Does Age of Patients Influence the Composition of Gene Mutations in Myeloid Neoplasms?

Abstract Introduction: Incidences of myeloid neoplasms, i.e. AML, MDS, MDS/MPN overlap, increase with age. Cytogenetic aberrations are still the hallmark for diagnosis and prognostication especially in AML and MDS. Different patterns of relations between chromosomal aberrations and age have been described. However, in recent years, gene mutations have been depicted to further discriminate patients with respect to their diagnosis and are increasingly used for prognostication. In parallel, recent studies (Jaiswal, NEJM 2014) have demonstrated that mutations in genes occurring in hematological neoplasms are also observed in healthy individuals and increase in frequency with age. Aim: To investigate if specific molecular markers, such as DNMT3A, ASXL1, and TET2, increase in frequency with age in myeloid neoplasms as recently shown for healthy individuals. Patients and methods: We investigated 1639 patients (pts) between 20 and 93 years (yrs), 578 with de novo AML (median age: 63 yrs), 846 with MDS (median age: 73 yrs), and 215 with CMML (median age: 75 yrs). In all cases, we followed the diagnostic criteria of the WHO classification based on morphology. All patients have also been investigated by cytogenetics and for disease-oriented molecular mutations (15-36 genes/pt: 15 in AML, 36 in MDS, and 21 in CMML). Analyses were performed by melting curve analysis, gene scan, Sanger sequencing, or next generation sequencing. Results: In total we detected 3089 mutations (range: 0-9/pt) spread over all except for seven analyzed genes. Grouping the entity-specific cohorts by age of the patients into decades revealed a steady increase of the prevalence of mutations with age in MDS (at least one mut/pt, 25% in 20-29 to 93% in >80 yrs; p<0.001), less prominent in AML (77% in 20-29 to 100% in >80 yrs, p=0.007), but not in CMML (96%-100% in all decades). However, the number of mutations per patient increased according to age in all three entities, significantly in MDS (p<0.001) and AML (p<0.001). Considering AML patients separated into three cytogenetic classes (Grimwade, Blood 2010) resulted in the same findings for the intermediate risk (p=0.012) and adverse risk group (p<0.001), while the good risk group showed no change in mutation numbers over decades (median: 1 mut/pt, range 0-3). This indicates that in good risk AML (PML-RARA, CBFB-MYH11, RUNX1 -RUNX1T1) only very few additional mutations are needed for AML initiation. In contrast, an age-dependent increasing incidence of gene mutations is specific in normal karyotype and in adverse cytogenetics. We next focused on specific gene mutations according to age <60 vs ≥60 yrs within all three entities. In addition, AML patients where again subgrouped by cytogenetics. In AML good and adverse risk groups no age-dependent significant increase of specific gene mutations occurred, while in the intermediate risk group mutations in ASXL1 (3/160 vs 32/114, p<0.001), MLL-PTD (3/160 vs 17/214, p=0.01), RUNX1 (32/160 vs 20/214, p=0.001), and TET2 (4/159 vs 26/214, p=0.001) were significantly more frequent at higher age. In contrast, NRAS mutations appeared more often in younger AML patients (32/160 vs 20/214, p=0.004). In MDS, mutations in SF3B1 (27/115 vs 253/731, p=0.019), SRSF2 (10/115 vs 133/730, p=0.011), TET2 (10/115 vs 250/731, p<0.001), and TP53 (2/115 vs 50/731, p=0.035) were more frequently observed in older patients. In CMML only TET2 mutations occurred more often in older patients (5/12 vs 135/190, p=0.026). Focusing on the genes recently described to be mutated in healthy individuals showed that all of the above mentioned mutations found in myeloid neoplasms (except MLL-PTD and RUNX1) are comprised in the 10 most frequently mutated genes in the healthy aging population. However, the fact that the frequencies of these mutations are not age-dependent in some entities, e.g. ASXL1 only age-dependent in AML but not in CMML and MDS, might indicate different roles of these mutations in the pathogenesis, i.e. driver mutations independent of age, as well as their contribution to accumulation of mutations and onset of a myeloid neoplasm. Conclusions: 1) The number of mutations significantly increase with age in AML and MDS and non-significantly in CMML. 2) Several genes show age-dependent frequencies, which differ between AML, MDS, and CMML and are also related to the cytogenetic background. 3) Based on molecular mutations healthy aging and myeloid neoplasms are neighbouring scenarios. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. 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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Genetic Patterns of Relapsed AML Differ Significantly from First Manifestation and Are Dependent on Cytogenetic Risk Groups at Diagnosis: Results in 175 Patients with Paired Samples

Blood ◽

10.1182/blood.v124.21.1029.1029 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1029-1029 ◽

Cited By ~ 1

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Karolina Perglerová ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

...

Keyword(s):

Risk Group ◽

Gene Mutations ◽

Primary Diagnosis ◽

Intermediate Risk ◽

Employment Equity ◽

Mutational Pattern ◽

Genetic Patterns ◽

Cytogenetic Changes ◽

Mutation Pattern ◽

Equity Ownership

Abstract Introduction: Genetic changes between diagnosis and relapse in AML have not been analyzed comprehensively yet. Especially in the favorable risk group (acute promyelocytic leukemia (APL) with PML-RARA, and core binding factor (CBF) leukemias with CBFB-MYH11 or RUNX1-RUNX1T1) data is scarce. Aim: To investigate genetic patterns in AML with favorable risk cytogenetics at diagnosis and at relapse in comparison to all other AML subtypes. Patients and Methods: We investigated 175 AML patients diagnosed by cytomorphology, immunophenotyping and cytogenetics following WHO criteria both at diagnosis and at relapse (350 samples). Cytogenetic risk stratification followed MRC as favorable, intermediate, and adverse (Grimwade, Blood 2010). 30 patients were diagnosed as APL or CBF leukemia (favorable), while 122 patients were intermediate, and 23 adverse risk. Data on molecular mutations was available for subsets of patients including ASXL1, CEBPA, DNMT3A, EZH2, FLT3-ITD, FLT3-TKD, KIT, IDH1, IDH2, MLL-PTD, NPM1, NRAS, KRAS, RUNX1, TET2, TP53, and WT1. Gene mutations were analyzed by Sanger sequencing, NGS, melting curve analysis, or gene scan. Cytogenetics was available for all 350 samples. Results: Changes in mutational and cytogenetic patterns of APL and CBF leukemias: 28 relapse samples revealed in total 22 gene mutations in 12 genes, with a median number of 0.8 mutations per patient. 7 patients (25%) showed no mutation, 20 (71%) showed 1 mutation, and 1 (4%) had 2 mutations. Most frequently FLT3-ITD was found (n=5), followed by mutations in KIT (n=3), EZH2, FLT3-TKD, NRAS, TET2 (for each n=2), and ASXL1, DNMT3A, IDH1, KRAS, TP53, and WT1(for each n=1). In 17/20 patients with molecular mutations identified either at relapse or primary diagnosis both samples were analyzed for the specific mutation. 22 mutations were present in this subset. Of these 10 (46%) mutations were already detected at primary diagnosis and thus remained stable, while 3/22 (14%) mutations were gained and 9/22 (41%) were lost at relapse. Including patients with no mutation revealed that 13/24 (54%) patients showed an unchanged mutation pattern while 11/24 (46%) gained or lost mutations. The karyotype was stable in 16/30 (53%) cases with favorable cytogenetics, while 14/30 (47%) showed cytogenetic changes: although the main cytogenetic aberration remained unchanged in all relapse samples the latter patients showed either clonal evolution (n=11), clonal regression (n=2) or both (n=1). Changes in mutational and cytogenetic patterns of AML with intermediate risk cytogenetics: In the intermediate risk group (97/122 patients (80%) had normal karyotype) the cytogenetic changes were slightly less frequent with 44/122 (36%). Interestingly, in this group 3 patients occurred with a totally different karyotype at relapse compared to primary diagnosis. In 2 cases, however, the molecular markers remained stable, while in 1 patient also the mutation pattern changed completely. Therefore, the latter one might be a t-AML, while the other two are most likely relapses. The molecular mutation patterns were unstable in this subgroup with 56/94 (60%) patients showing mutational gains as well as losses. Changes in mutational and cytogenetic patterns of AML with adverse cytogenetics: 11/23 patients (48%) showed cytogenetic changes. Also in this group 1 patient might have acquired a t-AML rather than a relapse as suggested by complete changes in cytogenetics and molecular markers. Only 5/18 (26%) showed changes in the mutational pattern at all. Of notice, while in favorable AML mutation patterns changed at relapse more by losses than by gains of mutations, in the intermediate and adverse groups gains of mutations were more frequent. The latter two groups had more mutations already at primary diagnosis and acquired additional mutations mostly in FLT3-ITD but also in the whole spectrum of analyzed genes, particularly in prognostically unfavorable genes such as TP53, ASXL1, RUNX1, DNMT3A, or WT1. Conclusions: 1) In AML with favorable cytogenetics the defining aberrations persist at relapse but changes in karyotype and mutational pattern occur in 47% and 46% of cases. 2) AML with intermediate risk cytogenetics is characterized by instability predominately on the molecular level. 3) AML with adverse cytogenetics shows only few changes in the molecular profile but high karyotype instability. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: 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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PPM1D and DNMT3A Mutations in Myelodysplasia and Clonal Hematopoiesis

Blood ◽

10.1182/blood-2019-122032 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1709-1709

Author(s):

Masanori Motomura ◽

Yoshikage Inoue ◽

Yasunobu Nagata ◽

Tetsuichi Yoshizato ◽

Constance Baer ◽

...

Keyword(s):

Research Funding ◽

Myeloproliferative Neoplasms ◽

Healthy Individuals ◽

Employment Equity ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Myeloid Neoplasms ◽

Age Related ◽

Increased Risk ◽

Equity Ownership

Introduction: Age-related clonal hematopoiesis (CH) has been implicated in an increased risk of myeloid neoplasms. While common driver genes mutated in CH largely overlap to those in myeloid neoplasms, a notable exception is protein phosphatase Mg2+/Mn2+dependent 1D gene (PPM1D), encoding a p53-targeting phosphatase. Although it is known to be involved in DNA damage response pathways and more frequently mutated in therapy-related myeloid neoplasms than in primary ones, its role in CH and myeloid neoplasms has not been fully understood. Aim: To identify genetic features associated with PPM1D mutations, we examined genetic profiles in the large cohorts of healthy elderly individuals and patients with myelodysplasia. Methods: We enrolled 10,826 healthy individuals (>60y) and 1,213 cases with myelodysplasia, including myelodysplastic syndromes (MDSs), myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) (n=1,080), and secondary acute myeloid leukemia (sAML) (n=133), of which 567 cases were treated by hematopoietic stem cell transplantation (HSCT) through the Japan Marrow Donor Program just after sampling, and 332 of them underwent any therapy before sampling. Samples from healthy individuals were subjected to multiplex-amplicon sequencing for 22 genes, including PPM1D and other genes, related to CH or myeloid neoplasms. Myelodysplasia samples had previously been sequenced for major myeloid drivers, except for PPM1D, which was newly sequenced in this study. Results: Frequency of PPM1D mutations in myelodysplasia and healthy individuals was 3.1% and 0.42%, with a median variant allele frequency (VAF) of 0.043 and 0.056, respectively. PPM1D mutations were more frequent in cases with previous treatment (4.8%) than in those without known history of therapy (2.3%) (P=0.038). In MDS and MDS/MPN cases, 59.5% of PPM1D mutations had accompanying mutations, in which DNMT3A mutations were the most frequently identified (16.2%, n=6). These 6 cases were diagnosed with RCUD (n=1), RCMD (n=2), RAEB-2 (n=2), or CMML (n=1). The association between PPM1D and DNMT3A mutations was also seen in 7 of 45 healthy individuals with PPM1D mutations, of which one had a DNMT3A-R882 mutation. In the HSCT cohort, 192 cases harbored ≥2 mutations of the 22 CH-related genes, and the relative temporal order of these mutations was investigated using Bradley-Terry model relying on their tumor cell fractions. The estimate of PPM1D mutations tended to be smaller than that of DNMT3A mutations. To further confirm chronological order of these mutations, VAF values were compared between them in the individuals with concurrent PPM1D and DNMT3A mutations (n=13; 6 myeloid neoplasms and 7 healthy donors). In the combined cohort, the VAFs of PPM1D and DNMT3A mutations were correlated (Spearman; correlation coefficient=0.87, P=1.2x10e-5). In both neoplastic and healthy cohort, the VAFs of DNMT3A-R882 mutations were larger than those of accompanying PPM1D mutations. These findings suggest that these mutations should be acquired in the same cell populations and that DNMT3A mutations might occur prior to PPM1D mutations. With regard to copy number alterations associated with PPM1D-mutated myelodysplasia, del(5q) (16.7%) and complex(-like) karyotypes (13.9%) were among the most frequent chromosomal abnormalities. Approximately 65% of PPM1D-mutated tumor samples had normal karyotype, which was similar to PPM1D-unmutated cases. PPM1D mutations did not significantly influence overall survival, although PPM1D mutations tended to negatively affect clinical outcome among patients who were treated with HSCT (Hazard ratio, 1.61; 95% confidence interval, 0.95 to 2.70). Conclusion: PPM1D mutations were more enriched in myelodysplasia than in CH, and the median value of VAF in PPM1D mutations in CH was not significantly different from that in myelodysplasia. The size of PPM1D-mutated clones tended to be relatively smaller compared with that of clones with other mutations in myelodysplasia. PPM1D and DNMT3A mutations might be cooperatively associated in the pathogenesis of myelodysplasia and CH. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership.

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SF3B1 Mutations in AML, MDS and MDS/MPN-RS-T Are Accompanied By Different Other Gene Mutations: Impact for Targeted Treatment Studies

Blood ◽

10.1182/blood.v128.22.1976.1976 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1976-1976

Author(s):

Manja Meggendorfer ◽

Sabine Jeromin ◽

Karolína Perglerová ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Specific Gene ◽

Prognostic Information ◽

Employment Equity ◽

Additional Mutation ◽

Potential Biomarker ◽

Leukemia Treatment ◽

Equity Ownership ◽

Relevant Gene

Abstract Introduction: Precision medicine aims at the molecular profiling of patients to specifically target gene mutations. Targeted therapies now enter leukemia treatment, e.g. by targeting FLT3-ITD or mutations in DNMT3A, TET2, IDH1/2 or JAK2. Recently, luspatercept, a fusion protein (ACE-536), was shown to inhibit different signaling cascades, resulting in differentiation and maturation of erythropoietic progenitors in anemic patients (Platzbecker et al., Haematologica 2015). Interestingly, only patients with myelodysplastic syndrome (MDS) and ring sideroblasts (RS) responded to luspatercept, suggesting SF3B1 to be a potential biomarker. Besides MDS, SF3B1 mutations occur also in acute myeloid leukemia (AML) and MDS/myeloproliferative neoplasms with RS and thrombocytosis (MDS/MPN-RS-T). However, concomitant gene mutations bearing prognostic information and/or also being therapeutic targets as well as the cytogenetic background may need to be addressed in addition before further investigation. Aim: To investigate the mutation pattern and cytogenetic background of patients with AML, MDS and MDS/MPN-RS-T carrying SF3B1 mutations. Patients and Methods: In a cohort of 365 patients - all showing SF3B1 mutations and the diagnosis of AML (n=51), MDS (n=263) or MDS/MPN-RS-T (n=51) - cytomorphology, cytogenetics and mutation status were available. The cohort comprised 145 females and 220 males, the median age was 75 yrs (range: 42-93 yrs). In all patients ASXL1, RUNX1, TP53 as important prognostic markers as well as DNMT3A, FLT3-TKD, IDH1/2, JAK2, K/NRAS and TET2 as optional targets were analysed for mutations. Furthermore, additional entity specific gene mutations were investigated in respective subcohorts (AML: CEBPA, FLT3-ITD, MLL-PTD, NPM1; MDS: ETV6, EZH2, SRSF2, U2AF1, ZRSR2; MDS/MPN-RS-T: MDS genes, CBL, MPL). Results: 73% of all patients (268/365) showed normal karyotypes. Addressing molecular genetics resulted in 370 mutations beside SF3B1 in 238 patients, leaving only 23% of patients (84/365) showing no other aberration than in SF3B1. The variant allele frequencies (VAF) of SF3B1 mutations were high in nearly all cases with only few (9/353) subclonal cases (VAF <10%). In AML the median VAF of SF3B1 was 45% (range: 5-70%) with 3 cases showing subclonal mutations, likewise in MDS with a VAF of 39% (range: 3-50%) and 6 subclonal cases, while in MDS/MPN-RS-T the VAF was also 39% (range: 15-50%) without any subclonal case. In detail, 63% of AML cases showed normal karyotypes. Looking at gene mutations revealed that 49/51 patients (96%) had additional gene mutations (median: 2, range: 0-4), while 28/51 cases (55%) showed mutations in at least one of the therapeutically relevant genes. Of note, 37/51 patients (73%) had a mutation known to be associated with adverse prognosis. Therefore, in AML just one patient had a sole SF3B1 mutation and only 3/51 cases (6%) showed only other targetable mutations beside SF3B1. In MDS 73% of patients showed normal karyotypes. MDS patients showed in median 1 additional mutation (range: 0-4), leaving 115/263 (44%) patients without additional mutations. Furthermore, 124/263 (47%) patients carried mutations in a therapeutically relevant gene, while only 32/263 cases (12%) had mutations worsening prognosis. This results in 74/263 MDS patients (28%) without any additional aberration and 85/263 patients (32%) showing only other targetable mutations beside SF3B1. Furthermore, 84% of MDS/MPN-RS-T showed normal karyotypes. In median 1 additional mutation (range: 0-7) was identified in MDS/MPN-RS-T patients, while 10/51 cases (20%) showed no additional mutation. Looking at therapeutically relevant gene mutations revealed in 39/51 patients a respective mutation, while 7/51 patients carried prognostically adverse mutations. Therefore, MDS/MPN-RS-T patients show also a high proportion of cases without additional aberration (9/51, 18%) and even 47% (24/51) of patients having only targetable gene mutations. Conclusion: 1) SF3B1 mutation is supposed to be in the main clone. 2) AML, MDS and MSD/MPN-RS-T differ in their respective patterns of molecular aberrations beside SF3B1 mutations. 3) MDS patients show most frequently SF3B1 mutations as sole abnormality and might therefore benefit best from SF3B1 targeting treatment. 4) Treatment decisions should in all cases consider additional targetable mutations but also those worsening prognosis. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.

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Patients with Therapy-Related Myeloid Disorders Share Genetic Features but Can Be Separated by Blast Counts and Cytogenetic Risk Groups Into Prognostically Relevant Subgroups,

Blood ◽

10.1182/blood.v118.21.3583.3583 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3583-3583

Author(s):

Ulrike Bacher ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Clinical Outcomes ◽

De Novo ◽

Risk Group ◽

Risk Groups ◽

Employment Equity ◽

Total Cohort ◽

Myeloid Neoplasms ◽

Equity Ownership ◽

De Novo Aml ◽

Group 1

Abstract Abstract 3583 Background: In the WHO classification of 2008, patients with therapy-related acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) following cytotoxic therapy/radiation are combined to the category “therapy-related myeloid neoplasms”. To contribute to the discussion whether blast percentage or other subclassification are prognostically relevant in therapy-related myeloid disorders, we evaluated this combined group for clinical/genetic aspects. Study design: A total of 520 pts (242 m/278 f; median, 67.4 years; r. 18.0–91.5 yrs) with therapy-related myeloid malignancies (253 pts with ≥20% bone marrow blasts termed “t-AML”, 267 with <20% BM blasts: “t-MDS”) were investigated by cytomorphology, chromosome banding analysis, molecular genetics, and for clinical outcomes. Results: When biological characteristics of pts with ≥20% (“t-AML”) and <20% (“t-MDS”) BM blasts were compared, t-AML had higher mean WBC counts (17.7 vs 5.8×10(9)/L; p<0.001) and lower Hb (94 vs 103 g/L; p=0.003) and platelet level (81 vs 115 x10(9)/L; p=0.027) than t-MDS. Mean age was equal in t-AML/t-MDS (64.5 vs 64.6 years). Male/female ratio was lower in t-AML (0.6 vs 1.3 in t-MDS; p<0.001). Aberrant karyotypes (KTs) were more frequent in t-AML than t-MDS (175/253; 69.2% vs 147/267; 55.1%; p=0.001). NPM1 mut were similar in t-AML and t-MDS (23/197; 11.7% vs 6/54 investigated; 11.1%; p=n.s.). When only normal KTs were considered, NPM1 mut were detected in 20/65 (30.8%) t-AML (which is less than the NPM1 mut rate known in de novo NKT-AML) and were similar in NKT t-MDS (6/17; 35.3%; p=n.s.). FLT3 -ITD were less frequent in t-AML (19/210; 9.0%) compared to data on de novo AML, but more frequent than in t-MDS (2/112; 1.8%; p=0.016). Frequencies of FLT3 -ITD in normal KT were 8/66 (12.1%) in t-AML and 2/36 (5.6%) in t-MDS (p=n.s.). RUNX1 mut (t-AML: 11/81; 13.6%; t-MDS: 5/69; 7.2%), CEBPA (t-AML: 6/104; 5.8%; t-MDS: 0/10; 0.0%), FLT3- TKD (t-AML: 3/120; 2.5%; t-MDS: 0/23; 0.0%), NRAS (7/64; 10.9% vs 6/82; 7.3%), IDH (11/86; 12.8% vs 1/13; 7.7%), and MLL -PTD (7/200; 3.5% vs 5/109; 4.6%) did not differ significantly between t-AML/t-MDS. Patients with <20% BM blasts had better overall survival (OS) than pts with ≥20% BM blasts (median 43.8 vs 18.2 months; p=0.003). According to MRC cytogenetic risk groups (Grimwade et al., 2010), only 35 (6.7%) of all pts had favorable, 303 (58.3%) intermediate (t-AML: 144/253; 56.9%; t-MDS: 159/267; 59.6%), but 182 (35.0%) had adverse KTs (t-AML: 79/253; 31.2%; t-MDS: 103/267; 38.6%). In the total cohort, favorable KTs (group 1) had better OS than intermediate (group 2) and adverse (group 3) KTs (median n.r. vs 43.8 vs 16.2 months; p=0.002 comparing all 3 groups; group 1 vs 2: p=0.011; 1 vs 3: p=0.001; 2 vs 3: p=0.048). This holds true as well when t-AML pts (p=0.002 comparing different MRC groups) or t-MDS (p=0.003) were investigated separately. OS of NPM1 mut/FLT3 -ITD-neg. pts did not differ significantly from other genotypes in the total cohort (median n.r. vs 20.9 months) nor in normal KT t-AML (16.9 vs 11.0 months). In t-AML, MLL -PTD+ had worse OS than MLL -PTD-neg. pts (median 3.8 vs 16.9 months; p=0.001). In the total cohort, univariable Cox regression for OS was significant for age (p<0.001), WBC counts (p=0.016), Hb level (p=0.025), BM blasts both by a threshold of ≥20% vs <20% (p=0.003) or as continuous parameter (p=0.002), and MRC risk group (p=0.001). No significant differences were found for gender, platelets, NPM1 mut/FLT3 -ITD-negative status, or MLL -PTD+. By multivariable Cox analysis for OS age (p<0.001), WBC count (p=0.042), BM blasts by a threshold of 20% (p<0.001), and MRC risk group (p=0.002) were significant. Conclusions: In this study, patients with t-AML or t-MDS separated by a BM blast threshold of 20% showed significant differences in clinical outcomes and biological parameters. This emphasizes maintaining both subtypes within the WHO defined combined cohort of “therapy-related myeloid neoplasms”. Within both morphologically defined subentities, the karyotype is an excellent parameter for further prognostic predictions and shows similar patterns. The underrepresentation of the FLT3 -ITD and NPM1 mut in t-AML compared to de novo AML, and the similar frequency of NPM1 mut in t-AML and t-MDS suggests common molecular characteristics of both categories irrespective of blast percentages and strongly supports to combine them as therapy-related myeloid neoplasms as suggested by the WHO. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger: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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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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Comprehensive Analysis Of U2AF1 Mutations In 843 Patients With Myeloid Neoplasms With Respect To Other Genetic Alterations

Blood ◽

10.1182/blood.v122.21.4973.4973 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4973-4973

Author(s):

Manja Meggendorfer ◽

Christiane Eder ◽

Sabine Jeromin ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hot Spots ◽

Melting Curve ◽

Gene Mutations ◽

Next Generation ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership ◽

Generation Sequencing ◽

Splicing Machinery

Abstract Introduction Genes affecting the splicing machinery have been found to be frequently mutated in MDS patients. U2AF1 codes for one of these splicing components, showing two distinct mutational hot spots at amino acids Ser34 and Gln157. Mutations in U2AF1 induce global abnormalities in RNA splicing, producing intron containing unspliced RNAs. U2AF1 has been shown to be most frequently mutated in MDS cases (7-11%), but was so far investigated only in small subsets of AML and MPN and was found rarely mutated. Aim To determine the frequency of U2AF1 mutations (U2AF1mut) in different myeloid entities and to evaluate the correlation of U2AF1mut with other gene mutations, cytogenetics and clinical features. Patients and Methods The total cohort consisted of 843 patients, whereof 74 were diagnosed as AML, 201 as MDS, 243 as MPN, and 325 as MDS/MPN overlap. 331 patients were female, 512 male. Cytogenetics was available in 830 patients and these were grouped by the following karyotypes: normal karyotype (n=561), +8 (n=39), -7 (n=15), del(20q) (n=95), -Y (n=29), other aberrations (n=59), and complex karyotype (n=32). Based on the previously described association of U2AF1mut with del(20q) there was an intended selection bias to this abnormality. Mutational analyses for U2AF1 were performed by either melting curve analyses or next generation sequencing. In subcohorts we investigated mutations in ASXL1 (n=505), CBL (n=647), CEBPA (n=68), CSF3R (n=213), DNMT3A (n=260), ETV6 (n=129), EZH2 (n=355), FLT3-ITD (n=352), FLT3-TKD (n=239), IDH1/2 (n=367 and 286, respectively), JAK2 (n=681), KITD816 (n=244), KRAS (n=393), MLL-PTD (n=384), MPLW515 (n=612), NPM1 (n=477), NRAS (n=509), RUNX1 (n=516), SETBP1 (n=336), SF3B1 (n=839), SRSF2 (n=784), TET2 (n=428), and TP53 (n=239) by Sanger sequencing, next generation sequencing, gene scan, or melting curve analysis. Results In the total cohort we detected U2AF1 mutations in 55/843 (6.5%) patients, the two mutational hot spots were equally affected with 29 p.Ser34 and 26 p.Gln157 mutations, respectively. Mutation frequencies were 10.9% in MDS, 9.5% in AML, 7.1% in MDS/MPN overlap and 1.2% in MPN. U2AF1mut patients were older (median: 72.6 vs. 71.8 years; p=0.012), the mutation was more frequent in males (42/512 (8.2%) vs. 13/331 (3.9%) in females; p=0.015) and associated with lower hemoglobin levels (median: 9.5 vs. 11.0g/dL; p<0.001), and platelet counts (median: 78x109/L vs. 179x109/L; p=0.002). Regarding cytogenetics we found a high association of U2AF1mut to del(20q): in 18 of 95 cases (18.9%) with del(20q) a U2AF1 mutation was detected compared to 37 U2AF1mut in 735 cases (5.0%) with any other karyotype (p<0.001). This was true for AML (5/16 vs. 2/56; p=0.005), MDS (11/49 vs. 11/150; p=0.007) and MDS/MPN overlap cases (1/8 vs. 21/309; p=0.441). In contrast in MPN none of the 21 del(20q) patients showed a U2AF1 mutation compared to 18/74 in all other entities (p=0.01). Mutations in the two other genes of the splicing machinery, SF3B1 and SRSF2, occurred in 122/839 (14.5%) and 198/784 (25.3%) cases and were mutually exclusive with U2AF1mut. Only one case each showed an U2AF1mut and a SF3B1 (p=0.002) or SRSF2 (p<0.001) mutation. We furthermore analyzed a number of other gene mutations frequently mutated in myeloid entities and their association to U2AF1mut. There was no correlation to mutations in NPM1, FLT3-ITD and FLT3-TKD, MLL-PTD, and CEBPA in AML patients. In MDS patients there was also no correlation to mutations in ASXL1,ETV6, EZH2, TP53, RUNX1, NRAS, and KRAS. This was also true for JAK2, MPL, CBL, and TET2 mutations in MPN. However in MDS/MPN overlap patients U2AF1mut were more frequently found in cases with ASXL1mut (14/115 (12.2%) in ASXL1mut vs. 7/179 (3.9%) in ASXL1wt; p=0.01) and together with KITD816mut (3/10 (30%) in KITD816mut vs. 15/212 (7%) in KITD816wt; p=0.038). Conclusion 1) U2AF1 is most frequently mutated in MDS, followed by AML and MDS/MPN overlap and in contrast rarely mutated in MPN. 2) U2AF1mut highly correlates with del(20q) in MDS, AML and MDS/MPN overlap but not in MPN cases. 3) In MDS/MPN overlap U2AF1mut associates significantly with ASXL1mut and KITD816mut. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin: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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Modulation of the Clonal Composition in Relapsed CLL: A Study Based on Targeted Deep-Sequencing of ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53

Blood ◽

10.1182/blood.v126.23.1721.1721 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1721-1721

Author(s):

Sabine Jeromin ◽

Wolfgang Kern ◽

Richard Schabath ◽

Tamara Alpermann ◽

Niroshan Nadarajah ◽

...

Keyword(s):

Deep Sequencing ◽

Clonal Evolution ◽

Gene Mutations ◽

Wild Type ◽

Mutation Load ◽

Employment Equity ◽

Time Points ◽

Mutational Status ◽

Equity Ownership ◽

Time Point

Abstract Background: Relapse or refractory disease is a challenging clinical problem in the majority of chronic lymphocytic leukemia (CLL) patients. Treatment influences the clonal composition by selection and eventually induction of additional genetic abnormalities. Aim: To characterize the clonal evolution in relapsed CLL patients by deep-sequencing analysis of mutations in ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53. Patients and Methods: Sequential samples of 20 relapsed CLL patients at three time-points were evaluated: A: at diagnosis (n=16) or in untreated state (n=4), B: at first relapse (n=20) and C: at second relapse (n=2). Patients were treated with diverse treatment schemes and had temporarily achieved either complete or partial remission during the course of the disease. The median time from diagnosis to first-line treatment was 13 months (1 - 169 months). All geneswere sequenced by a deep sequencing approach (Illumina, San Diego, CA). IGHV mutational status was determined by direct Sanger sequencing at time-point A. Chromosome banding analysis (CBA) and FISH data on del(17p), del(11q), trisomy 12 (+12), and del(13q) were available in 33/42 and 36/42 samples, respectively. Results: Initially, samples at first relapse were sequenced. Mutations in SF3B1 (6/20, 30%), TP53 (5/20, 25%), ATM (5/20, 25%), NOTCH1 (4/20, 20%), and SAMHD1 (3/20, 15%) were detected at high frequencies. No mutations were detected in BIRC3 and POT1. In total, 75% of cases presented with at least one mutation (Figure 1): 8 (40%) cases had one, 6 (30%) cases had two and one patient had three genes concomitantly mutated (mut). Patients were also analyzed for IGHV mutational status at diagnosis and presented with unmutated status at a frequency of 85% (17/20). Subsequently, samples from cases with mutations were analyzed at time-point A. In 12/15 (80%) cases the mutations at relapse were already detectable at time-point A with a similar load indicating presence of the main clone before and after chemotherapy. However, in 7/15 (47%) patients new gene mutations were acquired either additionally to existing mutations (n=4) or in previously wild-type cases (n=3). In 5/7 (71%) cases mutations were located in TP53. TP53 mut were the only mutations that were not detected in samples before treatment (sensitivity of 3%). Thus, TP53 mutations might have been initiated by chemotherapy or exist in a minor subclone subsequently selected by chemotherapy. Furthermore, only 4 cases had low-level mutations (3-6% mutation load) at diagnosis in either SAMHD1 or SF3B1 that eventually increased in their burden during disease course. Of note, in two patients a multibranching clonal evolution could be identified (#2, #9). For patient #2 three time-points were analyzed. At diagnosis 2 ATM mutations were detected with mutation loads of about 20%, each. In the course of the disease these mutations were lost, whereas SF3B1 mut showed a stable mutation load in all three time-points of about 40%. In contrast, mutation load of SAMHD1 increased over time from 4% to 87%. CBA was performed at diagnosis and detected independent clones with del(11q) and del(13q). Accordingly, del(11q) detected by FISH at diagnosis was lost and the percentage of cells with del(13q) increased from diagnosis to time-point C. Therefore, patient #2 shows different genetic subclones in parallel that were eradicated or selected by chemotherapy. In patient #9 two SF3B1 mutations were initially detected with the same mutation load of 10%. After treatment one mutation was lost, whereas the load of the second mutation increased indicating at least two different subclones with only one of them being sensitive to chemotherapy. This might be due to different additional aberrations. Indeed, CBA identified two clones: one with +12 alone and one in combination with del(13q). FISH revealed unchanged percentage of +12 at time-point B, whereas del(13q) positive cells were diminished. Conclusions: In 75% of relapsed CLL cases mutations in SF3B1, TP53, ATM, NOTCH1, and SAMHD1 are present at high frequencies. 80% of these mutations are already detectable before treatment initiation representing the main clone. Remarkably, TP53 mutations were the only mutations that were not detected before but only after chemotherapy. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. 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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Prevalence and Impact on Outcomes of Additional Karyotypic Abnormalities in Patients (Pts) with Myelodysplastic Syndromes (MDS) and Del(5q) from the MDS-003 and MDS-004 Studies

Blood ◽

10.1182/blood.v126.23.1680.1680 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1680-1680

Author(s):

Aristoteles Giagounidis ◽

Alan F. List ◽

Eva Hellström-Lindberg ◽

Ghulam J. Mufti ◽

Brigitte Schlegelberger ◽

...

Keyword(s):

Research Funding ◽

Employment Equity ◽

Multicenter Studies ◽

Free Survival ◽

Cytogenetic Abnormalities ◽

Poor Risk ◽

Increased Risk ◽

Equity Ownership ◽

Good Risk ◽

Rbc Transfusion

Abstract Introduction: Approximately 50% of pts with de novoMDS present with cytogenetic abnormalities at diagnosis (Haase D, et al. Ann Hematol. 1995;70:171); deletion (del)5q occurs in ~15% of pts (Haase D, et al. Blood. 2007;110:4385). Cytogenetic abnormalities in addition to del(5q) may be associated with shorter overall survival (OS) and increased risk of progression to acute myeloid leukemia (AML) versus del(5q) alone (Mallo M, et al. Leukemia. 2011;25:110). In 2 large multicenter studies (MDS-003 and MDS-004), lenalidomide (LEN) was evaluated in red blood cell (RBC) transfusion-dependent pts with IPSS Low- or Intermediate-1-risk MDS and del(5q) (List A, et al. N Engl J Med. 2006;355:1456; Fenaux P, et al. Blood. 2011;118:3765). Here, we examine specific cytogenetic abnormalities and outcomes in pts with MDS and del(5q) plus ≥ 2 additional cytogenetic abnormalities from MDS-003 and MDS-004. Methods: Of 353 pts enrolled, 281 had available cytogenetic data with ≥ 12 evaluable metaphases, and were included. Pts received either LEN 10 mg on days 1-21 of each 28-day cycle, LEN 5 mg or 10 mg continuously, or placebo (PBO). In MDS-004, at week (wk) 16, PBO pts could cross over to LEN 5 mg. Centrally reviewed cytogenetic studies were performed at baseline, and wks 24 and 48 (MDS-003); and at baseline, wks 12 and 24, and every 24 wks thereafter (MDS-004). RBC transfusion independence (TI) ≥ 26 wks, cytogenetic response (CyR), AML progression, OS, and AML-free survival were assessed by baseline cytogenetic complexity in LEN-treated pts with del(5q) plus ≥ 2 additional abnormalities. These patients did not fulfill IPSS lower-risk classification after central pathologic/cytogenetic evaluation. Results: Of 281 pts, 25 (8.9%) had del(5q) plus ≥ 2 additional abnormalities at baseline. In these pts, the most common additional abnormalities at baseline were -7 (20.0%), del(13q) (20.0%), +21 (16.0%), and del(11q) (16.0%). Baseline characteristics were comparable across the 24 LEN-treated pts with 2 (n = 9), 3 (n = 8), or ≥ 4 (n = 7) additional abnormalities. Rates of RBC-TI ≥ 26 wks were 44.4%, 50.0%, and 28.6% in pts with 2, 3, or ≥ 4 additional abnormalities (P = 0.77), respectively. In pts evaluable for CyR (n = 21), rates of CyR were 33.3%, 28.6%, and 20.0% (P = 1.00), respectively; all cytogenetic responders achieved RBC-TI ≥ 26 wks. The other pts who achieved RBC-TI ≥ 26 wks but did not meet the criteria for CyR showed reductions in the del(5q) clone. No PBO pts achieved CyR; however, 1 pt had a partial response (PR) after crossover to LEN 5 mg. Of the pts randomized to LEN, 4 achieved a complete response (CR) (5 mg, n = 1; 10 mg, n = 3) and 2 achieved a PR (5 mg and 10 mg). Median duration of CyR was 282 days (range 168-957). The median number of additional cytogenetic abnormalities in the subset of pts with poor-risk abnormalities (i.e. 17p, 3q, and monosomal abnormalities; n = 7) was 3 versus 2 in pts with good-risk abnormalities (i.e. all other abnormalities; n = 14). Rates of RBC-TI ≥ 26 wks were 28.6% versus 57.1% for the poor-risk versus good-risk groups, respectively. Rates of CyR were 14.3% versus 35.7%, respectively (all CR). In pts with 2, 3, or ≥ 4 additional abnormalities, the 2-year AML progression rates were 56.3% (95% confidence interval [CI] 25.8-89.9), 40.0% (95% CI 14.8-80.5), and 33.3% (95% CI 5.5-94.6), respectively. Median time to AML was 1.8 years (95% CI 0.6-not reached [NR]), 3.1 years (95% CI 0.4-4.8), and NR (95% CI 1.6-NR) (P = 0.75), respectively (Figure 1A). Of 10 pts who developed AML, 6 had involvement of chromosome 7 [del(7q) or -7] at baseline, but presence of -7 did not necessarily portend a poor response in all. Median OS was 1.8 years (95% CI 0.6-3.7), 3.6 years (95% CI 0.5-NR), and 1.6 years (95% CI 0.2-3.3) (P = 0.17) in pts with 2, 3, or ≥ 4 additional abnormalities (Figure 1B). Median AML-free survival was 1.5 years (95% CI 0.6-3.7), 2.5 years (95% CI 0.4-4.8), and 1.6 years (95% CI 0.2-3.3) (P = 0.36), respectively (Figure 1C). Conclusions: Although RBC-TI and CyR with LEN do occur in pts with del(5q) plus ≥ 2 additional abnormalities, the prognosis is generally dismal and less favorable versus isolated del(5q) and del(5q) plus 1 additional abnormality (Giagounidis A, et al. Blood. 2014;124:abstract 3270). Pts with del(5q) and complex karyotypes are generally associated with IPSS Intermediate-2- or High-risk MDS, which require more intensive treatment approaches, including azacitidine and stem cell transplantation, if feasible. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Giagounidis: Celgene Corporation: Honoraria. List:Celgene Corporation: Honoraria, Research Funding. Hellström-Lindberg:Celgene Corporation: Research Funding. Mufti:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Morrill:Celgene Corporation: Employment, Equity Ownership. Wu:Celgene Corporation: Employment, Equity Ownership. Skikne:Celgene Corporation: Employment, Equity Ownership. Fenaux:Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding.

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Two Novel Distinct Subtypes of Myeloid Neoplasms Molecularly Associated with Histone H3K36 Methylations

Blood ◽

10.1182/blood.v126.23.2841.2841 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2841-2841 ◽

Cited By ~ 1

Author(s):

Yosaku Watatani ◽

Yasunobu Nagata ◽

Vera Grossmann ◽

Yusuke Okuno ◽

Tetsuichi Yoshizato ◽

...

Keyword(s):

Gene Expression ◽

Copy Number ◽

Copy Number Variations ◽

Healthy Controls ◽

Employment Equity ◽

Gene Deletions ◽

Myeloid Neoplasms ◽

Frequent Mutations ◽

Equity Ownership

Abstract Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

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Analyzing the Transcriptome Discovers up-Regulation of HOXA Genes in Patients with Myeloid Neoplasms and Isochromosome 17q and Mutations in ASXL1, SETBP1 and SRSF2

Blood ◽

10.1182/blood.v128.22.2703.2703 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2703-2703

Author(s):

Manja Meggendorfer ◽

Niroshan Nadarajah ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Differential Expression ◽

Hox Genes ◽

Fold Change ◽

Cytogenetic Abnormality ◽

Differentially Expressed ◽

Employment Equity ◽

Log2 Fold Change ◽

Myeloid Neoplasms ◽

Hoxa Genes ◽

Equity Ownership

Abstract Introduction: Isochromosome 17 (i(17q)) is a rare cytogenetic abnormality reported in different myeloid neoplasms. I(17q) has been described as primary and as secondary chromosomal aberration, often acquired in the disease course. Recently, we have shown that patients with i(17q) show a distinct mutation profile with mutations in ASXL1, SETBP1 and SRSF2. Further data suggested a parallel acquisition of SETBP1 mutation and i(17q) (Meggendorfer et al., Leukemia, 2016). Of note, i(17q) results in three copies of the splicing factor SRSF2, potentially further influencing the transcriptome of affected cells. Aim: To characterize the transcriptome in myeloid neoplasms with i(17q) and the typical mutations in ASXL1, SETBP1 and SRSF2 (A/S/S/i17pos). Patients and Methods: In total 18 patients were selected based on the cytogenetic profile and the molecular mutations. All had the diagnosis of a myeloid neoplasm by cytomorphology according to the WHO. Chromosome banding and FISH analysis and mutation status of ASXL1, SETBP1 and SRSF2 was available in all cases. Three patient groups were defined: 1) ASXL1, SETBP1, SRSF2 mutated and a normal karyotype (A/S/S/-pos; n=5) and gain of i(17q) during follow up, 2) ASXL1, SETBP1, SRSF2 mutated and i(17q) as sole cytogenetic abnormality (A/S/S/i17pos; n=8), 3) ASXL1, SRSF2 mutated and i(17q) as sole cytogenetic abnormality (A/-/S/i17pos; n=5). In all cases RNA sequencing was performed (TruSeq RNA Sample Preparation V2, Illumina, San Diego, CA). A control RNA (Universal Human Reference RNA, Agilent Technologies, Santa Clara, CA) was investigated in triplicate for normalization and comparison. Expression analyses were performed with BaseSpace RNA Express app (Illumina, San Diego, CA). Results: In total 23,710 genes were annotated by RNA sequencing and at least 14,773 analyzed for differential expression, showing that all three patient groups show aberrant expression in comparison to the control RNA. In mean 4,930 genes/group (range: 3,603-6,711) were differentially expressed. The differential expression ranged from -12.7 to 11.2 log2(fold change). Comparing the two groups with all three gene mutations but different i(17q) status (A/S/S/-pos and A/S/S/i17pos) showed that the presence of i(17q) changes the expression pattern with 1,596/13,218 assessed genes differentially expressed. In detail, in A/S/S/i17pos cases 790 genes were significantly over expressed while 806 genes showed reduced expression compared to A/S/S/-pos, ranging from -2.84 to 2.78 log2(fold change). The expression of SRSF2 was not affected by i(17q), although i(17q) cases show three SRSF2 gene copies. Analyzing the most strongly affected genes (2<log2(fold change)<-2, n=48) showed that differential gene expression affected mostly the homeobox (HOX) genes clustering on chromosome 7p15 (HOXA1, HOXA5 and HOXA7) as well as MEIS1, an important cofactor of HOX genes. HOX genes represent a family of transcription factors, shown to be involved in hematopoiesis. Addressing specifically the differential expression of HOX genes showed that further 4 HOXA genes (HOXA2, HOXA4, HOXA9, HOXA10) and 3 HOXB genes (HOXB2, HOXB3, HOXB4) were significantly dysregulated, with HOXB clustering on 17q21. The expression of all HOX genes was up-regulated in cases with i(17q). Interestingly, the molecular mutation pattern A/S/Spos has also been shown to associate with patients having a monosomy 7 (Meggendorfer, #1364, ASH 2013), where the HOXA gene cluster is located. Comparing the HOXA and MEIS1 gene expression in all 18 samples to human control RNA revealed a significant lower expression level in A/S/S/-pos and an increased one in A/S/S/i17pos and A/-/S/i17pos patients, clearly differentiating i(17q) carrying patients. However, the additional SETBP1 mutation did not influence the expression pattern as seen by comparing A/S/S/i17pos and A/-/S/i17pos patients (0/13,398 assessed genes differentially expressed). Conclusion: 1) Transcriptome analysis of patients with myeloid malignancies, i(17q) and mutations in ASXL1, SETBP1 and SRSF2 show an up-regulation of HOXA genes. 2) Accompanying SETBP1 mutation does not further influence the transcriptome of A/-/S/i17pos patients. 3) The up-regulation of HOXA genes might indicate a pathogenic mechanism in patients with ASXL1, SETBP1, SRSF2 and i(17q). However, this finding has to be validated in a larger cohort and with an independent method. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah: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.

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