower FLT3-ITD/FLT3wt ratios compared to FLT3-ITD+ IDH1wt cases (mean: 0.16 vs. 0.72; p=0.005). All other mutations were distributed equally in IDHmut compared to IDHwt. For survival analysis only cases with de novo AML <65 years were included (n=164, IDHmut: n=37, n=, IDHwt: 127). In the total analysis there was no effect on overall survival or event free survival (EFS). However there was a trend for shorter EFS of the IDHmut vs. IDHwt (median: 439 days vs. not reached, p=0.080) in cases with NPM1+/FLT3-ITD- genotype. For IDH2 there was a significant adverse effect in the NPM1+/FLT3-ITD- group (median EFS: 397 vs. 679 days, p=0.045). Summary: IDH mutations belong to the most frequent mutations in NK AML and can occur together with all other known mutations. There is a high preponderance for the FAB M1 subtype and a more immature immunophenotype for both IDH mutations and a strong female preponderance for IDH1. In addition, an adverse prognostic impact of IDH mutations was shown for the NPM1+/FLT3-ITD- genotype. Further analyses should focus on the definition of the role and place of IDH mutations for therapeutic decisions in patients with AML. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann: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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Molecular Analyses of MDS/MPN Overlap Entities According to WHO Classification Reveal a Distinct Molecular Pattern for MDS/MPN, Unclassifiable

Blood ◽

10.1182/blood.v124.21.4618.4618 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4618-4618

Author(s):

Manja Meggendorfer ◽

Torsten Haferlach ◽

Sabine Jeromin ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Gene Mutation ◽

Who Classification ◽

Gene Mutations ◽

Normal Karyotype ◽

Employment Equity ◽

Ras Pathway ◽

Molecular Abnormalities ◽

Molecular Pattern ◽

Equity Ownership ◽

Stat Pathway

Abstract Introduction: The World Health Organization (WHO) classification defines myelodysplastic/myeloproliferative neoplasms (MDS/MPN) based on clinical, morphologic, and laboratory findings that show features of MDS and characteristics more consistent with MPN. This category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). In recent years RARS-T, CMML, and also aCML were deciphered by several molecular studies, while MDS/MPN, U cases warrant closer investigations. Aim: To comprehensively investigate mutations in 17 genes known to be mutated in aCML, CMML, MDS/MPN, U, and RARS-T and to define entity specific mutation patterns in comparison to cytogenetic and clinical data. Patients and Methods: We investigated 179 patients diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO criteria: 35 patients were diagnosed as aCML, 58 as CMML, 39 as MDS/MPN, U, and 47 as RARS-T. All patients underwent mutation analyses by a gene panel containing: ASXL1, TET2, DNMT3A, SRSF2, SF3B1, U2AF1, JAK2, CALR, MPL, NRAS, KRAS, CBL, BRAF, CSF3R, RUNX1, SETBP1, and NPM1. Gene mutations were analyzed by Sanger sequencing, next generation sequencing, melting curve analyses, or gene scan. Cytogenetics was available in 172/179 cases and was grouped as normal karyotype (n=128, 74%) or aberrant karyotype (n=44, 26%). Results: In the total cohort the most frequently mutated gene was ASXL1 (41%), followed by TET2 (40%), and the spliceosomal genes SF3B1 (31%) and SRSF2 (30%). Also frequently mutated were JAK2 (21%), NRAS (15%), RUNX1 (12%), and CBL (12%). All other investigated genes showed mutation frequencies below 10%. There were no significant differences between the 4 entities regarding frequencies of aberrant karyotypes (14-37%) and no correlation of the number of molecular mutations (0-6/patient) with any specific karyotype. Addressing the mutation patterns of these 4 entities showed that ASXL1 and TET2 are frequently mutated in all entities (19-60% and 26-53%, respectively), although significant differences between the entities exist (see figure): ASXL1 is less frequently mutated in RARS-T (19%) in comparison to aCML (60%; p<0.001) and CMML (52%; p=0.001), TET2 is more often mutated in CMML (53%) in comparison to MDS/MPN, U (26%; p=0.007) and RARS-T (32%; p=0.031). SRSF2 is more frequently mutated in CMML (53%) than in RARS-T (9%; p<0.001) and MDS/MPN, U (15%; p<0.001), SF3B1 is more often mutated in RARS-T (92%) than in all other entities (aCML: 11%, CMML: 5%, MDS/MPN, U: 13%; for all p<0.001). One important difference between aCML and CMML versus MDS/MPN, U and RARS-T was reflected by two different signaling pathways: i) JAK2/CALR/MPL (JAK/STAT pathway) were significantly more often affected in MDS/MPN, U (33%) and RARS-T (53%), (aCML: 9%, CMML: 7%; p<0.001). ii) NRAS/KRAS/CBL (RAS pathway) were more often mutated in aCML (37%) and CMML (52%), (MDS/MPN, U: 5%, RARS-T: 9%; p<0.001). The MDS/MPN, U cohort included most patients with no mutation in any analyzed gene (11/39, 28%) in contrast to aCML (2/23, 6%), CMML (5/58, 9%), and RARS-T (0/47, 0%). Furthermore all these MDS/MPN, U patients with no gene mutation had a normal karyotype. Looking at co-ocurrences of gene mutations in MDS/MPN, U revealed that SRSF2 and TET2 mutations occur together more frequently (4/10 vs. 2/29 in TET2wt; p=0.028). Of notice, in MDS/MPN, U U2AF1 (18%) was the most frequently mutated spliceosomal gene which was only rarely mutated in the other entities (5%, p=0.015). Conclusions: 1) ASXL1 and TET2 are the most frequently mutated genes found overall in MDS/MPN overlap. 2) SF3B1 mutations are specific for RARS-T. 3) SRSF2 is most frequently mutated in CMML, but also in aCML. 4) MDS/MPN, U is affected by mutations in all spliceosomal genes. 5) The JAK/STAT pathway is more often affected in MDS/MPN, U and RARS-T. 6) The RAS pathway is more often affected in aCML and CMML. 7) MDS/MPN, U shows a specific molecular pattern with characteristics reflecting a mixture of all other MDS/MPN entities. Red: gene mutation, orange: gene mutations combined, light grey: no mutation/normal karyotype, black: aberrant karyotype, white: not analyzed. Figure: Molecular abnormalities and cytogenetics in MDS/MPN entities. Figure:. Molecular abnormalities and cytogenetics in MDS/MPN entities. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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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 ◽

10.1182/blood.v116.21.1676.1676 ◽

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.

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Specific Molecular Mutations with Prognostic Relevance in AML with Normal Karyotype Are Also Associated with Outcome in AML with Aberrant Karyotype: A Study on 1981 Cases,

Blood ◽

10.1182/blood.v118.21.3517.3517 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3517-3517

Author(s):

Claudia Haferlach ◽

Torsten Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Susanne Schnittger

Keyword(s):

Important Implication ◽

Mutation Screening ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Equity Ownership ◽

The Impact ◽

Length Mutations ◽

Tandem Duplications

Abstract Abstract 3517 Background and Aim: The karyotype and molecular mutations are well established prognostic parameters in AML. However, the impact on outcome of molecular mutations has been evaluated mainly in the subset of AML with normal karyotype (NK). The aim of this study was 1. to determine the frequency of NPM1 mutations (NPM1 mut), partial tandem duplications within the MLL gene (MLL -PTD), length mutations within the FLT3 gene (FLT3 -ITD) and CEPBA mutations (CEPBA mut) in distinct cytogenetic subgroups and 2. to evaluate the prognostic impact of these mutations in relation to chromosome abnormalities. Patients and Methods: 1981 patients with AML and evaluable cytogenetics were included. Mutation data was available in the majority of cases: NPM1: 1646, CEPBA: 1324, FLT3 -ITD: 1726 and MLL -PTD: 1656. Based on the karyotype the cohort was subdivided according to revised MRC criteria (Grimwade et al. Blood 2010) and in addition into distinct cytogenetic subgroups. Results: According to cytogenetics 170 cases were assigned to the favorable MRC class (MRCF), 1414 to the intermediate MRC (MRCI) and 397 to the unfavorable MRC subset (MRCU). The frequency of NPM1 mut, CEPBA mut, MLL -PTD and FLT3 -ITD differed significantly between MRC classes and distinct cytogenetic groups. In the MRCI subset we evaluated overall survival (OS) between patients with normal or aberrant karyotype (AK) within the respective mutation groups. Within NPM1 mut, CEPBA mut, MLL -PTD+ and FLT3 -ITD+ patients no significant differences in OS were observed between patients with NK or AK. When separating CEPBA mut into biallelic (n=52) and monoallelic cases (n=40) also no difference in OS was observed between pats with NK or AK. Next we tested the prognostic impact of the respective molecular mutations within MRCI including 966 cases with NK and 448 cases with AK. OS was significantly longer in patients with NPM1 mut or CEPBA mut (median OS (mOS) 49.6 months (mo) vs 18.6 mo, p=0.003; not reached (n.r.) vs 21.1 mo, p=0.016) and significantly worse for patients with MLL -PTD or FLT3 -ITD (mOS 10.8 vs 23.0 mo, p=0.039; 13.8 vs 24.9 mo, p=0.003). Analyzing biallelic and monoallelic CEPA mut separately revealed that only biallelic CEPBA mut was associated with a longer OS (p=0.006). Restricting the analysis to MRCI patients with aberrant karyotype revealed a longer OS for patients with NPM1 mut/FLT3 -ITD-, and a shorter OS for patients with FLT3 -ITD (mOS n.r. vs 18.0 mo, p=0.033; 5.7 vs 23.0 mo, p=0.015). A trend towards better OS was observed for biallelic CEBPA mut. Conclusions: 1. The frequency of molecular mutations varies significantly between distinct cytogenetic subsets. They are particularly common in AK within MRCI. NPM1 mut, CEBPA mut and MLL -PTD were not observed in MRCF, in AML with 11q23/MLL -rearrangements, or in 3q26/EVI1 -rearrangements. Their frequency was below 5% in AML with complex karyotype. 2. The outcome of NPM1 mut, CEPBA mut and MLL -PTD+ cases was not different in AML with normal or aberrant karyotype within MRCI. 3. This data suggest to extend mutation screening for NPM1 mut, CEPBA mut, MLL -PTD and FLT3 -ITD to all AML with intermediate risk cytogenetics, as they are significantly associated with outcome not only in AML with normal karyotype but also in AML with cytogenetic abnormalities assigned to MRCI. This consequently will lead to better characterization of a reasonable number of cases from MRCI with important implication on treatment. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comparison Of Mutation Patterns Between Diagnosis and Relapse In 556 Adult Patients With AML Shows High Variability Of Stability

Blood ◽

10.1182/blood.v122.21.4978.4978 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4978-4978

Author(s):

Susanne Schnittger ◽

Niroshan Nadarajah ◽

Tamara Alpermann ◽

Christiane Eder ◽

Alexander Kohlmann ◽

...

Keyword(s):

Negative Impact ◽

Melting Curve ◽

Normal Karyotype ◽

Genetic Alterations ◽

Prognostic Impact ◽

Fusion Genes ◽

Employment Equity ◽

Disease Evolution ◽

Equity Ownership ◽

The Impact

Abstract Background In acute myeloid leukemia (AML) molecular mutations are becoming increasingly important as markers for classification, risk stratification and disease monitoring. Frequencies and prognostic impact of most of the currently known mutations have been widely studied. In contrast, the stability during disease evolution and the role of single markers at relapse are less clear. Aim 1) To compare the patterns of molecular mutations between diagnosis and relapse. 2) To analyze the impact of single mutations on time to relapse (TTR). Patients and Methods We investigated paired diagnostic and relapse samples in a cohort of 556 adult AML cases that were selected based on confirmed relapse with available samples from both time points (483 de novo, 33 t-AML, 40 s-AML). The cohort comprized 255 females and 301 males; median age: 63.0 years (range: 18.6-85.2 years). In total, 5,726 paired analyses were performed (mean: 10.3/pt, range: 2-21). Besides analyses for fusion genes (n=114) the following genes were analyzed in paired samples for mutations in respective numbers: ASXL1: n=464, CBL: n=99, CEBPA: n=374, DNMT3A: n=244, FLT3-ITD: n=534, FLT3-TKD: n=461, IDH1: n=469, IDH2: n=418, KIT: n=34, KRAS: n=49, MLL-PTD: n=165, NPM1: n=343, NRAS: n=105, RUNX1: n=356, TET2: n=243, WT1: n=153, others: n=1,101. Mutations were analyzed by amplicon deep-sequencing, direct Sanger sequencing, gene scan, conventional PCR, quantitative real time PCR or melting curve analyses. In addition, chromosome banding analysis data was available in 552 cases. Results 629 relapses were detected in the 556 patients (pts). 67 pts had a second and 6 even a third relapse. At diagnosis the subtypes according to cytogenetics were as follows: PML-RARA (n=10), RUNX1-RUNX1T1 (n=24), CBFB-MYH11 (n=23), MLL-translocations (n=30), DEK-CAN (n=3), other recurrent translocations (n=14), complex karyotype (n=30), other aberrations (n=107), normal karyotype (NK, n=311). In 515/556 (92.6%) pts at least one mutation (mut) was detected at diagnosis (mean: 2.3; range:1-5). In detail, the most frequent markers were mut in: NPM1: n=233, FLT3-ITD: n=217, fusion genes: n=114, DNMT3A: n=101, RUNX1: n=98, TET2: n=81, FLT3-TKD: n=58, ASXL1: n=54, IDH2R140: n=46, MLL-PTD: n=44, WT1: n=42, TP53: n=26, biallelic CEBPA(bi): n=28, monoallelic CEBPA(mono): n=23. At relapse a similar incidence of 88.6% (558/630) mutated pts (mean: 2.0: range 1-6) was detected. However, the mutational pattern changed at relapse in 381/629 (60.6%) of the pts. On the marker level a change was seen for 309 (24.5%) of the mut, with gain of 142/1,263 new mut (11.3%) and a loss of 167 (13.2%) mut that were detectable at diagnosis. Different degrees of stability were observed. Compared to all other markers a stable pattern was found for CEBPAbi: p=0.025; NPM1: p<0.001; MLL-PTD: p=0.006; and fusion genes: p<0.001. This is supporting the respective definition of entity defining mut in the WHO classification. Instead, a low stability (for gain and/or loss) compared to all others was seen for CEBPAmono: p=0.002, FLT3-ITD: p<0.001, FLT3-TKD: <0.001, NRAS: p<0.001, TP53: p<0.001 (only for gain), WT1: p<0.001. This is in accordance with the concept of typical “secondary” mutations, leading to acceleration of the disease. An intermediate stability was detected for TET2, DNMT3A, RUNX1, ASXL1,IDH2R140 (see figure). Of note, of 200 FLT3-ITD positive pts that retained an FLT3-ITD at relapse, only 6 (3%) were stable with respect to mutational load whereas 134 (67%) showed an increase of the FLT3-ITD/wildtype load, and 60 (30%) a decrease showing a further quality of genetic instability. Between the various disease entity defining groups there were no relevant differences in TTR. In contrast, an impact on shorter TTR was seen for mutations in DNMT3A (median TTR: 8.7 vs 12.1 months (m), p=0.058), FLT3-ITD (7.6 vs 12.2 m, p<0.001), RUNX1mut (10.1 vs 12.8 m, p=0.038) and TET2mut (9.9 vs 10.9 m, p=0.049). In the normal karyotype group an effect was only seen for FLT3-ITD (7.3 vs 13.6, p<0.001) and TET2mut (9.9 vs 12.3 m, p=0.025). Conclusions 1) Genetic alterations in AML can be subdivided into stable, intermediate and unstable markers. Entity defining markers according to WHO (fusion genes, NPM1mut, CEBPAbi) are stable between diagnosis and relapse. 2) A significant negative impact on TTR was shown only for secondary mutations (FLT3-ITD, TET2mut, DNMT3Amut and RUNX1 mut). Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern: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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Comprehensive Genetic Analyses Of 213 PMF Patients Revealed 13% Of SRSF2 Mutations Being Strongly Correlated To ASXL1 Mutations

Blood ◽

10.1182/blood.v122.21.2828.2828 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2828-2828

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Christiane Eder ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Marker ◽

Scoring System ◽

Sanger Sequencing ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Wild Type ◽

Employment Equity ◽

Cytogenetic Aberrations ◽

Equity Ownership

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm, characterized amongst others by stem-cell derived clonal myeloproliferation, bone marrow fibrosis, anemia, splenomegaly, constitutional symptoms and leukemic progression. Diagnosis is based in most cases on cytomorphology/histology demonstrating fibrosis as well as on mutations in JAK2 or MPL. The Dynamic Prognostic Scoring System (DIPSS)-plus is the current base for prognostication using different clinical parameters including karyotype. Furthermore, molecular genetic alterations are currently addressed to provide additional prognostic information. Recently, besides JAK2 and MPL further gene mutations have been described in a limited number of patients, including ASXL1 and SRSF2. Aim To analyze in a large cohort the frequency of SRSF2 and ASXL1 mutations in PMF patients, and to identify their prognostic impact in the context of other previously described gene mutations. Patients and Methods Diagnosis was done according to WHO classification. The cohort comprised 82 female and 131 male patients. In all cases a BCR-ABL rearrangement was excluded by RT-PCR or fluorescence in situ hybridization. JAK2V617F mutation was analyzed in all cases by melting curve analysis, MPLW515 mutation was subsequently analyzed in JAK2V617 wild type (wt) patients. In addition, we analyzed all patients for SRSF2 mutations by Sanger sequencing of the mutational hot spot region coding for amino acid Pro95. Cytogenetics was available in 139 patients. Patients were grouped in favorable (n=121) and unfavorable (n=18) karyotypes based on the DIPSS-plus-scoring system. Based on the previously described correlation of SRSF2mut with ASXL1mut and SETBP1mut in other myeloid entities, SRSF2 mutated cases were also analyzed for mutations in ASXL1 and SETBP1by Sanger sequencing. Follow-up data was available for 136 patients. Results 56% (120/213) of the patients showed JAK2V617F mutations and 18.0% (16/89) of JAK2wt patients carried a mutation in MPLW515 summing up to 65.1% of patients with an already established molecular marker. Of note, SRSF2 was mutated in 12.7% (27/213) of all PMF patients. Patients with SRSF2 mutation had higher white blood cell counts in comparison to SRSF2wt patients (20.00x109/L vs. 7.35x109/L; p=0.005), but there was no correlation to gender, age, hemoglobin level, platelet count or % of myeloblasts in the peripheral blood. In 17 SRSF2mut cases the karyotype was available, 12 were normal karyotype, while two cases showed an unfavorable karyotype according to DIPSS-plus with +8 and i(17)(q10), respectively. The remaining three aberrations belong to the favorable aberration group (del(20q), del(13q), and der(14)). There was no correlation of SRSF2 mutations to the cytogenetic subgroups normal karyotype (n=91) or DIPSS categories favorable and unfavorable aberrations. SRSF2 mutations were also equally distributed between both JAK2V617 or MPLW515 mutated and wild type cases. 18/27 SRSF2mut cases carried also either a JAK2 or MPL mutation, while 9 cases showed no additional JAK2 or MPL mutation. Therefore 30.6% patients remain that carry no mutation in at least one of the three genes investigated first. Interestingly, ASXL1 was frequently mutated in SRSF2 mutated patients (16/23 analyzed SRSF2mut patients) while none of the 24 analyzed SRSF2 mutated cases showed a mutation in SETBP1. To evaluate a potential influence of gene mutations on clinical outcome the overall survival (OS) was calculated. We could confirm that JAK2V617F had no prognostic impact. The same was true for MPLW515 mutations. In contrast to other studies we could not find any impact of SRSF2mutations on OS. Only cytogenetics, i.e. the normal karyotype showed a trend to a prognostic relevance: the median 3 year OS was 70.8% in patients with normal karyotype (n=56) but 58.8% in patients with cytogenetic aberrations (n=29; p=0.153). Conclusion 1) SRSF2 is mutated in 13% of PMF patients. 2) SRSF2 mutated patients show frequently an additional ASXL1 mutation but no coincidence with SETBP1. 3) The prognostic relevance of cytogenetic aberrations was confirmed, while the molecular marker SRSF2 shows no impact on prognosis. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Impact of Somatic Gene Mutations on Response to Lenalidomide (LEN) in IPSS Lower-Risk Myelodysplastic Syndromes (MDS) Patients (Pts) without Del(5q) and Ineligible for or Refractory to Erythropoiesis-Stimulating Agents (ESAs)

Blood ◽

10.1182/blood.v128.22.225.225 ◽

2016 ◽

Vol 128 (22) ◽

pp. 225-225 ◽

Cited By ~ 2

Author(s):

Valeria Santini ◽

Pierre Fenaux ◽

Aristoteles Giagounidis ◽

Uwe Platzbecker ◽

Alan F List ◽

...

Keyword(s):

Lower Risk ◽

Research Funding ◽

Response Rate ◽

Single Gene ◽

Gene Mutations ◽

Advisory Board ◽

Employment Equity ◽

Mutation Status ◽

Somatic Gene ◽

Equity Ownership

Abstract Background: Somatic gene mutations occur in the majority of MDS pts; specific mutations and high mutation frequency have prognostic relevance (Papaemmanuil et al. Blood. 2013;122:3616-27). Evaluation of somatic mutations may support the diagnosis of MDS and guide treatment (Tx) selection. The phase 3 randomized MDS-005 study compared LEN and placebo (PBO) Tx in red blood cell transfusion-dependent (RBC-TD) non-del(5q) lower-risk MDS pts ineligible for or refractory to ESAs. Deletions in chromosome 5q are associated with a high response rate to LEN in MDS pts; however, no mutations have been definitively associated with a predictable clinical response to LEN in non-del(5q) MDS. Aim:To investigate the relationship between somatic gene mutations detected by targeted next-generation sequencing (NGS) and response and overall survival (OS) in lower-risk non-del(5q) MDS pts treated with LEN in the MDS-005 study. Methods: Eligible pts were: RBC-TD (≥ 2 units packed RBCs/28 days 112 days immediately prior to randomization) with International Prognostic Scoring System defined Low-/Intermediate-1-risk non-del(5q) MDS; ineligible for ESA Tx (serum erythropoietin > 500 mU/mL); or unresponsive or refractory to ESAs (RBC-TD despite ESA Tx with adequate dose and duration). 239 pts were randomized 2:1 to oral LEN 10 mg once daily (5 mg for pts with creatinine clearance 40-60 mL/min) or PBO. DNA was isolated from bone marrow mononuclear cells or whole blood collected at screening from a subset of pts who gave informed consent for this exploratory biomarker analysis and had adequate tissue for analysis. Targeted NGS of 56 genes was performed at Munich Leukemia Laboratory; average sequencing coverage was 2,000-5,000-foldand the variant allele frequency detection cutoff was 3%. Target regions varied by gene, including all exons to hotspots. For association tests, mutant variants (heterozygous or homozygous) were scored as 1 (mutant) or 0 (wildtype) for gene-level analyses. A Fisher exact test was used to test association of mutation status with response. Median OS was calculated by the Kaplan-Meier method. Hazard ratios and 95% confidence intervals were determined by a non-stratified Cox proportional hazards model. A log-rank test was used to test treatment effect with OS for single gene mutation status. Results: The biomarker cohort included 198 of 239 pts (83%; LEN n = 130, PBO n = 68). At least 1 mutation was detected in 30/56 (54%) genes and 173/198 (87%) pts. The most frequently mutated genes were SF3B1 (59%), TET2 (33%), ASXL1 (23%), and DNMT3A (14%); the most frequent co-mutations were SF3B1/TET2 (23%), SF3B1/DNMT3A (10%), SF3B1/ASXL1 (10%), and TET2/ASXL1 (9%) (Figure). Of 116 pts with SF3B1 mutations, 115 (99%) had ≥ 5% ring sideroblasts. The 56-day RBC transfusion-independence (RBC-TI) response rate was significantly lower in LEN-treated ASXL1 mutant pts vs wildtype pts (10% vs 32%, respectively; P = 0.031). At 168 days, the RBC-TI response rate was still lower in LEN-treated ASXL1 mutant pts vs wildtype pts (7% vs 22%); however, the difference was not significant (P = 0.101). LEN-treated DNMT3A mutant pts had a higher 56-day RBC-TI response rate vs wildtype pts (44% vs 25%); however, this difference did not reach significance (P = 0.133) due to the small sample size. RBC-TI response rate with LEN was similar regardless of total number of mutations per pt. Higher numbers of mutations were significantly associated (P = 0.0005) with worse median OS. Mutation in any of the genes associated with a negative prognosis reported by Bejar et al. (N Engl J Med. 2011;346:2496-506) was also significantly associated (P = 0.0003) with worse median OS.However, OS was not significantly different in LEN- vs PBO-treated pts based on any single gene mutation status. Conclusions: In this group of lower-risk RBC-TD non-del(5q) MDS pts, somatic mutations in genes recurrently mutated in myeloid cancers were detected in 87% of pts. SF3B1 mutations (alone or in combination) were most frequent and not associated with response to LEN. ASXL1 mutant pts had a significantly lower LEN response rate vs wildtype pts, whereas DNMT3A mutant pts had a trend for improved LEN response. Median OS was influenced by mutations, but not significantly modified by LEN. Determining predictive clinical markers for Tx response in non-del(5q) MDS pts remains challenging; nevertheless, there is a significant need to identify pt subsets who may be responsive to LEN Tx. Figure. Figure. Disclosures Santini: Novartis: Consultancy, Honoraria; Amgen: Other: advisory board; Onconova: Other: advisory board; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astex: Other: advisory board. Fenaux:Celgene, Janssen, Novartis, Astex, Teva: Research Funding; Celgene, Novartis, Teva: Honoraria. Giagounidis:Celgene Corporation: Consultancy. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding. Zhong:Celgene Corporation: Employment, Equity Ownership. Wu:Celgene Corporation: Employment, Equity Ownership. Mavrommatis:Discitis DX: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Beach:Celgene Corporation: Employment, Equity Ownership. Hoenekopp:Celgene Corporation: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties, Research Funding.

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Characterization and Prognostic Impact of Multiple Productive IGHV Rearrangements in CLL

Blood ◽

10.1182/blood.v128.22.3207.3207 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3207-3207

Author(s):

Sabine Jeromin ◽

Claudia Haferlach ◽

Frank Dicker ◽

Manja Meggendorfer ◽

Torsten Haferlach ◽

...

Keyword(s):

Prognostic Factors ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Treatment Naïve ◽

Trisomy 12 ◽

Equity Ownership ◽

The Impact

Abstract Background: In chronic lymphocytic leukemia (CLL) one of the strongest prognostic factors is IGHV mutational status. Infrequently, patients present not only with a single IGHV rearrangement but with multiple productive rearrangements. In about 2% of all CLL patients analyzed on cDNA level multiple rearrangements display the same mutational status and are categorized accordingly following ERIC recommendations. In another 1% rearrangements with discordant IGHV mutational status are detected and preclude a definite risk assignment. Only limited data exist on these rare subgroups. Aim: To characterize treatment-naive CLL patients with multiple productive IGHV rearrangements and determine the impact on prognosis. Patients and Methods: Out of 8,016 treatment-naive CLL patients between 2005 and 2015 and with data on IGHV mutational status we identified 204 (3%) with multiple productive rearrangements. IGHV mutational status was analyzed on cDNA and in all cases according to ERIC recommendations. IGHV mutated status (M) was defined by sequence identity <98% and unmutated status (U) by ≥98%. Chromosome banding analysis was available in 102 cases and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 in 191. Male:female ratio was 3:1 and median age 68 years (range: 38-89). Additionally, data on SF3B1 and TP53 mutations was present in all cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 105 cases with a median follow-up of 4 years. For statistical comparison we used a cohort of 1,262 untreated CLL patients with single IGHV rearrangement (median age: 67 years; range: 30-91, median follow-up: 6 years). Results: Out of 204 patients with multiple, productive rearrangements 199 (98%) presented with two and 5 patients (2%) with three IGHV rearrangements. Concordant IGHV mutated status (MM) was present in 120 cases (59%), whereas concordant unmutated status (UU) was seen in 34 patients (17%). In 50 cases (25%) a mixed IGHV status (UM) was detected. We analyzed frequencies of complex karyotype by CBA, biclonality according to immunophenotype (concurrent kappa restricted and lambda restricted subpopulations) and/or CBA, TP53 disruption (TP53mut and/or del(17p)), SF3B1mut, del(11q), trisomy 12, and del(13q). Overall, a higher frequency of biclonality was detected in patients with multiple vs. single IGHV rearrangements (16% vs. 1%, p<0.001). However, association to neither MM, UU nor UM existed. MM presented with molecular and cytogenetic characteristics similar to M. Correspondingly, UU showed similar frequencies of mutations and aberrations to U, except for higher frequency of trisomy 12 in UU vs. U (42% vs. 19%, p=0.003). Interestingly, UM presented with characteristics similar to U and UU. UM was associated with TP53 disruption vs. M (16% vs. 5%, p=0.003) and vs. MM (5%, p=0.035) as well as with SF3B1mut vs. M (16% vs. 5%, p=0.008). Furthermore, UM cases showed high frequency of del(11q) vs. M (29% vs. 3%, p<0.001) and vs. MM (1%, p<0.001) and less frequently del(13q) sole vs. M (41% vs. 60%, p=0.011) and MM (41% vs. 69%, p=0.001). No significantly differences in TTT were observed between MM and M (median: 13 vs. 14 years) and between UU and U (6 vs. 4 years), respectively. However, the difference between MM vs. UU (p=0.022) and M vs. U (p<0.001) was significant. The UM subgroup presented with a TTT (median: 4 years) similar to U and UU, whereas it was significantly shorter vs. M (p=0.003) and MM (p=0.006), respectively. A similar picture emerged for survival. 5-year OS of MM was not different vs. M (94% vs. 90%) but vs. U (78%, p=0.001). The statistical analysis of OS in UU was hampered by low case numbers. UM presented again with similar 5-year OS vs. U (81% vs. 78%, n.s.) and significantly worse OS vs. M (90%, p=0.049) and vs. MM (94%, p=0.014). Conclusions: (1) Patients with multiple productive IGHV rearrangements and concordant IGHV status show similar prognosis and characteristics to patients with single rearrangement with the respective IGHV status. (2) Cases with mixed IGHV status show similar prognosis to patients with IGHV unmutated status and accordingly are characterized by high frequencies of adverse prognostic factors like TP53 disruption, SF3B1mut, and del(11q), whereas del(13q) sole is less frequent. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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