IDH1/2, TET2 and DNMT3A Mutations Are Not Mutually Exclusive in Secondary Acute Myeloid Leukemias,

Abstract Abstract 3556 IDH1/2, TET2 and DNMT3A mutations have been reported in myeloid malignancies including de novo AML. In this study, we have analyzed the frequency and prognostic impact of these mutations in a large retrospective cohort of patients (pts) with secondary AML (SA) which encompass myelodysplasia-related changes (MRC) AML and therapy-related (TR) AML according to the WHO classification. Bone marrow samples were collected from 247 pts at diagnosis with SA and the mutational status of IDH1/2, TET2 and DNMT3A genes together with other genes frequently mutated in AML (NPM1, FLT-3, N and K-RAS, WT1) was determined by Sanger sequencing or high resolution melting analysis. The cohort of 247 pts consisted in 201 MRC AML and in 46 TR AML, 39.5% of which with a normal karyotype (NK). The frequency of IDH1/2, TET2 and DNMT3A mutations was 12.6, 19.8 and 4.5%, respectively. Two pts had both TET2 and IDH1/2 mutations, 2 pts had TET2 and DNMT3A mutations and 5 pts had both IDH1/2 and DNMT3A mutations showing that these mutations were not mutually exclusive in SA. IDH1/2 and TET2 mutations were significantly more frequent in MRC AML (14.1 and 22.3%) than in TR AML (6.4 and 8.7%) (P =0.04 and P =0.03) while the frequency of DNMT3A mutations was identical in the two subgroups. The SA pts harbouring at least one IDH1/2 or TET2 or DNMT3A mutation were significantly older (P <0.0001) and presented higher leukocyte count and lower MCV (P <0.05) than unmutated pts. Percentage of blasts in the bone marrow was similar in the two groups. Karyotype was normal in 48% of the IDH1/2 or TET2 or DNMT3A mutated pts and 18% of the unmutated patients, indicating that these mutations were strongly associated with NK (P < 0.001). A statistically significant link was found between TET2 or IDH1/2 or DNMT3A mutations and NPM1 mutations, but not with FLT-3, N/K-RAS or WT1 mutations. Complete remission rate and overall survival were evaluated in a group of 158 pts which had received intensive chemotherapy at diagnosis, and were identical in the IDH1/2 or TET2 or DNMT3A mutated and unmutated groups. These mutations did significantly influence survival neither in the subgroup of pts with normal karyotype, nor in the subgroup of MRC-AML, or TR-AML which were of very poor prognosis. These data show that IDH1/2, TET2 or DNMT3A mutations could modify the clinical presentation without impact on prognosis. Disclosures: No relevant conflicts of interest to declare.

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Frequent Mutations and Their Influence on Prognosis in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.4949.4949 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4949-4949

Author(s):

Ekaterina Petrova ◽

Irina Martynkevich ◽

Luybov Polushkina ◽

Luydmila Martynenko ◽

Marina Ivanova ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Melting Curve ◽

Normal Karyotype ◽

Genetic Alterations ◽

Melting Curve Analysis ◽

Prognostic Impact ◽

Age Related ◽

Mutational Status

Abstract Background. Several genetic alterations such as translocations, gene mutations and deletions play an important role in myeloid leukemogenesis. The cytogenetic information is a very significant tool to classify pts at their initial diagnosis into prognostic categories. For pts with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3, CKIT, NRAS and DNMT3A. The aim of the research was to investigate the frequency and prognostic impact of FLT3, NPM1, CKIT, NRAS and DNMT3A mutations in AML pts and to analyze their interaction with other prognostic markers. Methods. This study was performed in 200 adult pts (190 pts with de novo and 10 pts with secondary AML), previously untreated, median age 55 years (18-86). According to the results of cytogenetic analyses pts were separated in four groups: with favourable (9,0%), unfavourable (14,0%) prognosis, with normal karyotype (NK) (48,5%) and other aberrations (28,5%). Mutations in FLT3, CKIT and NPM1 were analysed by PCR and in NRAS by sequencing. Mutation analysis of DNMT3A R882 was performed by high-resolution melting curve analysis. Cytogenetic studies were analysed on bone marrow samples using standard GTG-method. Results. Mutations in FLT3, CKIT, NRAS and NPM1 genes were detected in 105/200 (52,5%) pts. A total of 128 mutations were revealed in this group: 24,0% - FLT3-ITD, 6,5% - FLT3-TKD, 20,5% - in NPM1, 10,0% - in NRAS and 3,0% - in CKIT. 82 pts had single mutations and in 23 pts mutations occurred simultaneously: 17 with FLT3-ITD and in NPM1, 2 with FLT3-ITD and FLT3-TKD, 1 with FLT3-TKD and in NPM1, 3 with NPM1 and NRAS mutations. We found that mutations with the significantly higher incidence (p=0,001) were observed in the group of pts with NK (80/97), whereas there were only 8/28 pts with mutations in the group with complex karyotype. When analyzing the age-related features, it was shown that the majority of mutations were detected in the group of pts at the age from 60 to 69 years. Mutations FLT3-ITD and FLT3-TKD were associated with higher WBC count comparing with pts without mutations (p=0,001 and p=0,014, respectively). The median follow-up for overall (OS) and relapse-free (RFS) survival for pts with FLT3-ITD against ptswith FLT3-ITD- was: 5,4 vs 12,8 months and 4,9 vs 10,0 months (p=0,001 and p=0,001), respectively. Mutation FLT3-TKD was also found to be prognostically unfavorable, but only comparing with pts with FLT3-ITD- genotype. As the result of OS and RFS analyses in pts with and without NPM1 mutations we revealed the significant favorable influence of NPM1 mut on the prognosis (p=0,002 and p=0,020, respectively). However pts with genotype FLT3-ITD+/NPM1+ were found to get to the group with an intermediate risk. We detected the significant adverse influence of CKIT mut on RFS (p=0,041). Mutations in NRAS didn't impact on prognosis; we only showed the tendency towards worsening of OS and RFS in group of pts with favorable cytogenetics (p=0,214 and p=0,160, respectively). Mutations DNMT3A R882 were investigated in group of 143 AML pts and were detected in 23 (16,1%) pts. Pts with DNMT3A R882 had higher WBC (p=0,001) and platelets (p=0,020) count at diagnosis and more frequently belonged to FAB groups M5 (p=0,003), as compared with DNMT3A wt. Of 23 pts who had AML with DNMT3A mutations, 17 had tumors with NK profiles (24,3% of a total of 70 cytogenetically normal samples) (p=0,009). Pts with isolated DNMT3A mutations were seen in 4 cases, whereas in the rest of pts they were detected simultaneously with mutations in genes FLT3, NPM1, NRAS and CKIT. DNTM3A mutations were significantly more prevalent in NPM1 mut (p=0,005) and FLT3-ITD (p=0,005) positive cases than wild type. DNMT3A mutations were associated with negative influence on pts OS and risk of relapse, compared with DNMT3A wt (р = 0,031 and р = 0,045, respectively). Summary. Mutations in FLT3 and NPM1 had a significantly higher incidence in the group of pts with a normal karyotype. FLT3 mutations showed the adverse prognostic value. Insertions in NPM1 were shownto be the favorable factor, correlating with prolonged RFS in all pts excepting pts with FLT3-ITD+/ NPM1+ genotype. CKIT mut was associated with higher relapse incidence in AML pts, while NRAS mut showed lack of prognostic significance. AML with DNMT3A mut represent the group, homogeneous on a number of clinical and laboratory parameters, associated with adverse prognosis and a high risk of the relapse. Disclosures No relevant conflicts of interest to declare.

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Impact of Cytogenetics and New Molecular Markers On the Achievement of Complete Remission in Patients with Primary Acute Myeloid Leukemia. On Behalf of CETLAM Cooperative Group. Spain.

Blood ◽

10.1182/blood.v114.22.4711.4711 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4711-4711

Author(s):

Salut Brunet ◽

Mar Tormo ◽

Jordi Esteve ◽

Josep M. Ribera ◽

Montserrat Hoyos ◽

...

Keyword(s):

Complete Remission ◽

De Novo ◽

Conflicts Of Interest ◽

Molecular Profile ◽

Prognostic Impact ◽

Molecular Abnormalities ◽

Mutational Status ◽

De Novo Aml ◽

Cebpa Mutations ◽

Normal Cytogenetics

Abstract Abstract 4711 Aim To analyze the prognostic impact of cytogenetics and main molecular abnormalities on the achievement of complete remission (CR), in patients with primary (de novo) AML (M3 excluded). Patients and Methods Between december-2003 and july-2009, 605 patients up to 70 years-old were included in the CETLAM AML-03 protocol. Induction therapy consisted in one or two courses of idarubicin, intermediate dose ara-C and etoposide, in addition to G-CSF priming from day 0. Cytogenetics classification was as in the MRC studies: Favorable prognosis (FP), intermediate (IP) and adverse (AP). In the IP group, molecular analysis of NPM1 (NPM1+) mutations, CEBPA mutations and internal tandem duplication of FLT3 gene (ITD/FLT3) was performed. In the AP group, the absence (MK-) or presence of monosomal karyotype (MK+) was studied; MK+ was defined as 2 or more autosomal monosomies, or one monosomy and ≥1 structural alteration. Results Median age of the series was 53 years (range, 16-73). In 538 (89%) patients cytogenetic data were available; of them, 64 (10,5%) had FP, 375 (61,8) IP (included 255 with normal cytogenetics) and 99 (16,3%) AP. In the FP group, 33 (5,4%) had the AML1/ETO fusion and 31 (5,1%) the CBF/MYH11. In the IP group, 121 (31%) of 279 patients studied were NPM1+, 100 (26,7%) of 346 were DIT/FLT3+ and 22 (6%) of 235 analyzed were CEBPA+. In patients with AP, 47 were MK- and 33 MK+. Overall, 447 (73,8%) of 588 patients evaluable at the moment of the analysis achieved a CR. CR rates according cytogenetics were: FP 92,2%, IP 76,5% and AP 69,4%, p=0.01. In AML, with AML1-ETO+ and CBF/MYH11+, CR rates were 91% and 93.5%, respectively; of note no chemorefractory cases were observed. In the IP group, CR rates according to mutational status were: NPM1+/FLT3- 91.2%, CEBPA+/FLT3- 94.4%, no mutations 79.1% and DIT/FLT3+ 69.7% (p=0.003). Again, no refractoriness was observed in patients in IP patients belonging to the two groups with favourable molecular profile (p=0.003). In patients with AP, CR rate was 76.1% if MK- and 65.6% if MK+ (p=0.46). The prognostic impact of the herein described cytogenetic and molecular findings was confirmed in multivariate analyses. Comments Genetic characterization is nowadays mandatory in AML. CR rate is high and refractoriness exceptional in the presence of AML1-ETO or CBF/MYH11 rearrangements, or NPM1 or CEBPA mutations without DIT/FLT3. In contrast, CR probability is lower in AP group patients, mainly in those with MK+. These patients require new strategies within clinical trials. Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures: No relevant conflicts of interest to declare.

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AML with Recurrent Genetic Abnormalities and AML with Myelodysplasia-Related Changes Account for Three-Quarters of Previously Untreated Pediatric AML-the Results of a Nation-Wide Central Review in Japan-

Blood ◽

10.1182/blood.v118.21.4899.4899 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4899-4899

Author(s):

Akitoshi Kinoshita ◽

Hayato Miyachi ◽

Hiromichi Matsushita ◽

Tomohiko Taki ◽

Miharu Yabe ◽

...

Keyword(s):

Who Classification ◽

De Novo ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Prognostic Impact ◽

Genetic Abnormalities ◽

Precise Diagnosis ◽

De Novo Aml ◽

Pediatric Aml ◽

Mixed Phenotype

Abstract Abstract 4899 [Background] The WHO classification has been widely accepted among physicians who are engaged in treating pediatric AML patients. In 2008, the revised WHO classification has expanded the two categories in AML; AML with recurrent genetic abnormalities and AML with myelodysplasia-related changes. The epidemiology and prognostic significance of these refined categories remains to be explored in children. [Methods] JPLSG AML-05 is a nationwide clinical trial for children with de novo AML, excluding acute promyelocytic leukemia and myeloid leukemia with Down syndrome, which was conducted between November 2006 and December 2010 in Japan. A central review of diagnosis based on the WHO classification was prospectively performed on each case soon after morphological, cytogenetical and immunological data were submitted to data center. Regarding the cases with discrepant results among these parameters, further diagnostic tests including FISH and chimera gene analyses were underwent to confirm the diagnoses. [Results] Four hundred and eighty four patients were enrolled in the study. Thirty patients did not meet the criteria of AML. We could not collected suitable data for diagnosis in 6 patients. Regarding the rest 448 patients, diagnoses based on the WHO classification 2001 and 2008 were determined. According to the 2001 version, 227 (50.6%) had AML with recurrent genetic abnormalities:124 (27.7%) of AML with t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), and 33 (7.4%) of AML with the other11q23 (MLL) abnormalities, 36 (8.0%) had AML with multilineage dysplasia, and 185 (41.3%) had AML, not otherwise categorized. According to 2008 version, 235 (52.5%) had AML with recurrent genetic abnormalities: 124 (27.7%) of t(8;21)(q22;q22);(AML1/ETO ), 32 (7.1%) of AML with inv(16)(p13q22); (CBFβ/MYH11), 38 (8.5%) of AML with t(9;11)(p22;q23), 33 (7.4%) of AML with the other11q23 (MLL) abnormalities,4 of AML with t(6;9)(p23;q34);DEK-NUP214,2 of AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI13, and 2 of AML with t(1;22)(p13;q13);RBM15-MKL, 88 (19.6.7%) had AML with myelodysplasia-related changes (29 from morphological features of myelodysplasia and 59 from myelodysplasia-related cytogenetic abnormalities), 119 (26.6%) had AML, not otherwise categorized and 7(1.6%) had mixed phenotype acute leukemia (6 of T/myeloid and 1 of B/myeloid). [Discussion] Our comprehensive approach for diagnosis was a useful modality for precise diagnosis of uncertain cases, which might have been assigned to the category of AML, with not otherwise categorized, previously. As a result, the present study shows an increased prevalence of AML with recurrent genetic abnormalities or AML with myeloid dysplasia-related changes among pediatric patients with previously untreated AML. Analysis of the AML-05 trial will elucidate the prognostic impact of these categories. Disclosures: No relevant conflicts of interest to declare.

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EZH2 Mutations Are Associated with Low Blast Percentage in Bone Marrow and −7/Del(7q) Karyotype in De Novo Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2544.2544 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2544-2544

Author(s):

Xiuli Wang ◽

Haiping Dai ◽

Qian WANG ◽

Qinrong Wang ◽

Yang Xu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Myelogenous Leukemia ◽

Prognostic Impact ◽

Coding Region ◽

Acute Myeloid

Abstract Abstract 2544 Somatic mutation of the EZH2 gene is seen in myelodisplastic syndrome, myelofibrosis, and chronic myelomonocytic leukemia patients. The prevalence and prognostic impact of somatic mutations of EZH2 in patients with acute myelogenous leukemia (AML) remains unknown. In this study, we sought to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo AML by PCR amplification of the entire coding region followed by direct bidirectional DNA sequencing. EZH2 mutations were identified in 13/714 (1.8%) of AML patients and occurred almost exclusively in males (11/13, P=0.033). In univariate analysis, the presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P=0.0001) and −7/del(7q) (P=0.025). There was no difference in the incidence of mutations in 13 genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. Complete remission, event-free survival or overall survival was similar between AML patients with and without EZH2 mutation (p>0.05). These results demonstrated EZH2 mutation as a recurrent genetic abnormality associated with lower blast percentage in BM and −7/del(7q) in de novo acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Impact of the Interaction between DNMT3A mutation and Internal Tandem Duplication of the FLT3 Gene (FLT3-ITD) in Patients with De Novo Mutated NPM1 (NPM1mut) acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-115337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1492-1492

Author(s):

Guadalupe Oñate ◽

Ana Garrido ◽

Jordi Esteve ◽

Rosa Coll ◽

Montserrat Arnan Sangerman ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

De Novo ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

De Novo Aml ◽

Acute Myeloid

Abstract Introduction The association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio (<0.5, FLT3low) at diagnosis presented an overall survival and relapse rate similar to those with NPM1mut and FLT3wt. Therefore, in the CETLAM-2012 protocol, patients with FLT3low NPM1mut AML are not considered for allogenic hematopoietic stem cell transplant (allo-HSCT) in first complete remission (CR1). Recent studies suggest that the co-occurrence of DNMT3A mutation in FLT3-ITD NPM1mut AML patients confers a worse prognosis regardless of FLT3-ITD ratio. We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications. Methods and patients A total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1. Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used. Results Out of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234) When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A). In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B). Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012). Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B). Conclusions In this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. Disclosures No relevant conflicts of interest to declare.

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A 17-Gene Leukemia Stem Cell (LSC) Score in Adult Patients (Pts) with Acute Myeloid Leukemia (AML) Reveals a Distinct Mutational Landscape and Refines Current European Leukemianet (ELN) Genetic Risk Stratification

Blood ◽

10.1182/blood-2018-99-115114 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 289-289 ◽

Cited By ~ 1

Author(s):

Marius Bill ◽

Deedra Nicolet ◽

Ann-Kathrin Eisfeld ◽

Krzysztof Mrózek ◽

Christopher J. Walker ◽

...

Keyword(s):

De Novo ◽

Gene Mutations ◽

Disease Free Survival ◽

Prognostic Impact ◽

Expression Data ◽

Age Related ◽

Mutational Status ◽

Multivariable Analyses ◽

De Novo Aml ◽

Favorable Group

Abstract Introduction: Prognosis of AML pts is still poor mainly because of refractoriness to or relapse after intensive chemotherapy. High rates of relapse are also attributed to LSCs, which are a small subset of cells with acquired abnormal self-renewal capacity and increased resistance to chemotherapy. A better understanding of LSCs is critical to improve outcomes of pts with AML. Ng et al. (Nature 2016;540:433) defined a 17 stemness-associated gene score that was highly prognostic. Aims: The aim of this study was to validate the prognostic relevance of the 17-gene LSC score and explore its utility in the context of the ELN classification. We also examined gene mutations associated with the 17-gene LSC score. Methods: We analyzed a total of 934 pts [729 aged <60 years (y) and 205 aged ≥60 y] with de novo AML. We used whole transcriptome expression data (RNAseq) to calculate the aforementioned 17-gene LSC score for each pt in our cohort. Similar to Ng et al., we used the median of the whole cohort to discriminate between pts with LSChigh and LSClow scores. The mutational status of 80 cancer- and leukemia-associated genes (Eisfeld et al. Leukemia 2017;31:2211) were determined using a targeted next-generation sequencing panel, CEBPA mutations using Sanger sequencing, and an internal tandem duplication (ITD) of the FLT3 gene using fragment analysis in pretreatment bone marrow or blood samples. All pts were treated on frontline Cancer and Leukemia Group B/Alliance protocols. Results: A comparison of pretreatment clinical and genetic features revealed that LSChigh pts were older (P<.001; median age, 53 vs 46 y) and had higher platelet counts (P<.001; median, 63 vs 50x109/L) than LSClow pts. Pts with a LSChigh score more frequently had FLT3-ITD (P<.001) and mutations in the ASXL1 (P=.001), DNMT3A (P<.001), RUNX1 (P=.002), SRSF2 (P=.02), STAG2 (P=.009), TET2 (P=.008) and TP53 (P<.001) genes. Conversely, these pts had a lower frequency of biallelic CEBPA (P<.001), GATA2 (P=.008) and KIT (P<.001) mutations. Because of differences in treatment intensity, we analyzed outcomes of younger and older pts separately. Younger pts with a LSChigh score had a lower complete remission (CR) rate (P<.001; 63% vs 87%), shorter disease-free survival (DFS; P<.001; 3-y rates, 26% vs 48%; Figure 1A) and overall survival (OS; P<.001; 3-y rates, 27% vs 59%; Figure 1B) compared to those of LSClow pts. In multivariable analyses including clinical and genetic factors that impact on outcome, a LSChigh score associated with lower remission rates (P<.001; HR: 0.36), shorter DFS (P<.001; HR: 1.67) and OS (P<.001; HR: 1.88) after adjusting for other co-variates. We also analyzed the prognostic impact of the LSC score with respect to the 2017 ELN classification. We found that LSC score associated with different ELN groups (P<.001), with LSChigh pts being more often classified in the Adverse or Intermediate group and less often in the Favorable group. Within the ELN Favorable and Adverse groups, LSChigh score retained its prognostic impact and identified pts with a lower CR rate and shorter DFS and OS (Table1). In older pts, a LSChigh score also associated with lower CR rate (P=.004; 50% vs 72%), shorter DFS (P=.04; 3-y rates, 6% vs 17%; Figure 1C) and OS (P<.001; 3-y rates, 9% vs 27%; Figure 1D). In multivariable analyses, LSC score remained significant only for OS (P<.003; HR: 1.70) after adjusting for other co-variates. Regarding the ELN classification, pts with LSChigh score in the Favorable group had shorter OS (P=.05; 3-y rates, 17% vs 50%) and, by trend, shorter DFS (P=.09; 3-y rates, 17% vs 39%); no significant differences were found in Intermediate or Adverse groups. Conclusions: We used RNAseq expression data and applied the previously established 17-gene LSC signature to score 934 de novo AML pts. We detected distinct mutational differences between LSChigh and LSClow pts, with LSChigh pts more often carrying gene mutations associated with age-related clonal hematopoiesis (i.e., ASXL1, DNMT3A, TET2, SRSF2 and TP53 mutations). Moreover, this score, derived from the expression of stemness-associated genes, has not only a prognostic impact on its own but also in the context of the current 2017 ELN classification. Disclosures Kolitz: Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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About 17% of AML with NPM1 mutations Show a Specific Pattern of Chromosome Aberrations but These Cases Do Not Differ Prognostically from AML with NPM1 Mutations Carrying a Normal Karyotype

Blood ◽

10.1182/blood.v112.11.2527.2527 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2527-2527

Author(s):

Claudia Haferlach ◽

Susanne Schnittger ◽

Tamara Weiss ◽

Wolfgang Kern ◽

Brunangelo Falini ◽

...

Keyword(s):

Chromosome Aberrations ◽

Chromosome Banding ◽

Who Classification ◽

De Novo ◽

Normal Karyotype ◽

Prognostic Impact ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Additional Chromosome ◽

De Novo Aml

Abstract AML with mutated nucleophosmin gene (AML NPM1mut) usually carries a normal karyotype and will be suggested as a provisional entity in the new WHO classification. Thus far, the impact of chromosome aberrations in AML NPM1mut has not been evaluated in detail. Aim of this study was to determine the incidence and prognostic impact of clonal chromosome aberrations in NPM1mut. We further compared this pattern to additional aberrations in AML with recurrent genetic aberrations: t(8;21)(q22;q22), inv(16)(p13q22)/t(16;16)(p13;q22), t(15;17)(q22;q12) and 11q23-abnormalities leading to an MLL-rearrangement. In total 415 AML (de novo AML: 392, s-AML: 11, t-AML: 12) showing an NPM1 mutation were analyzed by chromosome banding analysis. 71 of these showed clonal chromosome aberrations (17.1%; de novo AML: 63 (16.1%), s-AML: 5 (45.5%), t-AML: 3 (25%); de novo AML vs. s-AML: p=0.024). Overall, 111 chromosome aberrations were observed. The most frequent abnormalities were +8 (n=30), −Y (n=10), +4 (n=9), del(9q) (n=5), +21 (n=4), −7 (n=3), +5 (n=2), +10 (n=2), +13 (n=2),+18 (n=2), del(12p) (n=2), del(20q) (n=2), other non-recurrent balanced aberrations (n=6), other non-recurrent unbalanced aberrations (n=32). For comparison 63 AML with t(8;21), 37 cases with inv(16)/t(16;16), 83 patients with t(15;17) and 83 AML showing a 11q23/MLL-rearrangement were evaluated. 44 (69.7%), 13 (35.1%), 39 (47%), and 28 (43.1%) cases showed clonal chromosome aberrations in addition, respectively. Therefore, additional chromosomal aberrations are more frequent in all these subgroups than in the AML NPM1mut. Similar to NPM1mut cases +8 (n=2), −X/Y (n=32), +4 (n=2), and del(9q) (n=10) were observed. The only other recurrent additional aberrations was del(11q) (n=2). In inv(16)/t(16;16) we also found +8 (n=5) and −Y (n=1). The only other recurring additional aberrations were +22 (n=6) and del(7q) (n=2). In AML with t(15;17) recurring additional abnormalities were +8 (n=12), −Y (n=3), del(9q) (n=2), ider(17)(q10) t(15;17) (n=7). AML with 11q23/MLL-rearrangement showed +4 (n=2), +8 (n=8), +13 (n=2), +19 (n=4), +21(n=4), +22 (n=2), −Y (n=1). Thus, chromosome aberration in AML NPM1mut share many overlaps to those in AML with recurrent aberrations. Furthermore, the prognostic impact of chromosome aberrations in AML NPM1mut was evaluated. No difference with respect to overall survival (OS) and event-free survival (EFS) was observed between AML NPM1mut with a normal (n=344) and an aberrant karyotype (n=71) (OS at 2 yrs 78% vs. 81%, p=0.969; EFS at 2 yrs 40% vs. 50%, median EFS 544 days vs. 522 days, p=0.253). The FLT3-ITD status was available in 400 cases. 127 (38%) of 334 cases with a normal karyotype showed a FLT3-ITD, while in only 16 (24%) of 66 cases with an aberrant karyotype a FLT3-ITD was observed (p=0.035). While the negative prognostic impact of additional FLT3-ITD was confirmed in our cohort, the presence of chromosome aberrations did not influence prognosis neither in the FLT3-ITD negative nor in the FLT3-ITD positive subgroup. In addition, 31 patients with AML NPM1mut were analyzed by chromosome banding analysis at diagnosis and at relapse (median time diagnosis to relapse: 301 days (range: 71–986). 22 cases (71%) showed a normal karyotype both at diagnosis and relapse. In 4 cases a normal karyotype was observed at diagnosis and an aberrant karyotype at relapse (del(9q) (n=2), t(2;11) (n=1), inv(12) (n=1)). One case with +8 at diagnosis showed +8 also at relapse. One case with +4 at diagnosis showed +4 and additional aberrations at relapse. In 1 case clonal regression was observed (+21 -> normal). One case with an unbalanced 1;3-translocation at diagnosis showed a der(17;18) (q10;q10) at relapse and one case with −Y at diagnosis showed a del(3p) at relapse. In conclusion: 1. Frequency of additional chromosome aberrations is low in AML NPM1mut as compared to other genetically defined WHO entities. 2. The pattern of additional chromosome aberrations is overlapping between the 5 groups analyzed. 3. Chromosome aberrations observed at diagnosis in AML NPM1mut do not influence prognosis in comparison to AML NPM1mut with normal karyotype. 4. The karyotype is stable in most AML NPM1mut patients at diagnosis and at relapse. These results point to chromosomal aberrations occurring in AML NPM1mut as secondary events and further support inclusion of AML NPM1mut as a provisional entity in the new WHO classification.

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Analysis of Clonal Cytogenetic Abnormalities in 143 Patients with Secondary AML/MDS

Blood ◽

10.1182/blood.v118.21.1473.1473 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1473-1473

Author(s):

Elena V Domracheva ◽

Elena A Aseeva ◽

Galina A Alimova ◽

Olga S Kremenetskaya ◽

Liubov A Shishigina ◽

...

Keyword(s):

Bone Marrow ◽

De Novo ◽

Conflicts Of Interest ◽

Chromosome 7 ◽

Secondary Neoplasms ◽

Monosomy 7 ◽

Cytogenetic Abnormalities ◽

De Novo Aml ◽

Secondary Mds ◽

Balanced Translocations

Abstract Abstract 1473 The incidence of the secondary neoplasms has increased because of the rising numbers of long-term survivors of tumours. Secondary leukemias (sL) and secondary MDS (sMDS) are among the most common types of secondary tumours. Until recently prognosis in cases of sL and sMDS was considered less favorable than in leukemias de novo. Age at presentation and identified clonal cytogenetic abnormalities are among the most important independent prognostic factors in adult patients with leukemias. It is obvious today that the presence of t(15;17)(q22;q12), t(8;21)(q22;q22), inv(16)(p13;q22)/t(16,16)(p13;q22) predicts a relatively favorable outcome, and in contrast the presence of inv(3)(q21q26)/t(3,3)(q21;q26), del(5q), −5, −7 or a complex karyotype (CK) with 3 or more abnormalities generally suggests a very poor prognosis. The monosomal karyotype (MK) defined as two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in combination with at least one structural chromosomal abnormality is also considered as an adverse prognostic factor according to Breems D.A. et al., 2008; Medeiros B.C. et al., 2010 4-year overall survival in AML patients with MK is very low – 3–4%. Therefore, the purpose of our analysis was to determine the frequency of “unfavorable” and “highly unfavorable” (according to Breems D.A. et al.) clonal cytogenetic abnormalities, identified in our laboratory in bone marrow samples of 143 patients with sL/sMDS and to compare it with the frequency of MK in leukemias and MDS de novo according to a published multicenter study (Haase D. et al., 2007; Medeiros B.C. et al., 2010; Grimwade D. et al., 2010). All examined patients with sL/sMDS had solid tumors or lymphomas in anamnesis, for which they received chemotherapy and/or radiotherapy. sMDS was identified in 81 patients (54 patients – ≤5% blasts in bone marrow; in 27 patients – >5%). sAML was identified in 56 patients, sALL – in 1 patient, sCML – in 5 patients. Abnormal karyotypes were observed in 42 (52%) sMDS patients, in 37 (66%) sAML patients, in all 5 sCML patients, in the only sALL patient. The most frequent abnormality in sMDS was isolated monosomy 7: it was observed in 24.4% of the tested abnormal karyotypes. CK and MK are considerably more frequent in sMDS than in de novo MDS. CK occurred in 12 (30.9%) sMDS patients with abnormal karyotypes. Monosomies or deletions of the long arm of chromosome 7 were detected in 8 of 12 identified CK. Balanced translocations in sMDS were detected in only 9 (21%) of 42 karyotypes; no rearrangements involving 3q26, rather frequently occurred in de novo MDS, were registered. Very rare for de novo MDS t(1;7)(q10;q10) was found in 5 of these 9 cases. In general, chromosome 7 abnormalities (translocations, monosomies and/or deletions) were observed in 58.5% of sMDS cases with abnormal karyotypes. In de novo MDS chromosome 7 abnormalities were detected only in 21% of cases. On the contrary, del(5q) occurred more frequently in de novo MDS than in sMDS (30% versus 12.2%). Monosomic karyotypes occurred in 23.8% of sMDS patients with abnormal karyotypes. “Favorable” anomalies were presented in 5 of 37 sAML cases (13,5%) abnormal karyotypes. t(15;17), as a single anomaly, was detected in 3 patients; t(8;21) was detected in 2 cases. “Unfavorable” abnormalities, such as inv(3)(q21;q26)/t(3;3)(q21;q26) in complex karyotypes were observed in 4 cases. Chromosome 5 deletions in complex karyotypes were found in 5 cases, and only in 1 case - as a single anomaly. Other deletions, del(11)(q23), del(12)(p11), del(13)(q12), were found only as isolated anomalies. Complex karyotypes in sAML were observed in 40% (15 of 37) of cases with abnormal karyotypes, whereas in de novo AML CK occurred only in 18% of patients with abnormal karyotypes. Monosomic karyotypes occurred more often in patients with sAML - 27% compared to 13% of cases in de novo AML. In conclusion, prognostically “unfavorable” and “highly unfavorable” cytogenetic abnormalities account for 60% and 25% of all cases with karyotype abnormalities in sAML/sMDS. Thus, our study shows that “unfavorable” and “highly unfavorable” cytogenetic abnormalities in leukemic clone occur more often in sAML/sMDS than in de novo AML/MDS. Disclosures: No relevant conflicts of interest to declare.

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Clinical Implications of Reticulin Fibrosis of Bone Marrow in De Novo Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2585.2585 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2585-2585

Author(s):

Tzung-Chih Tang ◽

Hung Chang ◽

Chien-Feng Sun ◽

Lee-Yung Shih ◽

Po Dunn ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

De Novo ◽

Risk Groups ◽

Prognostic Impact ◽

Recovery Duration ◽

De Novo Aml ◽

Acute Myeloid

Abstract Abstract 2585 Background: Microenvironment of bone marrow (BM) plays an important role to support proliferation, renewal and differentiation of hematopoietic stem cells. Whether the stroma of BM affects leukemic cells with the same manner, or impacts on the prognosis in leukemia patients, has not been fully investigated. Previous studies have described that increased reticulin content in the BM is associated with poor outcome in patients with acute lymphoblastic leukemia, chronic myeloid leukemia and primary myelofibrosis, but there is no cohort study to determine the clinical correlation between degree of reticulin fibrosis of BM and acute myeloid leukemia (AML). To investigate prognostic impact of reticulin fibrosis on de novo AML, 881 patients diagnosed between Jun 1999 to Dec 2011 in Chang Gung Memorial Hospital and treated with anthracycline-containing induction chemotherapy were retrospectively reviewed. Patients and methods: According to the grading of reticulin content in the bone marrow, we categorized the 881 patients into four groups: A. BM easily aspirated without biopsy, n = 698; B. Reticulin grade 0, n = 99; C. Reticulin grade 1–2, n = 51; D. Reticulin grade 3–4, n = 33. The induction failure (IF) rate after treatment with induction chemotherapy, the recovery duration of absolute neutrophil count (ANC) greater than 0.5 × 109/L in patients who achieved the first complete remission, the overall survival (OS) and relapse-free survival (RFS) in four groups were analyzed. Based on the cytogenetic or molecular features, 648 of the patients were stratified into unfavorable, intermediate and favorable risk groups, and the clinical significance of reticulin fibrosis of BM were also examined for various risk groups. Results: Of the 881 patients, the patients in group D had a statistically higher IF rate (P = 0.0108) and longer ANC recovery duration (P = 0.0008). But the OS and RFS between four groups were not significantly different (P = 0.5146 and 0.3853, respectively). After risk stratified by cytogenetic and molecular analysis, increased reticulin content of BM (group C or D) had an adverse impact on OS in the intermediate and favorable risk groups (P = 0.006 and 0.0215, respectively). Conclusion: Reticulin content of BM influences the IF rate and myeloid recovery for the patients of de novo AML, and affects OS in patients with intermediate or favorable risk factors. Disclosures: No relevant conflicts of interest to declare.

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WT1 Levels At Diagnosis and POST-Induction Provide Prognostic Information in Adult De Novo AML. Results From the Spanish Cetlam Group.

Blood ◽

10.1182/blood.v120.21.2524.2524 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2524-2524

Author(s):

Josep F Nomdedeu ◽

Montserrat Hoyos ◽

Maite Carricondo ◽

Elena Bussaglia ◽

Camino Estivill ◽

...

Keyword(s):

Bone Marrow ◽

De Novo ◽

High Dose ◽

Prognostic Impact ◽

Post Induction ◽

Group 3 ◽

Group 2 ◽

De Novo Aml ◽

Intermediate Dose ◽

Group 1

Abstract Abstract 2524 WT1 monitoring is an almost universal target to follow de novo AML. Its exppression in myeloid malignancies is upregulated in parallel to the blast percentage. Recently, WT1 determination has been standardized as result of an European Leukemia Net initiative. Early reports have demonstrated that the best results are obtained when peripheral blood is used to establish clinical predictions. Pediatric studies in AML have shown that raised WT1 levels after induction associate with unfavourable outcome. Despite all the mentioned, WT1 quantitation has not yet gained widespread use, in part because some AML show normal WT1 levels at diagnosis. To investigate the prognostic impact of the normalized bone marrow WT1 levels at diagnosis and post-induction in a consecutive series of de novo AML patients enrolled in the CETLAM group trials. Available bone marrow samples at diagnosis (586 cases) and post induction (367 cases) were obtained in each participating center and sent to the CETLAM repository center at the Hospital de la Santa Creu i Sant Pau for complete immunophenotype and molecular analyses. One μg of RNA was reverse transcribed to cDNA in a total reaction volume of 20μl containing Cl2Mg 5mM, 10× Buffer, DTT 10mM, dNTP's 10mM each, random hexamers 15μM, RNAsin 20 units (Promega) and 200 units of MMLV enzyme. WT1 expression levels were determined by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI PRISM 7700® Genetic Analyzer (Applied Biosystems, Foster City, CA) using the primers and conditions described by the ELN group (Cilloni et al J. Clin. Oncol 2009;27:5195-201). For WT1 copy number titration, the IPSOGEN® (Marseille, France) plasmid was employed. Results were expressed as copies and four normal bone marrow samples were used as test controls. Patients were treated between 2004 and 2011 according to the CETLAM03 protocol. Adults up to 70 years of age received induction chemotherapy with idarubicin, intermediate-dose cytarabine and etoposide, followed by consolidation with mitoxantrone and intermediate-dose ara-C. Subsequently, patients with favourable cytogenetics at diagnosis received one cycle of high-dose cytarabine.G-CSF priming during induction and consolidation was used. Patients with favorable cytogenetics and high leukocyte counts at diagnosis were treated with autologous transplantation instead of high-dose cytarabine. Furthermore, patients with a normal karyotype but an adverse molecular profile (FLT3 mutations or MLL rearrangements) were allocated to the treatment for unfavorable cases; this included allogeneic transplantation from an HLA-identical donor. Overall survival (OS) was measured from the date of enrolment until the date of death. Leukemia-free survival (LFS) for patients who achieved a CR was calculated from the date of CR to relapse or death. OS and LFS were plotted by the Kaplan-Meier method; differences between curves were analyzed by the log-rank test. The probability of relapse was calculated using cumulative incidence estimates and taking into account the competing risk of death in remission. A WT1 cut-off value of 5065.2 copies at diagnosis was obtained. Two hundred and four samples had WT1 levels greater than this value, whereas 382 samples showed levels below this cut-off. These groups had statistically different OS 55±3 vs 33±5 p<0.001, LFS 52±3 vs 30±6 p:0.004 and CIR 34±3 vs 56±6 p<0.001. As regards the post-induction results, four groups were established: Group 0 (135 patients) with WT1 levels between 0 and 17.5 copies, Group 1 (107 patients) with WT1 values ranging from 17.6 to 76 copies, Group 2 (54 patients) with WT1 between 76.1 and 170.5 copies and Group 3 (71 patients) with WT1 levels after induction greater than>170.6 copies. These groups showed statistically significant differences(p<0.001) in terms of OS: Group 0 59±4 months, Group 1 50±5 months, Group 2 45±7 months and Group 3 23±6 months. LFS was also statiscally different: Group 0: 58±4, Group 1: 46±5, Group 2: 39±8 and Group 3:19±8 (all p<0.001). Lastlly, CIR was markedly different between the four groups: Group 0:25±4, Group 1: 44±5, Group 2: 46±8 and Group 3: 68±8(p<0.001) . WT1 quantitation at diagnosis and post-induction provide a simple and well standardized measurement of the prognostic risk of adult AML patiens. Larger series need to be analyzed to ascertain whether this determination could be incorporated to initial AML risk stratification. Disclosures: No relevant conflicts of interest to declare.

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