Myeloid Malignancies with Isolated 7q Deletion Can be Further Characterized By Their Accompanying Molecular Mutations

Abstract Background: 7q deletions (del(7q)) are recurrent cytogenetic abnormalities. They occur either as the sole abnormality or accompanied by additional chromosome aberrations in AML, MDS, MDS/MPN and MPN. Cases with del(7q) as the sole abnormality are rare and poorly characterized. Aim: In patients with myeloid malignancies and del(7q) as the sole abnormality we determined 1. Type and size of the del(7q) 2. Spectrum of accompanying molecular mutations and their impact on the phenotype. Patients and Methods: 81 cases with myeloid malignancies and del(7q) as the sole abnormality were included in this study. Of these 38 had AML (27 de novo, 7 secondary, 4 therapy-related), 17 MDS (14 de novo, 3 therapy-related), 10 MDS/MPN (9 CMML, 1 MDS/MPN unclassifiable) and 16 MPN. The median age was 72 years (range: 29-89 years). All cases were investigated by array CGH (Agilent, Waldbronn, Germany) and for mutations in ASXL1, CALR, CBL, DNMT3A, ETV6, EZH2, JAK2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Results: Array CGH revealed an interstitial del(7q) in 67 cases, while 14 cases showed terminal del(7q). Further characterization of these deletions using 24 color FISH revealed unbalanced translocations in 10 of the 14 cases with terminal deletion. Partner chromosomes were X, 8, 9, 12, 13, 17 (n=2), 19 (n=2), and 22. The breakpoints on chromosome 7 were diverse ranging from 7q11 to 7q32. In two cases the breakpoint was within the CDK6 gene. In two cases with terminal del(7q) the complete loss of 7q was due to an idic(7)(q11.21). In the remaining two cases the terminal deletion could not be further resolved. In the 67 cases with interstitial del(7q) the size of the del(7q) varied between 1.8 and 158.9 Mb (median: 52.6 Mb). No commonly deleted region could be identified for all cases. However, in 57 cases the deleted region encompassed genomic position 101,912.442 (7q22.1) to 119,608.824 (7q31.31) including 111 genes. The size of the 7q deletion was smaller in cases with interstitial deletion as compared to terminal deletion (57.7 MB vs 70.9 MB, p=0.04) and in MPN as compared to all other entities (48.7 MB vs 62.8 MB, p<0.001). The mutation analyses revealed mutations in TET2 37% (25/67), ASXL1 35% (27/78), RUNX1 26% (18/69), DNMT3A 21% (14/68), SRSF2 18% (13/73), JAK2 V617F 14% (11/79), CBL 9% (7/75), NRAS 9% (7/77), MLL -PTD 5% (4/80), KRAS 5% (3/66), EZH2 4% (3/72), TP53 4% (3/74), SF3B1 4% (3/75), ETV6 3% (2/73), NPM1 3% (2/77), CALR 1% (1/77), MPL 1% (1/76). ASXL1 and TET2 were frequently co-mutated as 56% of ASXL1 mutated cases also harbored a TET2 mutation (p=0.02). 39 cases were analysed for all 16 molecular mutations. The majority of patients (n=27, 69%) had more than one mutation (range: 2-4), 9 patients (23%) had one mutation and in 3 patients (8%) no mutation was detected. The number of mutations per patient was lower in patients <70 years as compared to patients ≥70 years (0, 1,2,3,4 mutations detected in: 23%, 15%, 15%, 46%, and 0% vs 0%, 27%, 27%, 31%, and 15%, p=0.05). CBL mutations were most frequent in CMML (44%) but rare in all other subtypes (5%, p=0.003), while RUNX1 mutations were most frequent in AML (43% vs 9%; p=0.002) and JAK2 V617F mutations most frequent in MPN (50% vs 5%, p<0.001). DNMT3A mutations and MLL -PTD were significantly more frequent in de novo AML than in all other entities (43% vs 11%, p=0.007; 15% vs 0%, p=0.009), while no significant differences in frequency were observed between the different entities for any of the other mutations or the number of mutations per case. In CMML CBL mutations were associated with del(7q) (44%) as CBL mutations were present in only 17% of non del(7q) CMML (n=101, p=0.07). The frequency of RUNX1 mutations was significantly higher in AML with del(7q) as the sole abnormality (43%) as compared to all other AML (n=2273, 21%; p=0.001). Median overall survival (OS) for the total cohort was 25 months and did not differ significantly between AML, MDS, MDS/MPN and MPN (26, 27, not reached, 15 months, respectively). Conclusions: 1. Sizes and localisations of the del(7q) largely overlapped between AML, MDS, MDS/MPN and MPN. 2. 92% of all patients with 7q deletion harbored at least 1 molecular mutation. 3. TET2 and ASXL1 were the most frequently mutated genes and were present at comparable frequencies in all subtypes. 4. AML with del(7q) is closely associated with RUNX1 mutations while CMML with del(7q) frequently harbored CBL mutations suggesting a cooperative leukemogenic potential in these entities. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. 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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Mutations In SETBP1 Occur In 3.1% Of De Novo AML and Show a Distinct Genetic Pattern That Highly Resembles Atypical CML

Blood ◽

10.1182/blood.v122.21.2560.2560 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2560-2560

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Elisabeth Sirch ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Sanger Sequencing ◽

De Novo ◽

Complex Karyotype ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Myeloid Malignancies ◽

Genetic Pattern ◽

Equity Ownership ◽

De Novo Aml ◽

Atypical Cml

Abstract Introduction Recently, mutations in SETBP1 (SETBP1mut) have been identified in different myeloid malignancies. We previously determined mutation frequencies in the range of 5-10% in MPN and MDS/MPN overlap, while we found SETBP1 more frequently mutated in atypical CML (32%). SETBP1mut has been shown to associate with CBL and ASXL1 mutations, as well as the cytogenetic abnormalities -7 and i(17)(q10). While SETBP1 mutations have been detected in 3% of s-AML cases, so far no mutations of SETBP1 in de novo AML have been described. Aim To analyze the mutation frequency of SETBP1 mutations in de novo AML with corresponding cytogenetic abnormalities and their respective correlation to clinical data and other gene mutations. Patients and Methods We investigated 422 adult de novo AML patients, diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO classification. SETBP1 was analyzed by Sanger sequencing of the coding region for amino acids 800 to 935. The cohort comprised 229 males and 193 females, the median age was 65.8 years (range: 19.3 – 89.0). Cytogenetics was available in all 422 cases. Based on the previously described association of SETBP1mut with -7 and i(17)(q10) in other myeloid malignancies there was a selection bias to these karyotypes. Cases were grouped according to cytogenetic abnormalities: normal karyotype (n=88) and aberrant karyotype (n=334), i.e. i(17)(q10) (n=15), +14 (n=20), -7 (n=100), other abnormalities (n=129), and complex karyotype (n=114; 44 contained i(17)(q10), +14 or -7). Within the SETBP1mut cases the following molecular markers were analyzed: ASXL1, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1/2, KRAS, NRAS, NPM1, MLL-PTD, RUNX1, SRSF2, TP53 and WT1 by Sanger sequencing, next generation sequencing, gene scan or melting curve analyses. These data were also available in sub-cohorts of SETBP1 negative cases. Results In the total cohort mutations in SETBP1 were detected in 3.1% (13/422) of all cases. SETBP1mut patients were older (median age: 73.5 vs. 65.7 years; p=0.05) and showed a slightly higher white blood cell count (14.5 vs. 13.8x109/L; p<0.001). There was no correlation to gender, hemoglobin level and platelet count. However, analyzing the cytogenetic groups SETBP1mut showed, like in other myeloid malignancies, a strong co-occurrence with -7 and i(17)(q10), since 4/13 SETBP1 positive cases carried a monosomy 7, and 7/13 an i(17)(q10). The two remaining cases showed a trisomy 14 or a complex karyotype that also contained a i(17)(q10). No SETBP1mut was found in any other cytogenetic subgroup. Therefore, SETBP1mut correlated significantly with i(17)(q10) (8/15 i(17)(q10) were SETBP1mut vs. 5/407 in all other karyotypes; p<0.001). Further, we analyzed the association of SETBP1 mutations with other molecular markers. SETBP1mut correlated with ASXL1mut, 9/33 (27%) ASXL1mut patients showed a mutation in SETBP1, while only 2 (1%) showed a SETBP1 mutation in 229 ASXL1 wild type (wt) patients (p<0.001). This was also true for CBLmut, where 4/8 (50%) CBLmut cases were SETBP1mut, while only 8/158 (5%) were SETBP1mut in the group of CBLwt (p=0.001). This was even more prominent in SRSF2mut patients, where all 9 SRSF2mut were also SETBP1mut, while only 4/8 (50%) patients carried a SETBP1 mutation within the SRSF2wt group (p=0.029). In contrast, SETBP1mut were mutually exclusive of mutations in TP53 (0/67 in TP53mut vs. 12/194 in TP53wt; p=0.04), possibly reflecting the exclusiveness of TP53mut in i(17)(q10) patients. There was no correlation to any other analyzed gene mutation. Remarkably, while there was a high coincidence of SETBP1mut, SRSF2mut (9/13) and ASXL1mut (9/11), none of these patients showed mutations in the typical AML markers NPM1, FLT3-ITD, CEBPA, MLL-PTD, or WT1. Comparing the mutational loads of SETBP1, ASXL1 and SRSF2 resulted in SRSF2 having in most cases the highest mutational loads (range: 30-70%) while ASXL1 and SETBP1 showed equal or lower mutational loads (15-50% and 10-50%, respectively), possibly indicating that SRSF2 mutation is a former event followed by ASXL1 and SETBP1 mutation. Conclusions 1) For the first time we describe, that SETBP1 mutations occur in de novo AML. 2) SETBP1 mutations are correlated with a distinct genetic pattern with high association to i(17)(q10), ASXL1 and SRSF2 mutations and are mutually exclusive of TP53mut. 3) Thus, the genetic pattern of SETBP1 mutated AML highly resembles that of atypical CML. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Sirch: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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ETV6 Rearrangements Are Recurrent Genetic Events In Myeloid Malignancies and In AML Are Associated with NPM1- and RUNX1-Mutations and An Intermediate Prognosis

Blood ◽

10.1182/blood.v116.21.2758.2758 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2758-2758

Author(s):

Claudia Haferlach ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Normal Karyotype ◽

Employment Equity ◽

Childhood All ◽

Myeloid Malignancies ◽

Equity Ownership ◽

De Novo Aml ◽

Diagnostic Work ◽

Work Up

Abstract Abstract 2758 Introduction: The ETV6 gene (formerly TEL) is located in the chromosomal band 12p13 and is a frequent target of deletions and chromosomal translocations in both myeloid and lymphoid leukemias. In ALL the most frequent partner gene of ETV6 is RUNX1. ALL with ETV6-RUNX1 fusions are observed in 20% of childhood ALL and are associated with favorable outcome. In contrast ETV6 rearrangements are less frequent and not well described in myeloid malignancies. Therefore, the aim of this study was to analyze ETV6 rearrangements in myeloid malignancies with respect to frequency, partner genes and impact on prognosis. Patients/Methods: 55 cases with ETV6 rearrangements were identified in a total cohort of 9,550 cases (0.5%) with myeloid malignancies (de novo AML: n=3,090, s-AML: 486, t-AML: 222, MDS: n=3,375, MDS/MPN overlap: n=210, CMML: n=447, MPN: n=1,720) which had been sent to our laboratory between 08/2005 and 07/2010 for diagnostic work-up. In all cases chromosome banding analysis was performed and in cases with abnormalities involving 12p13 FISH was carried out in addition to verify the ETV6 rearrangement. Results: ETV6 rearrangements were observed in 31 patients with de novo AML (1.0% of investigated cases), 8 with s-AML (1.7%), 5 with t-AML (2.3%), 6 with MDS (0.2%) and 5 with MPN (0.3%). No ETV6 rearrangements were detected in the cohorts of MDS/MPN or CMML. ETV6 rearrangements were significantly more frequent in s-AML and t-AML as compared to de novo AML (p<0.001). Median age in AML was 59.9 years. In 15 cases with de novo AML FAB-subtypes were available: M0: n=8, M1: n=4, M2: n=1, M4: n=1, and M7: n=1. Thus, ETV6 rearrangements are closely related to immature AML subtypes. In 25/55 cases (45.5%) the ETV6 rearrangement was the sole abnormality. Recurrent additional abnormalities were 7q-/-7 in 10 cases and del(5q) in 8 cases. 36 different partners of ETV6 were observed, recurrent partners were located on 3q26 (EVI1, n=11), 5q33 (PDGFRB, n=4), 22q12 (n=3), 2q31 (n=2), 5q31 (ACSL6, n=2), 12p12 (n=2), 17q11 (n=2). Molecular analysis was performed in addition in AML with ETV6 rearrangements for mutations in NPM1 (n=26 investigated), FLT3-ITD (n=33), FLT3-TKD (n=11), MLL-PTD (n=25) and RUNX1 (n=7). NPM1-mutations were observed in 5 cases (19.2%), FLT3-ITD in 3 cases (9.1%), FLT3-TKD in 2 cases (18.2%), MLL-PTD in 1 case (4%) and RUNX1 mutations in 4 cases (57.1%), respectively. Clinical follow-up data was available of 47 cases. No differences in overall survival (OS) and event-free survival (EFS) were observed in cases with ETV6 rearrangement whether or not additional cytogenetic abnormalities or 7q-/-7 or del(5q) were present. Next 30 de novo AML with ETV6 rearrangement were compared to 819 AML without ETV6 rearrangement. Based on cytogenetics cases were assigned into 9 subgroups: 1) t(15;17)(q22;q21), n=48; 2) t(8;21)(q22;q22), n=29; 3) inv(16)(p13q22)/t(16;16)(p13;q22), n=19; 4) 11q23/MLL abnormalities, n=28; 5) inv(3)(q21q26)/t(3;3)(q21;q26), n=6; 6) normal karyotype, n=424; 7) complex karyotype, n=71; 8) other abnormalities, n=194 and 9) ETV6 rearrangements, n=30. Median OS was not reached for groups 1, 2, 3, 4, and 6 and was 10.6 mo, 11.8 mo, 32.2 and 26.3 mo for groups 5, 7, 8, and 9 respectively. OS at 2 yrs was 95.6%, 96.3%, 76.6%, 64.9%, 26.7%, 63.3%, 23.9%, 58.5% and 60.1% for groups 1–9, respectively. The respective data for median EFS were: not reached for groups 1 and 2 and 15.9 mo, 13.5 mo, 5.1 mo, 16.6 mo, 7.5 mo, 12.5 mo and 14.0 mo for groups 3–9, respectively. Conclusions: ETV6 rearrangements are rare in myeloid malignancies. ETV6 is rearranged with a large variety of partner genes. The highest frequency of ETV6 rearrangements was observed in s-AML and t-AML. OS and EFS of AML with ETV6 rearrangements are comparable to AML with normal karyotype. Thus, the detection of ETV6 rearrangements is associated with in intermediate prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. 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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FISH Analysis In 843 Cases with Myeloid Malignancies Revealed TET2 deletions In 6% of Patients Which Were Accompanied by TET2 Mutations In 51% of Cases

Blood ◽

10.1182/blood.v116.21.1706.1706 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1706-1706

Author(s):

Claudia Haferlach ◽

Sandra Wille ◽

Alexander Kohlmann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Copy Number ◽

De Novo ◽

Snp Array ◽

Suppressor Gene ◽

Employment Equity ◽

Myeloid Malignancies ◽

Tet2 Mutation ◽

Snp Array Analysis ◽

Equity Ownership ◽

De Novo Aml

Abstract Abstract 1706 TET2 (tet oncogene family member 2) on chromosome 4q24 was identified as a candidate tumor suppressor gene. Recurring submicroscopic deletions and copy-neutral loss of heterozygosity (CN-LOH) involving 4q in MDS patients detected by SNP microarray analyses suggested TET2 as an interesting candidate gene. Subsequent sequencing studies revealed TET2 mutations in 10–25% of patients with AML, MDS, and MPNs, while a mutation frequency of up to 42% was reported in CMML. Only a subset of studies evaluated both TET2 mutation status and TET2 copy number status, although this might be of pathophysiological and even of prognostic relevance if TET2 functions as a classical tumor suppressor gene. In the majority of studies copy number status was determined by SNP array analysis, although being expensive and time consuming. Here, in order to investigate TET2 deletions in a large cohort of patients an easy to perform FISH assay was developed applying BACs covering the TET2 gene (RP11-351K6 and RP11-16G16; BlueGnome, Cambridge, UK). This assay was validated on samples with TET2 deletions proven by SNP array analysis. With these FISH probes we analyzed 843 cases with myeloid malignancies (404 AML (323 de novo AML, 68 s-AML, 13 t-AML), 166 MDS, 201 CMML, and 72 MPN). Overall 50 (5.9%) cases with TET2 deletion were identified. These included 22 AML (5.0%), in detail 14 de novo AML (4.3%), 6 s-AML (8.8%), and 2 t-AML (15.4%) as well as 15 CMML (7.5%), 9 MDS (5.4%) and 4 MPN (5.6%). Patients with TET2 deletions showed the following karyotypes: normal: n=15, cytogenetically balanced rearrangements involving 4q24: n=3, 4q deletion as the sole abnormality: n=2, complex: n=25, other abnormalities: n=5. In 25/50 (50%) cases the TET2 deletion was cytogenetically cryptic. In patients with complex aberrant karyotype loss of 4q material was due to interstitial deletion in 7/25 cases and due to unbalanced rearrangements in 16/25 cases, while in 2/25 cases chromosomes 4 were normal in chromosome banding analysis. Furthermore, in patients with TET2 deletions mutation analyses was performed for mutations in TET2 (n=37 investigated), RUNX1 (n=13), NPM1 (n=18), JAK2V617F (n=18), CBL (n=36), NRAS (n=17), KRAS (n=36), FLT3-ITD (n=26), FLT3-TKD (n=8), IDH1 (n=9) and MLL-PTD (n=24). Mutations in TET2 were detected in 19/37 cases (51%), in RUNX1 in 1/13 (8%), in JAK2V617F in 6/18 (33.3%), in CBL in 2/36 (5.6%), in NRAS in 1/17 (6%), in KRAS in 1/36 (2.8%) and in NPM1 in 1/18 (6%) cases, whereas no mutations within the other genes analyzed were found. In the cohort of cases with TET2 deletion and concomitantly TET2 mutation (n=19) 10 had a normal karyotype (52.6%), 5 a complex karyotype (26.3%) and 4 had other abnormalities (21.1%). Importantly, in the cohort of CMML, in 10 of 14 cases (71.4%) both a TET2 deletion and TET2 mutation was detected. Overall, TET2 mutations were significantly more frequent in patients with cytogenetically cryptic TET2 deletion as compared to cytogenetically visible 4q deletions (68.2% vs. 26.7%, p=0.020). In addition FISH screening identified 2 cases (one CMML, one t-MDS) which showed a translocation involving the TET2 locus not leading to a deletion of the BAC signals but a separation suggesting a fusion with yet unidentified partner genes. In conclusion, FISH analyses identified TET2 deletions in 6% of myeloid malignancies. 50% of these deletions were submicroscopic and therefore not detectable by chromosome banding analysis. TET2 deletions were accompanied by TET2 mutations in 51% of respective cases. FISH is a reliable and efficient method to determine the copy number state of TET2. Still, the prognostic impact of TET2 deletions with and without additional TET2 mutations in the various myeloid malignancies has to be evaluated in future investigations. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Wille:MLL Munich Leukemia Laboratory: Employment. Kohlmann: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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Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

Blood ◽

10.1182/blood.v122.21.2582.2582 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2582-2582 ◽

Cited By ~ 1

Author(s):

Frauke Bellos ◽

Bruce H. Davis ◽

Naomi B. Culp ◽

Birgitte Booij ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Myeloid Leukemia ◽

De Novo ◽

Molecular Genetic ◽

Newly Diagnosed ◽

Employment Equity ◽

Equity Ownership ◽

De Novo Aml ◽

Very High ◽

Acute Myeloid

Abstract Background Nucleoside analogs depend on cellular hENT1 expression for entry into cells and cytotoxic activity. Studies suggest low cellular hENT1 levels correlate with poor response to such chemotherapies in solid tumors, data on AML and MDS is scarce. Aim To examine hENT1 expression by multiparameter flow cytometry (MFC) in newly diagnosed AML and MDS and correlate results to morphologic, cytogenetic (CG) and molecular genetic (MG) findings. To examine hENT1 expression with respect to clinical outcome in AML patients (pts) treated with intensive cytarabine-based chemotherapy (CHT). Methods We studied pts with newly diagnosed AML (n=145) and MDS (n=96), 133/108 male/female, median age 67.3 (AML) and 73.3 years (MDS). CG was done in 130 AML and 86 MDS. Pts included 107 de novo AML, 9 t-AML, 29 s-AML; FAB: 9 M0, 27 M1, 50 M2, 9 M3, 21 M4, 8 M4eo, 7 M5, 14 not classified; by CG (MRC): 21 favorable, 75 intermediate, 34 adverse. 91 were de novo MDS, 5 t-MDS; 1 RARS, 17 RCMD-RS, 37 RCMD, 3 5q- syndrome, 3 RAEB-1, 5 RAEB-2, 1 CMML, 24 not classified; 2 IPSS-R very low, 55 IPSS-R low, 8 IPSS-R intermediate, 8 IPSS-R high, 13 IPSS-R very high. hENT1 expression was quantified by a novel four color intracellular staining assay using monoclonal antibodies against hENT1, CD45, CD64 and myeloperoxidase. Median fluorescence intensities (MFI) of hENT1 were determined in myeloid progenitors (MP), granulocytes (G) and monocytic cells (Mo) and correlated to hENT1 MFI in lymphocytes to derive hENT1 index (index). Results No correlation of index to age, gender, hemoglobin level or counts for blasts, WBC or platelets was detected. In AML, we generally saw higher index by trend in the more favorable prognostic subgroups. M3/t(15;17)/PML-RARA+ displayed higher index in MP than non-M3 AML (4.24 vs 2.56, p<0.001). G index was lower in M0 (3.01) vs M1, M2, M4 and M4eo (5.66, 4.34, 5.35, 4.77; p=0.01, 0.028, 0.004, 0.043, respectively) and in M2 compared to M1 and M4 (4.34. vs 5.66 and 5.35, p=0.01 and 0.033, respectively). M2 showed lower MP index than M5 (2.42 vs 2.99, p=0.016). Considering CG, index in MP was higher in favorable vs intermediate and adverse pts (3.05 vs 2.58 and 2.53, p=0.034 and 0.023, respectively), Mo index was higher ín favorable vs adverse pts (3.17 vs 2.71, p=0.044). By MG, higher index in Mo and G was observed in RUNX1-RUNX1T1+ AML (4/83, 4.32 vs 3.04, p=0.01; 8.16 vs 4.60, p=0.002, respectively). Higher index for MP was found in FLT3-ITD mutated (mut) (18/111; 3.19 vs 2.62, p=0.012), CEPBA mut (4/26, 3.15 vs 2.35, p=0.004) and for Mo in NPM1 mut AML (23/104; 3.72 vs 2.84, p=0.02), whereas lower index for MP was found in RUNX1mut pts (13/65; 2.17 vs 2.59, p=0.031). De novo AML displayed higher MP index than s-AML (2.7 vs 2.28, p=0.008). Using lowest quartile of index for MP (2.1185) as cut-off, AML pts in the MRC intermediate group treated with CHT (n=38) had inferior OS if MP index was below vs above this cut-off (OS at 6 months 63% vs. 95%, p=0.017, median follow up 4.6 months). MDS showed lower Mo and MP index than AML (2.68 vs 2.96, p=0.021, 1.84 vs 2.65, p<0.001, respectively). By IPSS-R, significance was reached for higher index in Mo and MP in very low risk compared to low risk pts (3.39 vs 2.54, p=0.013 and 4.07 vs 1.78, p<0.001, respectively), MP in very low compared to intermediate and high risk pts (4.07 vs 1.95, p=0.004; 4.07 vs 1.76, p=0.002), and MP and G in very low vs very high risk pts (4.07 vs 1.71, p=0.005; 5.86 vs 3.85, p=0.001, respectively). IPSS-R intermediate vs poor and very poor showed lower G index (5.47 vs 3.59, p=0.018 and vs 3.85, p=0.034 respectively). Conclusion AML with genetic and molecular genetic good risk profile had higher hENT1 expression in MP, G and Mo, suggesting a causal mechanism for better response to CHT and better outcome. Consequently, AML with poor risk molecular genetics (RUNX1 mut) showed lower levels of hENT1 in MP. The detection of higher levels in FLT3-ITD mut pts is in line with reportedly good response to CHT, overall worse outcome being mostly due to early relapses. Strikingly, we saw differences in outcome in pts treated with CHT according to hENT1 expression with shorter OS in pts with low index for MP. Higher index in de novo AML than s-AML and MDS may be causal for better response to nucleoside-based CHT in de novo AML. Data for MDS may be interpreted accordingly, lower risk cases showing higher index in MP, G and Mo. Further analyses are needed to explore hENT1 expression in AML and MDS more comprehensively. Disclosures: Bellos: MLL Munich Leukemia Laboratory: Employment. Davis:Trillium Diagnostics, LLC: Equity Ownership. Culp:Trillium Diagnostics, LLC: 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. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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The RUNX1 Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion

Blood ◽

10.1182/blood.v124.21.123.123 ◽

2014 ◽

Vol 124 (21) ◽

pp. 123-123

Author(s):

Claudia Haferlach ◽

Niroshan Nadarajah ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Intermediate Risk ◽

Missense Mutations ◽

Employment Equity ◽

Runx1 Gene ◽

Genetic Abnormalities ◽

Partner Gene ◽

Different Types ◽

Equity Ownership ◽

De Novo Aml

Abstract Background: In AML four types of acquired alterations of the RUNX1 gene have been described: 1. translocations involving RUNX1 leading to fusion genes such as RUNX1-RUNX1T1, 2. molecular mutations, 3. amplifications of RUNX1, 4. Partial or complete deletions of the RUNX1 gene. Aim: To determine the frequency of different RUNX1 alterations and to characterize the spectrum of accompanying genetic abnormalities. Patients and Methods: We screened 726 de novo AML patients (pts) for RUNX1 deletions (del) and translocations using a dual color break-apart probe covering the 5' and 3' part of RUNX1 (MetaSystems, Altlussheim, Germany) and in addition evaluated RUNX1 mutations (mut) by Sanger or next-generation amplicon deep-sequencing. Median age was 67 yrs (range: 18 to 100 yrs). For all patients cytogenetics was available and categorized according to MRC criteria (Grimwade et al. Blood 2010). Partial deletions of RUNX1 as detected by FISH were confirmed by array CGH (Agilent Technologies, Santa Clara, CA). Results: In 89/726 pts (12.3%) abnormalities of the RUNX1 gene were detected by FISH: 10 pts (1.4%) showed a deletion encompassing the whole RUNX1 gene while additional 9 pts (1.2%) showed a partial loss of one RUNX1 copy. A gain of one RUNX1 copy was present in 45/726 (6.2%) pts. In 3 pts a gain of the 5' part of RUNX1 was accompanied by a loss of the 3' part while in 2 pts one copy of the 3' part was gained accompanied by a loss of the 5' part. A translocation affecting the RUNX1 gene was detected in 31 pts (4.3%). The partner gene was RUNX1T1 in 29 pts and located on 16q13 and 18p11 in one pt each. One pt with a RUNX1 translocation also showed a 5' RUNX1 deletion. In 110/726 pts (15.2%) a RUNX1mut was detected. Of these, 16 pts showed two and 5 pts three mutations in RUNX1. Thus, in total 136 mutations were detected in 110 pts: 58 (42.6%) were frameshift, 42 (30.9%) missense, 21 (15.4%) nonsense, 9 (6.6%) splice-site and 6 (4.4%) in-frame insertions/deletions. The RUNX1mut was homozygous in 15 pts, these were predominantly missense mutations (9/15; 60%). Within the subset of pts with RUNX1mut 2 harbored an additional RUNX1del and 9 pts a gain of a RUNX1 copy, while no RUNX1 translocation was present. In AML FAB type M0 both RUNX1mut and RUNX1del showed the highest frequencies (33.3% and 14.8%). 48.4% and 45.2% of cases with RUNX1 translocations were FAB type M1 and M2. While RUNX1mut were most frequent in the cytogenetic intermediate risk group (19.1%; favorable: 2.2%, adverse: 9.7%), RUNX1del were most frequent in pts with adverse risk cytogenetics (9.7%; favorable: 1.1%, intermediate: 0.8%). A comparable distribution was observed for a gain of RUNX1 copies (adverse: 19.4%, favorable: 4.5%, intermediate: 2.6%). With respect to additional molecular mutations all types of RUNX1 alterations were mutually exclusive of NPM1mut. Further, the frequency of DNMT3Amut and CEBPAmut was significantly lower in pts with RUNX1 alterations as compared to those without (14.3% vs. 34.3%; p<0.0001 and 6.4% vs. 13.4%; p=0.012). However, some striking differences between the different types of RUNX1 alterations were detected: ASXL1mut were significantly more frequent in pts with RUNX1mut (36.7%) but rather infrequent in pts with RUNX1del, gain and translocation (12.5%, 6.1%, and 6.7%). A comparable association was noticed for SF3B1mut which were frequent in RUNX1mut pts (23.8%) and rather infrequent in pts with RUNX1del, gain and translocations (0%, 10.5%, and 0%). In contrast, pts with either RUNX1del or RUNX1 gains showed a significantly higher TP53mut frequency (66.7% and 35.3%) as compared to RUNX1mut or RUNX1 translocated pts (7.1% and 4.8%). In the total cohort median overall survival (OS) was 18.7 months and differed significantly between the different types of RUNX1 alterations: for RUNX1 translocations, mutations, gains and deletions it was 35.5, 14.1, 12.4 and 4.3 months. Conclusions: 1. The RUNX1 gene is altered in 26% of AML. 2. All types of RUNX1 alterations predominantly occur in AML M0, M1 and M2 and are rare in the remainder AML. 3. They are mutually exclusive of NPM1 mutations and show a negative association with DNMT3A mutations. 4. While RUNX1 mutations were most frequent in patients with intermediate risk cytogenetics, RUNX1 deletions and gains were most frequent in patients with adverse cytogenetics. 5. Outcome differs significantly and is best in patients with RUNX1 translocations and worst in cases with RUNX1 deletions. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah: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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MDS with Deletions in the Long Arm of Chromosome 11 Are Associated with a High Frequency of SF3B1 Mutations

Blood ◽

10.1182/blood.v128.22.4289.4289 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4289-4289

Author(s):

Anna Stengel ◽

Wolfgang Kern ◽

Manja Meggendorfer ◽

Torsten Haferlach ◽

Claudia Haferlach

Keyword(s):

Chromosomal Aberrations ◽

High Frequency ◽

Array Cgh ◽

De Novo ◽

Good Prognosis ◽

International Prognostic Scoring System ◽

Chromosome 11 ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership

Background: According to the revised International Prognostic Scoring System (IPSS-R), patients with MDS and deletions in the long arm of chromosome 11 (del(11q)) as sole abnormality are categorized as very good prognosis, even better than patients with a normal karyotype. Molecular data on this rare MDS subset is still limited. Aims: Comprehensive cytogenetic and molecular genetic characterization of MDS patients with del(11q) with and without additional aberrations and evaluation of prognosis. Patient cohorts and methods: Within 9225 unselected cases with de novo MDS, 33 cases with isolated del(11q) (del(11q) sole) and 23 cases with del(11q) accompanied by additional chromosomal aberrations (del(11q) other) were detected. All 56 cases were investigated using chromosome banding analysis (CBA) and interphase FISH. For 46 patients (33 of del(11)q sole, 13 of del(11q) other) material was available for investigation by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). In 44 cases amplicon sequencing was performed to detect mutations in ASXL1, CALR, CBL, CSF3R, CSNK1A1, DNMT3A, ETNK1, ETV6, EZH2, FLT3-TKD GATA1, GATA2, IDH1, IDH2, JAK, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Variants of unknown significance were excluded from statistical analysis. The cohort comprised 23 male and 33 female patients, median age was 75 years (range: 47-89 years). Results: 33/9925 cases with del(11q) sole were found by CBA (0.4%). In the 23 del(11q) other cases additional aberrations included del(5q) (12/23 cases, 52%), del(20q) (3/23 cases, 13%), del(12p) (2/23 cases, 9%) and +8 (2/23 cases, 9%) as recurrent abnormalities. In 6/23 cases (26%), a complex karyotype (>3 aberrations) was observed. In 13/33 cases with del(11q) sole by CBA, array CGH identified additional chromosomal aberrations (all non-recurrent, submicroscopic). In the total cohort analyzed by array CGH, the size of del(11q) varied between 12 - 58 Mb with a median of 39 Mb. In more detail, in cases with del(11q) sole the size ranged from 12 - 58 Mb (median: 38 Mb), in cases with del(11q) other from 21 - 56 Mb (median: 40 Mb). While the minimal deleted region for cases with del(11q) other was identified between genomic position 107 251 381 - 120 088 150, for cases with del(11q) sole no common minimal deleted region was determined due to the strong variation between the respective deleted regions. Mutation analyses revealed a high frequency of SF3B1 mutations in the total cohort (47%). Mutation frequencies >10% were also detected for ASXL1 (17%), SRSF2 (13%), TET2 (13%) and U2AF1 (10%). The median number of mutations was 1 (range 0-4) without differences between both subgroups. However, the high frequency of SF3B1 mutations was even more prominent when only cases with del(11q) sole were analyzed (56% vs. 23% in del(11q) other). Moreover, mutations in U2AF1 were exclusively found in cases with del(11q) sole (14% vs. 0%). By contrast, mutations in ASXL1 were more frequent in del(11q) other (23% vs. 14%). However, these correlations were not found to be statistically significant due to the low number of cases. Comparison of the SF3B1 mutation loads with the proportion of cells with del(11q) observed by FISH revealed that in most cases, del(11q) is found in the main clone. Ring sideroblasts (≥15%) were detected in 11/45 cases (24%), 10 of them harbored an SF3B1 mutation. The minimal deleted region of del(11q) for SF3B1 mutated cases was identified between 107 789 141 - 119 437 544. In the total cohort, the overall survival (OS) at 5 years was 91% and is thus in line with the very good prognosis as defined by the IPSS-R. A lower OS at 5 years was observed for patients with del(11q) other compared to del(11q) sole, although it was not found to be statistically significant (94% vs. 85%). Conclusion: In 0.4% of MDS patients a del(11q) sole was observed. This incidence is in line with data used for calculation of the IPSS-R (0.7% in Schanz et al., 2012). Especially in cases with del(11q) sole, but also in del(11q) other, a high frequency of SF3B1 mutations was observed, which might explain in part the very good prognosis as stated in the IPSS-R and also observed in the present study. Moreover, these patients were found to show a low number of other molecular mutations. The role of U2AF1 mutations, which were exclusively detected in cases with del(11q) sole, needs further investigation. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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AML with Inv(3)(q21q26) or t(3;3)(q21;q26) Are Frequently Accompanied by Mutations in RUNX1 and NRAS and Show a High Incidence of NF1 Deletions: a Study On 40 Cases.

Blood ◽

10.1182/blood.v114.22.273.273 ◽

2009 ◽

Vol 114 (22) ◽

pp. 273-273

Author(s):

Claudia Haferlach, ◽

Torsten Haferlach ◽

Frank Dicker ◽

Tamara Weiss ◽

Wolfgang Kern ◽

...

Keyword(s):

Cell Count ◽

Chromosome Aberrations ◽

De Novo ◽

Blast Cell ◽

Splice Mutation ◽

Acute Leukemias ◽

Genetic Abnormalities ◽

Additional Chromosome ◽

Equity Ownership ◽

De Novo Aml

Abstract Abstract 273 AML with inv(3)(q21q26) or t(3;3)(q21;q26) have been defined as a distinct entity in the WHO classification in the category of “acute myeloid leukemia with recurrent genetic abnormalities”. Whereas cases with t(8;21)(q22;q22), inv(16)(p13q22/t(16;16)(p13;q22) or t(15;17)(q22;q12) are considered as acute leukemias regardless of the percentage of blasts in the bone marrow, it is not clear whether cases with inv(3)(q21q26)/t(3;3)(q21;q26) should be categorized as such if blast cell count is <20%. To analyze the spectrum of myeloid neoplasms in which inv(3)/t(3;3) occurs, all cases with these abnormalities as diagnosed since 2005 in our laboratory were identified. CML cases showing a t(9;22) and inv(3)/t(3;3) were not included as here CML blast crisis was evident. To further decipher accompanying genetic lesions in AML with inv(3)(q21q26)/t(3;3)(q21;q26) we performed in addition to chromosome banding analyses, FISH for the detection of NF1 deletions and mutation screening of NPM1, MLL (PTD), FLT3 (ITD, TKD), RUNX1, KIT (D816), NRAS (codon12/13/61), CBL (exon8/9 splice mutation), MPL, and JAK2 (V617F, exon12). The study cohort included 20 males and 20 females. Median age was 64.8 years (range: 36.3–91.3), median WBC was 3.9G/l (range: 1.1–75.0G/l) and median platelet count 133G/l (range: 5–799G/l). Based on cytomorphology 23 cases were classified as AML (de novo: 18, t-AML: 2, s-AML: 3 (2 after MPN, 1 after MDS), 15 as MDS and 2 as MPN. 27 showed an inv(3)(q21q26) and 13 a t(3;3)(q21;q26). Additional chromosome aberrations were observed in 23/40 (57.5%) cases (AML: 16/23, 69.6%; MDS 7/17, 41.2%): one, two or more than two additional aberrations in 13, 6 and 4 cases, respectively. Recurrent abnormalities were −7 (n=17), del(5q) (n=4). The following molecular mutations were detected: FLT3-TKD (1/32, 3.1%), NRAS (7/29, 24%), RUNX1 (6/29, 20.7%), CBL exon8/9 splice mutation (n=2/24, 8.3%) and JAK2V617F (2/27, 7.4%). One case each with JAK2V617F was diagnosed with MPN and s-AML after MPN, respectively. No mutations were detected for: NPM1 (0/20), MLL-PTD (0/22), FLT3-ITD (0/35), KIT (0/25), JAK2exon12 (0/13) and MPL (0/13). One copy of the NF1 (neurofibromin 1) gene, which negatively regulates the RAS pathway, was found to be deleted in 4/26 (15.4%) cases using FISH (NF1/MPO probe from Kreatech, Amsterdam, The Netherlands). Overall, 22 molecular alterations were observed in the analyzed genes (15 in AML cases, 7 in MDS/MPN cases). Taking also additional cytogenetic aberrations into account 31/40 patients (20/23 AML, 11/17 MDS/MPN) showed further genetic abnormalities in addition to inv(3)/t(3;3). Survival data were available in 28 cases. No significant differences were observed with respect to overall survival (OS) and event-free survival (EFS) between cases diagnosed as MDS or MPN vs AML. Whereas no impact of additional chromosome aberrations or presence of molecular mutations on OS was observed, a trend for a shorter OS in cases with RUNX1 mutation was found (2 months vs 21.8 months, p=0.07). In addition 17 cases with inv(3)(q21q26)/t(3;3)(q21;q26) and de novo AML were compared to 814 de novo AML without inv(3)(q21q26)/t(3;3)(q21;q26). The median OS for the total cohort was 48.2 months (mo), the median EFS 16.3 mo. Based on cytogenetics cases were assigned into 8 subgroups: 1. t(15;17)(q22;q21), 2. t(8;21)(q22;q22), 3. inv(16)(p13q22)/t(16;16)(p13;q22), 4. 11q23/MLL abnormalities, 5. inv(3)(q21q26)/t(3;3)(q21;q26), 6. normal karyotype, 7. complex karyotype, 8. other abnormalities. Median OS was not reached for groups 1, 2, 3, 4, and 6 and was 23.4 mo, 11.8 mo and 32.2 mo for groups 5, 7, and 8, respectively. OS at 2 yrs was 95.6%, 96.3%, 76.6%, 64.9%, 47.5%, 63.5%, 23.9% and 58.5% for groups 1–8, respectively. The respective data for median EFS were: not reached for groups 1 and 2 and 15.9 mo, 13.5 mo, 6.3 mo, 16.9 mo, 7.5 mo and 12.5 mo for groups 3–8, respectively. In summary, inv(3)/t(3;3) is observed in MDS, MPN, de novo AML, s-AML and t-AML and frequently accompanied by additional cytogenetic or molecular genetic abnormalities. Especially frequent were mutations in RUNX1, NRAS and deletions of NF1. Prognosis of patients with inv(3)/t(3;3) is unfavourable irrespective of the cytomorphological diagnosis. These data suggest to consider cases with inv(3)/t(3;3) as one entity regardless of blast cell count. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: 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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ERG Overexpression Is Highly Associated With ERG Gene Amplifications In Patients With Myeloid Malignancies

Blood ◽

10.1182/blood.v122.21.3744.3744 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3744-3744

Author(s):

Simone Weber ◽

Claudia Haferlach ◽

Louisa Noel ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Markers ◽

Mrna Expression ◽

De Novo ◽

Chromosome 21 ◽

Adverse Impact ◽

Employment Equity ◽

Expression Levels ◽

Myeloid Malignancies ◽

Copy Numbers ◽

Equity Ownership

Abstract Background The proto-oncogene ERG is located on chromosome 21q22 and overexpression of ERG was shown to have an adverse impact on outcome in cytogenetically normal AML (CN-AML). Acquired gain of chromosome 21 (+21) is a recurrent cytogenetic abnormality in AML, however, the pathogenetic impact remains elusive. Data indicate that +21 could be the underlying mechanism for altered expression of genes located in the respective regions, such as ERG, which might contribute to the pathogenesis in myeloid malignancies with +21. Aims 1) To investigate a possible relationship between ERG copy numbers and variations in ERG mRNA expression levels in patients (pts) with myeloid malignancies. 2) Analysis of ERG expression in CN-AML to reveal a possible association between ERG overexpression and other relevant molecular markers and to ascertain the prognostic impact of ERGin context of these markers. Methods ERG mRNA expression and ERG copy numbers were analyzed using a hydrolysis probe based real-time PCR assay. 1) To address ERG expression in relation to +21 the following cohorts were analyzed: 62 AML pts with a complex karyotype including +21 (CK+21); 44 AML pts with a non-complex aberrant karyotype including +21 (AK+21); 19 pts with various myeloid malignancies (10 de novo AML, 3 MDS, 1 s-AML, 2 t-AML, 1 MDS/AML, 1 t-MDS, 1 MDS/MPN) all of them showing ERG amplification by interphase FISH and array CGH (ERG-Amp). In addition, 32 CK-AML and 330 CN-AML pts without +21 or ERGamplification were analyzed. Results were expressed as mean values±SEM. Expression levels/DNA copy numbers were compared by t-test. 2)ERG expression was assessed in a cohort of 330 patients (<65 years) with de novo CN-AML. Female/male ratio was 169/161. To distinguish low from high ERG expressers the median %ERG/ABL1 level was used. BAALC expression levels were analyzed accordingly. Expression levels were correlated with clinical outcome and with the presence of mutations (mut) in ASXL1 (n=330), CEBPA (n=330), DNMT3A (n=261), FLT3-TKD (n=330), IDH1R132 (n=328), IDH2R140 (n=328), IDH2R172 (n=328), NPM1 (n=330), NRAS (n=330), RUNX1 (n=329), TET2 (n=166), WT1 (n=329) and FLT3-ITD (n=330), and with MLL-PTD (n=330) and BAALCexpression (n=328). Results 1) Analysis of pts with +21 or ERG-Amp revealed significantly higher expression levels of ERG in pts with +21 (AK+21 and CK+21 combined, 331 ± 28) and ERG-Amp (606 ± 127) as compared to pts with CN-AML (229 ± 10; p=0.001, p=0.008, respectively) or CK-AML (177 ± 36; p=0.001, p=0.004, respectively). Mean ERG expression was even higher in pts with ERG-Amp (606 ± 127) as compared to pts with +21 (331 ± 28, p=0.047). Quantification of ERG copy numbers on DNA level showed good correlation to ERG mRNA expression, with abundantly higher ERG DNA amount in +21 (3.25 ±0.14) and moreover in ERG-Amp (9.21 ±1.05) as compared to CN-AML and CK-AML (combined: 2.20 ± 0.05; p<0.001 and p<0.001, respectively). 2) In CN-AML, %ERG/ABL1 levels ranged from 0.078 to 1,016.027 (median: 188.904). High ERG expression was associated with lower age (mean: 48.7 vs. 52.4 years, p=0.002), higher white blood cell (WBC) count (mean: 64.8 vs. 44.4 x 109/L, p=0.019), FLT3-ITDmut/wt ratio≥0.5 (51/165, 30.9% vs. 24/165, 14.5%, p=0.001), IDH2R172mut (6/164, 3.7% vs. 0/164, 0.0%, p=0.030) and high BAALC expression (104/164, 63.4% vs. 59/164, 36.0%, p<0.001), as compared to low ERG expression. In contrast, NPM1mut (91/165, 55.2% vs. 118/165, 71.5%, p=0.003), IDH1R132mut (10/165, 6.1% vs. 29/163, 17.8%, p=0.001) and TET2mut (10/76, 13.2% vs. 22/90, 24.4%, p=0.077) were less frequent in high ERG expressers. Survival analysis revealed inferior overall survival (OS at 3 years: 52.2% vs. 68.7%, p=0.021) and event free survival (EFS at 3 years: 34.6% vs. 43.1%, p=0.052) for high ERG expressers as compared to low ERG expressers. In a multivariate analysis adjusted for age, WBC, BAALC expression, FLT3-ITDmut/wt ratio≥0.5, MLL-PTD and WT1mut, high ERG expression revealed a trend towards an adverse impact on OS (p=0.069), while no impact on EFS was observed. Conclusions 1) Gain of chromosome 21 and especially amplification of chromosomal band 21q22 is a mechanism for ERG overexpression. This might indicate ERG as an important factor contributing to the pathogenesis and progression of myeloid malignancies with gain of chromosome 21. 2) In CN-AML, ERG overexpression is associated to several molecular markers and has a negative impact on OS and EFS. Disclosures: Weber: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Noel: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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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RUNX1 Mutated AML Are Associated with a Distinct Pattern of Cytogenetic and Additional Molecular Genetic Abnormalities

Blood ◽

10.1182/blood.v124.21.124.124 ◽

2014 ◽

Vol 124 (21) ◽

pp. 124-124

Author(s):

Niroshan Nadarajah ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

Claudia Haferlach

Keyword(s):

High Frequency ◽

De Novo ◽

Male Gender ◽

The Other ◽

Typical Pattern ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Genetic Abnormalities ◽

Equity Ownership ◽

De Novo Aml

Abstract Background: Mutations in RUNX1 have been reported in 5 to 20% of AML. The aim of this study was to analyse a large cohort of RUNX1 mutated AML in detail with respect to accompanying cytogenetic and moleculargenetic abnormalities, mutation type and mutation load. Patients and Methods: We investigated 468 AML with RUNX1 mutations (mut) all identified during diagnostic work-up in our laboratory. Sequencing was performed by either Sanger or next-generation sequencing. Median age was 71.9 yrs (range 18-91 yrs), male : female ratio 297 : 171. 369 patients had de novo AML, 75 s-AML following MDS, 24 t-AML. For all patients (pts) cytogenetics was available and categorized according to MRC (Grimwade et al. Blood 2010). Mutation data was available for NPM1 (n=455), MLL-PTD (n=454), CEBPA (n=449), FLT3-TKD (n=422), WT1 (n=394), FLT3-ITD (n=362), ASXL1 (n=293), TP53 (n=204), DNMT3A (n=143), TET2 (n=143), and SF3B1 (n=99). Data on FAB subtype was available in 342 cases (73.1%). Results: The most frequent FAB subtype in RUNX1mut AML was AML M2 (145/342; 42.4%), followed by M1 (23.1%), M4 (15.8%), M0 (15.5%), other subtypes were rare (<2%). Cytogenetics were favorable in 2 (0.4%), intermediate in 397 (84.8%) and adverse in 69 (14.7%) pts. Most frequent cytogenetic abnormalities were +8 (76; 34.7%), +13 (46; 21.0%), +21 (15; 6.8%), +11 (12; 5.5%), -7 (22; 10%), del(7q) (18; 8.2%), del(5q) (12; 5.5%). Only 12 pts (5.5%) showed a complex karyotype (> 3 aberrations). The frequency of all other abnormalities was <5%. Two pts showed a t(15;17)(q24;q21) and 3 MLL rearrangements (partner genes on 7q32, 17q21, 19p13). None of the other recurrent cytogenetic abnormalities according to WHO classification was present. 249 (53.2%) pts had a normal karyotype. ASXL1mut were the most frequent accompanying mutations (36.5%). Mutation frequencies for the other genes were: SF3B1 (27.3%), TET2 (26.6%), FLT3-ITD (17.7%), DNMT3A (16.1%), MLL-PTD (10.8%), CEBPA (6.9%; single mutated (sm): 5.3%, double mutated (dm): 1.6%), WT1 (5.8%), FLT3-TKD (5.5%), TP53 (4.4%), and NPM1 (1.1%). While mutations in ASXL1 and TET2 frequently occurred concomitantly (37.5% of ASXL1mut cases also harboured a TET2mut, p=0.032), ASXL1mut and SF3B1mut rarely co-occurred (only 2 ASXL1mut were SF3B1mut, p=0.001). There were no differences in mutation frequencies and cytogenetic abnormalities observed between de novo AML, s-AML and t-AML. In 368 cases (78.6%) one RUNX1 mutation was detected, 81 pts (17.3%) showed two, 16 cases (3.4%) three, and 3 cases (0.6%) four. The difference in mutation loads between the 2 mutations with the highest load was ≤10% in 55/100 pts, suggesting that both mutations were present in the same clone. In total 592 RUNX1 mutations were detected, 242 (40.9%) were frameshift, 206 (34.8%) missense, 83 (14.0%) nonsense, 37 (6.3%) in frame insertion/deletions, 23 (3.9%) splice-site and 1 no stop change. No association between the types of 1st and 2nd mutations was observed. The mutations were homozygous in 65 pts (13.9%), these were predominantly missense mutations (38; 58.5%). A significantly higher frequency of homozygous mutations was observed in pts with +13 (28.3%, p=0.006), +21 (46.7%; p=0.002), AML M0 (35.8%; p<0.001) and M1 (22.8%; p=0.014) while they were less frequent in M2 (4.8%; p<0.001) and M4 (3.7%, p=0.017). Survival analyses were restricted to 203 de novo AML pts who were treated with intensive chemotherapy (median overall survival (OS) 20.4 months (mo)). Median OS was significantly longer in female than in male pts (45.6 vs 16.3 mo; p=0.003) and in pts ≤ 60 vs >60 yrs (44.4 vs 16.1 mo; p<0.001). Shorter OS was observed for pts with del(5q) (1.8 vs 21.1 mo; p=0.002) and adverse cytogenetics (12.4 vs 23.3 mo, p=0.016). Including these 4 parameters into a multivariate Cox regression analysis revealed that age, male gender and del(5q) were independently associated with shorter OS (relative risk: 1.8, 1.6, 3.4; p: 0.01, 0.038, 0.028) Conclusions: RUNX1 mutated AML: 1. is associated with a myeloid rather than monocytic differentiation, 2. shows a typical pattern of cytogenetic abnormalities with a high frequency of +8 and +13, 3. has a typical pattern of additional molecular mutations with a high frequency of accompanying ASXL1 und SF3B1 mutations, 4. is nearly mutually exclusive of NPM1 and CEBPAdm mutations and other entity defining genetic abnormalities, 5. Male gender, age >60 yrs and del(5q) were negative prognostic factors. Disclosures Nadarajah: 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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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