WT1 SNP Rs16754 Research Articles

WT1 and BCORL1 Identified By Computational Biology Modeling Analysis of Patient Genomics Are Novel Predictors of Response to Azacitidine (AZA) and Lenalidomide (LEN) Treatment in Acute Myeloid Leukemia (AML)

Introduction: AML patients with relapsed/refractory (R/R) disease have few effective treatment options. LEN+AZA may be an active and better tolerated regimen compared with conventional chemotherapy. This combination has been tested in a phase 2 pilot study of LEN+AZA in 37 R/R older patients with a 49% overall response rate (4 complete remission (CR) / CR with incomplete recovery (CRi) and 14 with morphologic leukemia free state (MLFS). The objective of this study was to utilize the genomics of these patients to retrospectively predict response to AZA+LEN treatment using CBM workflow and identify key genomic characteristics of the responder profiles.Methods: We analyzed the clinical and genomic (NGS WES, cytogenetics) data from pre-treatment sample for a cohort of R/R AML patients. (Blood 2017 130:1337) All available genomic data was input into the CBM predictive workflow (Cellworks Group). Customized, individualized AML patient models - patient specific disease network maps of activated and inactivated dysregulated pathways were created.Digital drug models of AZA and LEN were created for CBM by programming their mechanism of action and effects on specific protein targets and pathways determined from published literature. The digital drug models were simulated individually and in combination on every patient's disease at varying doses. Drug impact was assessed by quantitatively measuring a disease inhibition score (DIS), the degree to which cancer phenotypes (proliferation, viability, apoptosis) were repressed, along with impact on the identified patient specific biomarkers. CBM predictions were compared with clinical outcomes in a retrospective blinded manner. Clinical response was defined as partial response or better as per International Working Group 2006 criteria.Results: 32 of 37 patients were analyzed by CBM based on availability of genomic profiling. 24 of the 32 patients were clinically evaluated, of which 4 had CR or CRi and 9 had MLFS response. CBM predictions matched for 20 of 24 profiles. The statistics of prediction accuracy against clinical outcome are presented in Table 1.A common aberration associating with sensitivity discovered in 7 profiles was a synonymous WT1 SNP rs16754. It was found in 1 CR and 5 MLFS cases and in the 1 non-responder case that along with WT1 SNP also had mutations in EZH2 and NOTCH1 that caused drug resistance. This WT1 SNP rs16754 correlated with higher mRNA expression of Wt1 and was associated with significantly improved outcome in pAML (PMID: 21189390) WT1 directly transactivates DNMT3A expression, thus improving response to hypomethylating agent. (PMID: 23042785)Another surprising find was the LOF mutation in BCORL1 (BCL6 Corepressor Like 1) L622P, that was the only common aberration found in all the 4 CR profiles. BCORL1 is a transcriptional corepressor that can also bind and regulate class II HDACs. LOF mutations in BCORL1 have been found in AML patients with abnormal karyotypes (PMID:22210327) In aplastic anemia, clones with BCORL1 mutations correlated with better response to immunosuppressive therapy and higher OS (PMID: 26132940), but no such association has been established in AML and may be worth exploring further in larger datasets.Conclusion: The CBM prediction of AZA+LEN in R/R AML patients based on genomic abnormalities and clinical data showed high predictive accuracy of 83.33%. The study validates the approach to a priori predict response and identify the right therapy option for the patient and could be used to establish criteria for precision enrollment in drug development trials. In addition, analysis uncovered novel predictors of response to AZA+LEN therapy that include presence of WT1 SNP rs16754 and possibly BCORL1 LOF, that were found in the responder profiles and linked to enhancing the drug efficacy network. This clinical decision support technology and these biomarkers may improve patient selection for AZA+LEN treatment in R/R AML. [Display omitted] DisclosuresAbbasi:Cell Works Group Inc.: Employment. Singh:Cellworks Research India Private Limited: Employment. Ullal:Cellworks Research India Private Limited: Employment. Kumari:Cellworks Research India Private Limited: Employment. Tyagi:Cellworks Research India Private Limited: Employment. Alam:Cellworks Research India Private Limited: Employment. Lunkad:Cellworks Research India Private Limited: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry. Vali:Cell Works Group Inc.: Employment. Pollyea:AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Curis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Wilms tumor gene single nucleotide polymorphism rs16754 predicts a favorable outcome in children with acute lymphoblastic leukemia.

Wilms tumor gene single nucleotide polymorphism (WT1 SNP) rs16754 has been described as a favorable risk marker in patients with acute myeloid leukemia. Subsequent studies revealed inconsistent results in both adult and pediatric patients. We analyzed its impact on clinical outcome in children with acute lymphoblastic leukemia (ALL) for the first time. WT1 SNP rs16754 of 158 children with ALL treated according to ALL Berlin-Frankfurt-Münster treatment trials from 1990 to 2009 and 43 hematopoietic stem cell donors was analyzed by allelic discrimination. WT1 SNP status was correlated with disease characteristics and clinical outcome comparing SNP (WT1(GG/AG)) and wildtype (WT1(AA)). At least one minor allele was found in 23.4 % of patients and 34.9 % of donors (P = 0.07). Distribution of patient and disease characteristics was similar between WT1(GG/AG) and WT1(AA). In multivariate analyses, WT1 SNP was an independent good prognostic marker for cumulative incidence of relapse (CIR WT1(AA) vs. WT1(GG/AG) HR = 3.384, P = 0.021) and event-free survival (EFS; event risk WT1(AA) vs. WT1(GG/AG) HR = 2.503, P = 0.036). Univariate subanalyses of patients who underwent an allogeneic hematopoietic stem cell transplantation revealed more significant differences in CIR (P = 0.017), EFS (P = 0.012), and overall survival (OS; P = 0.017). Donor's WT1 SNP status did not affect outcome. We found no correlation between WT1 SNP and WT1 expression level at diagnosis (P = 0.634). WT1 SNP rs16754 predicts improved CIR and EFS. Outcome differences were more prominent in transplanted children. Our findings identify WT1 SNP rs16754 as a favorable risk marker in pediatric ALL which is independent from known risk factors.

The Clinical and Prognostic Influence Of Mutations In The Cohesin Complex In Acute Myeloid Leukemia

BackgroundThe cohesin complex presents a ring structure that regulates chromosome segregation during meiosis and mitosis and is thus essential for cell division. Genes that belong to the cohesin complex in somatic vertebrate cells are SMC1A, SMC3, RAD21 (SCC1), STAG2 (SA-2) and STAG1 (SA-1). Recently, mutations in the cohesin complex have been reported to occur in AML. The aim of this study was to evaluate the clinical and prognostic implications of cohesin mutations in 389 uniformly treated younger AML patients. Patients and MethodsDiagnostic bone marrow or peripheral blood samples were analyzed from 389 younger adult patients with de novo (n=348) or secondary AML (n=41) with French-American-British (FAB) classification M0-M2 or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 (n=276) or AML SHG 0295 (n=113). Patient samples were assessed for frequently occurring mutations including FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. In the subgroup of cytogenetically normal AML (CN-AML), additional mutation analyses were performed for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS, and expression levels of BAALC, ERG, EVI1, MN1, MLL and WT1. Sequencing of all coding exons of the cohesin genes was performed with the SOLiDTM system (Life Technologie, Darmstadt, Germany). Sequences were analyzed twice separately using the DNAnexus software and a separate pipeline of bioinformatics software. Identified mutations were validated by Sanger sequencing. Results27 patients (7%) were identified to carry a mutation in one of the cohesin genes. 10 patients harboured a mutation in STAG1. The second most frequently mutated gene in the cohesin complex was SMC3 with 7 patients showing this mutation. 5 patients harboured a mutation in RAD21, 3 patients in STAG2 and 2 patients in SMC1A. All mutations were mutually exclusive. The majority of mutations were missense mutations, apart from 2 frameshift mutations and 3 nonsense mutations. All evaluable patients lost their cohesin mutation during remission and with the exception of one patient regained their mutation during relapse.Patients with mutations in the cohesin genes were older (47 vs 44 years, P=.04), had a higher white blood cell count (WBC) (P=.046) and a higher peripheral blast count (P=.048). We observed a strong correlation between cohesin gene and NPM1 mutations (P=.007). BAALC expression was lower in patients with a mutation compared to patients without a mutation in the cohesin genes (P=.026).In order to get a better understanding of when mutations in the cohesin complex occur during clonal evolution we evaluated the allelic burden of mutations in the cohesin complex. Because of the strong association between NPM1 and cohesin mutations, the allelic ratio of mutated and wildtype NPM1 was compared to the allelic ratio of mutated and wildtype cohesin genes. Interestingly, we found a similar mutation burden between NPM1 and genes of the cohesin complex (Pearson correlation coefficient R=0.67), suggesting that cohesin gene mutations occurred in the same clone as NPM1 mutations.When considering all mutations in the cohesin complex as one group, overall survival (OS), relapse free survival (RFS) and complete remission rate (CR) were not influenced by the presence of cohesin mutations (OS: HR 1.23; 95%CI 0.77-1.99; P=.4; RFS: HR 0.92; 95%CI 0.51-1.65; P=.77; CR: mutated 77% vs wildtype 76%, P=.83). We next evaluated the prognostic influence of each gene in the cohesin complex separately in all patients. STAG1, SMC3 and RAD21 mutations had no influence on OS and RFS. A trend for a reduced OS was observed for STAG2 mutated patients (OS: HR 3.02; 95%CI 0.97-9.46; P=.057), although the analysis is limited by the small number of mutated patients. In the subgroup of patients with cytogenetically normal AML we did not identify a difference in OS, RFS or CR rates for patients with (n=18) or without mutations (283) in the cohesin complex (OS: HR 1.00; 95%CI 0.52-1.92; P=.99; RFS: HR 0.64; 95%CI 0.30-1.39; P=.26; CR rates: mutated 80% vs wildtype 73%, P=.64). ConclusionMutations in the cohesin genes represent novel, recurrent mutations in AML. We found a strong association between these mutations and mutations in NPM1. Cohesin complex mutations as a group had no prognostic impact in all and in cytogenetically normal AML patients. Disclosures:Schlenk:Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

The prognostic effect of high diagnostic WT1 gene expression in pediatric AML depends on WT1 SNP rs16754 status: Report from the Children's Oncology Group

WT1 is aberrantly over-expressed in most cases of AML. We recently demonstrated that WT1 SNP rs16754 correlates with favorable outcome and high diagnostic WT1 expression in childhood AML. We examined the clinical correlates of diagnostic WT1 expression within a contemporary COG trial and determined whether its prognostic impact differs between SNP+ and SNP- patients. WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on COG-AAML03P1. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and outcome. Patients were categorized into four groups (quartiles: Q1 through Q4) based on diagnostic WT1 expression for analysis. FLT3/ITD (P = 0.017) and WT1 mutations (P < 0.001) both occurred more frequently in patients with the highest WT1 expression. SNP rs16754 frequency did not vary significantly among the quartiles. When all patients were considered, survival outcomes were similar between quartiles. However, when only SNP- patients (n = 150) were analyzed, those with highest WT1 expression (Q4) had the poorest OS (51% vs. 72% for Q1-Q3, P = 0.006) and EFS (35% vs. 54% for Q1-Q3, P = 0.031). Among SNP+ patients (n = 75), survival did not vary significantly between WT1 expression quartiles. Although WT1 expression was not prognostic when all patients were considered together, stratifying patients by SNP rs16754 genotype revealed significant differences in outcome. In SNP- patients, high WT1 expression predicted decreased survival in univariate, but not multivariate, analysis, due to a preponderance of high-risk cyto/molecular abnormalities in the highest expression quartile.

Clinical Impact of TERT A1062T Mutations in Younger Patients with Acute Myeloblastic Leukemia

AbstractAbstract 1381▪▪This icon denotes a clinically relevant abstract Background:Acute myeloid leukemia (AML) is a heterogenous disease. In addition to cytogenetic aberrations, somatic mutations in several genes (e.g. NPM1, FLT3, DNMT3A, WT1) have been identified which are involved in the pathogenesis of AML and affect the prognosis of these patients. Recently, it was shown that also germline mutations in the gene encoding the reverse transcriptase component of the human telomerase complex (TERT) are associated with AML and occur with a frequency of approximately 3–5% (Calado et al. Proc Natl Acad Sci USA 2009; 106:1187–92). These mutations lead to a reduced enzymatic activity of the telomerase complex. The most frequent of these mutations is an G>A conversion in codon 1062 in exon 15 of TERT which leads to an alanine>threonine substitution (A1062T). Telomerase complex mutations had previously been described in patients with bone marrow failure or other organ dysfunction such as liver cirrhosis or pulmonary fibrosis. The impact of such mutations on the clinical course and prognosis of AML patients, however, is unknown. Aims:We wanted to analyze the clinical and prognostic impact of the most common TERT mutation A1062T in younger AML patients treated within two prospective multicenter trials. Methods:The mutational hotspot in exon 15 of the TERT gene was analyzed by direct sequencing in 420 patients (age 16–60 years) with AML treated with intensive double induction and consolidation therapy within the AMLSG 295 and 0199 trials (NCT00209833). The patients were also analyzed for mutations/aberrations in the genes NPM1, FLT3, WT1 and DNMT3A. Median follow up was 79 months. Results:Fifteen of the 420 analyzed patients (3.3%) carried a TERT A1062T mutation. In the four patients of whom remission bone marrow or blood samples were available, the mutation was also detected in complete remission suggesting a germline origin of the mutation. Patients with TERT mutations had a trend (p = 0.06) towards less favorable (13% vs. 42%) and more intermediate-2/adverse karyotypes/genotypes (60% vs. 31%) according to the ELN classification. Other clinical and molecular parameters did not differ from wildtype patients. In univariate analysis, patients with TERT mutations had a significantly inferior overall survival (OS) compared to wildtype patients (HR 2.31; 95% CI 1.29 – 4.13, p = 0.005; 3year OS 20% vs. 46%). Also, in multivariate analysis TERT mutations were an independent negative factor for OS when analyzed together with age, leukocytes/peripheral blasts, platelets, extramedullary disease, de novo vs. secondary AML, ELN risk classification, WT1 SNP rs16754 and mutations in NPM1, FLT3 and DNMT3A. In addition to a high relapse rate, TERT mutated patients also showed a high rate of treatment related mortality: 5/15 (33%) of the mutated patients died during induction therapy or in CR as compared to 62/405 (15%) of the wildtype patients (p = 0.07). Of note, three of four TERT mutated patients who received an allogeneic stem cell transplantation in first CR died in CR. Therefore, we also analyzed treatment related toxicity during induction therapy. In the TERT mutated patients, 14/15 (93%) suffered from non-hematological/non-infectious grade 3 or 4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wildtype patients (p = 0.006). In multivariate analysis, TERT mutations were an independent risk factor predicting for adverse events during induction (OR 9.6; 95% CI 1.24 – 75.23, p = 0.031) when analyzed together with age, ELN risk classification, other gene mutations, blood counts, extramedullary disease, ECOG performance status, response to first induction therapy, and de novo vs. secondary AML. Conclusions:TERT A1062T mutations are an independent negative prognostic factor in younger patients with AML. These mutations seem to predispose AML patients to treatment-related toxicity and mortality. Disclosures:No relevant conflicts of interest to declare.

Frequency and Prognostic Impact of NUP98/NSD1 Translocations in Adult AML and MDS Patients

AbstractAbstract 1402Over 20 different fusion partner genes of NUP98 have been reported in hematological malignancies with the majority of NUP98 fusions occurring in acute myeloid leukemia (AML). Recently, a specific fusion of NUP98 with Nuclear receptor-binding SET domain protein 1 (NSD1) has been analyzed in a large cohort of pediatric and adult AML patients. In this report, 16.1% of pediatric AML samples and 2.3% of adult cytogentically normal (CN)-AML cases were found to be NUP98/NSD1-positive. NUP98/NSD1-positive patients had an adverse outcome. The aim of our study was to investigate the frequency, clinical features and the prognostic impact of NUP98/NSD1 in another large, uniformily treated adult AML cohort, and in patients with myelodysplastic syndromes, which frequently precede overt leukemia.We studied 504 younger AML patients (<60 years) and 193 MDS patients for the presence of NUP98/NSD1 translocations.Analysis in the AML cohort was also performed for mutations in FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. Additional mutation analyses were performed in the subgroup of CN-AML for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS. The NUP98/NSD1 fusion was identified by a nested RT-PCR using patient-derived cDNA. cDNA from a patient with proven NUP98-NSD1 fusion was used as a positive control. NUP98/NSD1 fusions were identified in 7 out of 504 younger adult AML patients (1.4%) while the NUP98/NSD1 fusion was not found in any of the 193 MDS patients. In the AML cohort, NUP98/NSD1 positive patients showed a higher number of peripheral blood blasts (P=.002), while other clinical characteristics such as FAB-subtype, type of AML, hemoglobin levels, white cell count or platelet count did not significantly differ between patients with or without the NUP98/NSD1 fusion. The majority of NUP98/NSD1 positive patients (5 out of 7) showed a normal karyotype while one patient was found to have a del(9) and another patient an inv(3)(q21q26) with monosomy 7. We identified an association between FLT3-ITD and NUP98/NSD1 fusion in our cohort (P=.007, 5 patients with NUP98/NSD1 also had a FLT3-ITD). This finding confirms the data by Hollink et al. and suggests a possible functional link between FLT3-ITD and the NUP98/NSD1 fusion in leukemogenesis. NUP98/NSD1 fusions were not associated with other mutations like those suggested in epigenetic regulation (DNMT3A, IDH1 and IDH2). In CN-AML, we also looked for an association between BAALC, ERG, EVI1, MN1, MLL5 and WT1 expression but did not find a significant difference between the expression of these genes and NUP98/NSD1 fusion genes. Due to the low frequency of the aberration outcome analysis was performed for explorative purposes. Considering the whole AML cohort we did not detect a significant difference for OS and for RFS between NUP98/NSD1 positive and negative patients (OS, HR 1.6; 95%CI 0.66–3.88; P=.3; RFS, HR 2.33; 95%CI 0.74–7.30; P=.147). However, NUP98/NSD1 positive patients had significantly lower complete remission (CR) rates (43 vs. 77 percent, P=.037). When considering only patients with CN-AML the NUP98/NSD1 positive patients (n=5) had no significantly different OS and RFS (OS, HR 2.08; 95%CI 0.77–5.64; P=.15; RFS, HR 2.88; 95%CI 0.71–11.71; P=.14). Again, a significantly lower CR rate was observed in the NUP98/NSD1 positive patients compared to NUP98/NSD1 negative patients with CN-AML (40 vs. 80 percent, P=.03). Because of the association between FLT3-ITD and NUP98/NSD1 we also investigated the prognostic impact in the subgroup of CN-AML patients also positive for FLT3-ITD. Again, OS and RFS did not differ between NUP98/NSD1 positive and negative patients (OS, HR 1.31; 95%CI 0.47–3.64; P=.61; RFS, HR 2.04; 95%CI 0.49–8.57; P=.33). For this subgroup a trend towards a lower CR rate was observed for NUP98/NSD1 positive FLT3-ITD positive CN-AML patients (40 vs 75 percent, P=.08).In summary, our analysis confirms the presence but low incidence of the NUP98/NSD1 fusion gene in adult AML patients and the strong association with FLT3-ITD. The specific association of NUP98/NSD1 with FLT3-ITD mutations warrants clinical investigation with FLT3 inhibitors in these patients. Disclosures:No relevant conflicts of interest to declare.

WT1 snp rs16754 Genotype Predicts Treatment Related Mortality (TRM) in African-American and Asian Pediatric AML Patients: A Report From the Children's Oncology Group

AbstractAbstract 1385▪▪This icon denotes a clinically relevant abstract Background:Successful treatment of childhood acute myeloid leukemia (AML) requires multi-agent chemotherapy, beginning with intensive induction regimens. Treatment-related mortality (TRM) remains a significant concern, and may be influenced by patient-specific factors. Outcomes in adult and pediatric AML differ among ethnicities. We reported that the minor (G) allele of the WT1 synonymous SNP rs16754 is associated with improved outcome. Racial frequencies of SNP rs16754 vary significantly. Here we explore rs16754 genotype and 5-year rates of overall survival (OS), complete remission (CR), relapse risk (RR) and TRM in the Children's Cancer Group trial CCG-2961, in the context of ethnicity. Methods:To assess SNP rs16754 genotype, we directly sequenced WT1 exon 7 on available diagnostic specimens (N=492) from pediatric AML patients enrolled on CCG-2961. Patients with at least one minor (G) allele were considered SNP+, while patients homozygous for the major (A) allele were considered SNP−. We stratified SNP genotype by ethnicity and analyzed outcomes. In the 2961 trial, Induction Course 1 consisted of an intensively-timed hybrid regimen (Ida-DCTER: idarubicin/daunorubicin, dexamethasone, cytarabine, thioguanine, and etoposide). Patients with at least partial remission (<30% marrow blasts) were randomized to receive Induction Course 2 consisting of either a repeat course of Ida-DCTER (Regimen A) or Ida-FLAG (idarubicin, fludarabine, cytarabine; Regimen B). As most TRM events occurred within the first two cycles of chemotherapy, TRM was further examined separately on each of these two study arms. Results:Twenty-eight percent of patients were SNP+; SNP positivity correlated with improved overall survival (5 year OS, 61% vs. 44%, p=0.009), although other outcome measures (CR, RR, DFS) were not significantly different between SNP+ and SNP− groups in the 2961 study. Prevalence of the SNP G allele varied significantly across ethnic groups (p=0.034): Asian (n=8/15, 53%), Hispanic (n=28/82, 34%), Caucasian (n=81/322, 25%), and African-American (n=10/47, 21%). When examining rs16754 genotype within ethnic subgroups, SNP+ patients had higher 5 year OS in each population (Hispanic: 49% vs. 37%, p=0.433; African-American: 56% vs. 30%, p=0.207; Asian: 88% vs. 29%, p=0.037; and Caucasian: 65% vs. 48%, p =0.051); statistical significance was approached in the Caucasian group and was achieved only in the Asian group. When patients of all ethnicities were considered together, TRM rates did not vary by rs16754 genotype. However, TRM rate was significantly lower in SNP+ African-American and Asian patients taken together compared to their SNP− counterparts (p=0.020) with a trend to significance taken individually (Asian: 0% vs. 43%, p=0.063; African-American: 0% vs. 25%, p=0.105). Further, for patients who survived Induction 1 and underwent Induction 2 randomization, SNP− Asian and African-American patients experienced higher rates of TRM from randomization disproportionately on Arm B (p=0.107), where TRM in SNP+ vs. SNP− patients was 0% vs 60% in Asian patients (Arm B n=9, p=0.110) and 0% vs. 14% in African-American patients (Arm B n=21, p=0.305). Conclusions:As CCG-2961 comprised blocks of intensive inpatient therapy, this clinical trial provides a setting in which to study the effects of ethnicity on outcome while minimizing confounding non-biologic factors. African-American and Asian patients who were negative for SNP rs16754 experienced disproportionate TRM, particularly on experimental Arm B, which tested the then-novel agent fludarabine; conversely, the TRM rate was 0% for SNP+ patients of these two ethnicities. The biologic mechanism for this observation bears further study, as biomarkers predictive of drug-specific toxicity may inform treatment decisions in certain ethnic populations. Disclosures:No relevant conflicts of interest to declare.

ID1 Expression Correlates with CEBPA Mutational Status and Is Not An Independent Risk Factor in Cytogenetically Normal AML,

Abstract 3554 Background:Acute myeloid leukemia with normal karyotype (CN-AML) is a heterogenous disease. During the last years, mutations in several genes (e.g. NPM1, FLT3, CEBPA, WT1, IDH1, IDH2) have been identified which are involved in the pathogenesis of AML and affect the prognosis of these patients. Moreover, deregulated expression of genes such as MN1, BAALC, ERG and WT1 was demonstrated to be predictive of outcome in CN-AML. Recently, high expression of the ID1 gene was described as a negative prognostic factor in AML (Tang et al. Blood 2009, 114:2993–3000). Aims:We have shown that C/EBPα, a transcription factor encoded by the CEBPA gene, binds to a regulatory element in the promoter region of the ID1 gene and regulates ID1 expression in leukemic cells (Wagner et al. Proc Natl Acad Sci USA 2006, 103:6338–6343). Therefore, we wanted to analyze the prognostic impact of ID1 expression in CN-AML in the context of other molecular markers, in particular CEBPA mutations. Methods:ID1 expression was quantified normalized to ABL by real time RT-PCR in 269 patients (age 16–60 years) with CN-AML treated with intensive double induction and consolidation therapy within the AMLSG 295 and 0199 trials (NCT00209833). The patients were also analyzed for mutations in the genes NPM1, FLT3, CEBPA, WT1, IDH1 and IDH2. Median follow up was 79 months. Results:Expression of ID1 varied over a 3-log range. High expression of ID1 (ID1high, defined as > median expression level) was significantly associated with the presence of a FLT3 -ITD or an IDH2 mutation and WT1 wildtype. Moreover, ID1 expression was closely associated with CEBPA mutational status. Altogether, 41 patients (15%) harboured a CEBPA mutation (24 monoallelic and 17 biallelic mutations). ID1 expression in the CEBPA wildtype patients was significantly higher than in patients with monoallelic CEBPA mutations and these patients had a significantly higher ID1 expression compared to patients with biallelic CEBPA mutations (p = 0.001). ID1high patients had a trend to a lower complete remission (CR) rate (74% vs. 84%; p = 0.07), but in multivariate analysis only blast clearance on day 15 after induction 1, age and WT1 SNP rs16754 were independent predictors for the achievement of CR. In univariate analysis, ID1high patients had an inferior overall survival (OS) compared to patients with low expression (median OS 29 vs. 78 months, 5 year OS 39% vs. 53%, p = 0.026). ID1high status was an independent negative prognostic factor in multivariate analysis when analyzed together with NPM1, FLT3 -ITD, WT1, IDH1, IDH2, extramedullary disease and platelet counts (HR 1.51; 95% CI 1.06–2.19). However, when also CEBPA mutational status was entered into the model, ID1 expression lost its prognostic impact and the only independent prognostic factors were age, platelets, CEBPA mutations, NPM1 /FLT3 -ITD risk group and WT1 SNP rs16754. Likewise, ID1high patients had a trend to an inferior relapse-free survival (RFS; HR 1.36, 95% CI 0.96–1.93, p = 0.086) in univariate analysis. However, in multivariate analysis including CEBPA mutational status, ID1 expression had no impact on RFS and the only prognostic factors for RFS were NPM1 and CEBPA mutations and WT1 SNP rs16754. In CEBPA wildtype patients, ID1 expression had no impact on CR-rate, OS or RFS in univariate or multivariate analysis. Conclusions:CEBPA mutations seem to deregulate ID1 expression in CN-AML. Therefore, ID1 expression is not an independent prognostic factor in CN-AML. Disclosures:No relevant conflicts of interest to declare.

The Prognostic Effect of High WT1 Gene Expression in Pediatric AML Depends on WT1 SNP rs16754 Status: A Report From the Children's Oncology Group (COG)

Abstract 1444 Background:WT1 is aberrantly over-expressed in the blast cells of most AML patients. Several adult AML groups have reported poor outcome in association with high WT1 expression. In contrast, in previously the largest pediatric study, COG investigators found no association between diagnostic WT1 expression and survival in 155 patients treated on the legacy POG-9421 trial. We recently demonstrated that the WT1 synonymous SNP rs16754 correlates with favorable outcome in childhood AML; further, SNP+ patients had higher median WT1 expression. In the present study we examine the clinical correlates of diagnostic WT1 expression within a contemporary COG trial, accounting for SNP rs16754 genotypes. Methods:WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on the pediatric trial COG-AAML03P1. Expression levels were normalized against pooled normal bone marrow (NBM) controls. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and clinical outcome. Results:WT1 expression varied over 4 log folds across the study cohort (range: 0.00 to 3148.27 fold NBM, median: 75.91). In 29 (13%) patients, WT1 expression was undetectable. These patients had distinct biologic characteristics compared to the remainder of the cohort, as they were predominantly FAB class M5 (56%, p<0.001) and the majority (52%, p<0.001) had 11q23 rearrangements. This group was also younger (median age 5.1 years), and rarely harbored favorable cytogenetics (Inv(16): 1 patient; t(8;21): 1 patient) or molecular abnormalities (FLT3/ ITD: 1 patient; NPM1- mutated: 1 patient; no patients with mutated CEBPA or WT1). Survival outcomes for this group did not differ significantly from the study cohort at large. WT1 expression higher than NBM was detected in 183/196 of the remaining patients. We grouped patients into WT1 expression quartiles (Q1-Q4) for comparison of clinical characteristics and outcome. FLT3/ ITD (23% vs. 9%, p=0.017) and WT1 mutations (22% vs. 4%, p<0.001) occurred more frequently in those within the highest quartile (Q4; WT1 expression >302.97 fold NBM) compared to the lower 3 quartiles. Infant patients were also less common in Q4 (p=0.010). WT1 SNP rs16754 was equally distributed among quartile groups. Survival outcomes did not differ significantly between the quartiles, confirming that diagnostic WT1 expression has no prognostic impact in contemporarily-treated pediatric AML patients, when all patients were considered. However, given our recent findings regarding the clinical relevance of the WT1 SNP, we examined the prognostic impact of WT1 expression in SNP+ and SNP- subgroups. Median WT1 expression for SNP+ and SNP- patients was 149.76 (range: 0 – 2104.44) vs. 74.40 (range: 0 – 3148.27) respectively (p=0.299). When restricting analysis to SNP rs16754 negative patients (n=150), those in the highest quartile had significantly worse 3-year overall survival (OS) from study entry (51% vs. 72%, p=0.034) and event-free survival (EFS, 35% vs. 54%, p=0.033) than those in lower quartiles. When comparing SNP- patients with higher vs. lower than median WT1 expression (Q3 + Q4 vs. Q1 + Q2), patients with higher expression again had inferior EFS (39% vs 59%, p=0.008) with a corresponding relapse rate from CR of 45% vs. 25% (p=0.011). Notably, similar comparisons in WT1 SNP+ patients with high vs. low WT1 expression did not demonstrate significant outcome differences. Surprisingly, SNP+ patients within the lowest expression quartile had the poorest outcome, while those in the highest quartile had no relapses from CR. Conclusion:WT1 expression levels vary across cyto-molecular subgroups of AML. WT1 expression was not prognostic when all AML patients were considered; however, stratifying patients by SNP rs16754 genotype revealed that high WT1 expression was significantly associated with poor clinical outcome in SNP- patients. Conversely, although the small sample size of SNP+ patients precluded significant comparisons, increasing WT1 expression trended towards improved outcome in the SNP+ group. The clinical and biologic significance of WT1 over-expression appears to differ depending on the presence or absence of SNP rs16754. WT1 expression must be considered in the context of SNP status, to determine which patients would most benefit from novel WT1 -targeted therapies. Disclosures:No relevant conflicts of interest to declare.

ERG, BAALC, and MN1 RNA Levels Are Correlatively Over-Expressed, and Relate to Risk-Defining Prognostic Mutations in Intermediate-Risk Acute Myeloid Leukemia

Abstract 1471 Introduction:A variety of prognostic molecular markers have been proposed for risk stratification of intermediate-risk AML, which includes patients with normal cytogenetics (CN) and patients with cytogenetic abnormalities other than the recurrent prognostically informative cytogenetic aberrations (CA). Acquired mutations of FLT3 ITD, NPM1, and CEBPA have well-established prognostic significance, and have been incorporated into consensus classification schemes. In addition, several gene expression markers (WT1, ERG, BAALC, and MN1) have been proposed as prognostic indicators, but with less extensive validation. Whether these new gene expression markers should be incorporated into proposed “molecular risk scores” for AML has not been well refined. In this study, we have determined the transcript levels of WT1, ERG, BAALC, and MN1 in AML patients, and correlated these quantitative levels with other validated clinical and laboratory prognostic indicators. Methods: Diagnostic blood or bone marrow samples were analyzed from 56 pre-treatment AML patients from our institution (48% male; median age 59). Metaphase cytogenetics showed 4 patients with inv(16), 4 with a monosomal karyotype, 25 with a CN karyotype and 23 with a CA karyotype (intermediate-risk cytogenetics). The expression levels of WT1, ERG, BAALC, and MN1 were quantified with TaqMan real-time PCR and normalized to ABL. The FLT3 ITD, NPM1, DNMT3A R882, CEBPA, and MLL PTD mutations were detected with either HRM, direct sequencing, and/or PCR fragment analysis. The WT1 SNP rs16754 was determined with a TaqMan genotyping assay. Results: In the 56 AML patients, the prevalence of the FLT3 ITD, NPM1, MLL-PTD, and DNMT3A R882 mutations were similar to previously reported values at 37%, 27%, 6%, and 13%, respectively. The frequency of patients carrying the minor allele of the WT1 SNP (combined heterozygous and homozygous) was 30%. Compared with their expression level in 29 normal control individuals, all four genes were significantly over-expressed in the AML patients, with mean RNA level differences of 1.98-log for WT1 (P<0.0001), 1.18-log for ERG (P<0.0001), 1.02-log for BAALC (P<0.0001), and 0.53-log for MN1 (P = 0.005). ERG, BAALC, and MN1 were expressed at low but detectable levels in 100% of the normal control samples, while WT1 was not detectably expressed in 48% of the controls. For all 4 genes, there were no significant differences in the expression levels between the CA & CN cytogenetic subclasses. As high transcript levels of WT1, ERG, BAALC, and MN1 have been proposed to impart a high risk AML phenotype, one might predict that the RNA levels of each of these 4 genes would correlate with one another. To confirm that hypothesis, we found a highly significant correlation of quantitative RNA levels among the ERG, BAALC, and MN1 genes (P <.004; R=0.42–0.72). WT1 RNA levels were highly correlated with ERG (P<0.0001; R=0.59), but less highly correlated with BAALC & MN1 (P=0.04; R=0.3). Categorization of the gene expression level as “high” or “low” (above or below the median) also revealed a similar relationship, in which ERG, BAALC, and MN1 RNA were all significantly correlated (P<0.01), but there was no significant correlation of WT1 RNA with the other 3 genes (P>0.9). Consistent with its role as a prognostic risk marker, ERG transcript levels were significantly lower in CN-AML patients with the low-risk NPM+/FLT3- genotype than in those with other genotypes (0.42 log expression difference; P<0.04). Consistent with WT1’s role as a risk marker, in the CA-AML patients, WT1 RNA levels were significantly higher in patients with the high-risk FLT3+ genotype than in those with other genotypes (1.3 log expression difference; P<0.04). Conclusions: To better define prognostic risk markers in AML, we have performed comprehensive mutation and gene expression analysis on 56 diagnostic AML samples. WT1, ERG, BAALC, and MN1 RNA were significantly over-expressed in AML patients compared to normal controls. ERG, BAALC, and MN1 RNA levels significantly correlated with one another. The prevalence of FLT3, NPM, DNMT3A, and MLL mutations in these AML patients was predictably high, and some of these well-characterized prognostic mutations were significantly associated with gene expression levels. Additional details on the relationship of these new prognostic RNA markers to the clinical outcome of this AML cohort will be reported at the meeting. Disclosures:No relevant conflicts of interest to declare.

The Role of MSI2 Expression Levels on Outcome of MDS and AML Patients

Abstract 1734Musashi (MSI) represents an evolutionarily conserved family of RNA-binding proteins. MSI2 is the predominant form in hematopoetic stem cells (HSC). Recently, overexpression of MSI2 in a mouse model was found to increase cell cycle progression and to cooperate with BCR-ABL1 to induce aggressive leukemia. The aim of this study was to analyze the prognostic importance of MSI2 expression in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML). Diagnostic bone marrow or peripheral blood samples were analyzed from 454 adult patients (aged 17–60 years) with de novo (n=406) or secondary AML (n=48) with French-American-British (FAB] classification M0-M2, or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 or AML SHG 0295. Additionally, we analyzed a cohort of 148 patients (aged 36–92 years) with MDS with low to high IPSS scores. Real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed using patient-derived cDNA and ABL as an endogenous control. MSI-2 expression levels were quantified using the TaqMan Gene Expression Assay (Applied Biosystems, Assay ID MSI2: Hs01592567_m1). To define the MSI2 expression status patient cohorts were divided into four quartiles (Q) according to levels of MSI2/ABL expression and subsequently dichotomized into two groups including the three lower Qs (Q1, Q2, and Q3) and the upper Q (Q4) of MSI2/ABL values. Patients of the quartile with highest MSI2 expression levels were classified as high MSI2 expressing patients.In MDS, MSI2 high versus low expression was independent of transfusions (P=.55), cytogenetic risk (P=.6), and ASXL1 mutations (P=.99), but was associated with a higher blast percentage (p=.034). In AML, higher MSI2 expression correlated with increased peripheral blood blasts (P=.036), and inversely correlated with the favourable cytogenetic risk group defined by inv(16) and t(8;21) according to Medical Research Council criteria (P<.001). Additionally, patients with higher MSI2 expression were likely to be FLT3-ITD positive (P<.001), NPM1 (P<.001), DNMT3A (P=.003), NRAS (P=.018) and CEBPA double mutated (P=.043). No association was found with other molecular markers including IDH1, IDH2 and WT1 mutations.Next, we evaluated the effect of MSI2 expression levels on patient outcome in MDS and AML. In MDS, high MSI2 expression predicted an increased likelihood of and more rapid progression to AML (HR 2.95, 95%CI 1.55–5.64, P=.001), but MSI2 expression levels did not affect overall survival (OS, HR 1.34, 95%CI 0.81–2.23, P=.25). In multivariate analysis when adjusting for karyotype, transfusion dependence, percentage of bone marrow blasts, ASXL1 frameshift mutation status and IDH1 mutation status, high MSI2 expression remained an independent prognostic factor for AML transformation in MDS (HR 2.33, 95%CI 1.16–4.67, P=.018).In AML, OS was shorter in AML patients with higher MSI2 expression as compared to low expression (HR 1.48, 95%CI 1.13–1.95, P=.005). However, relapse free survival (RFS) and complete remission (CR) rates were not influenced by high versus low MSI2 expression (RFS, HR 1.27, 95%CI.92–1.76, P=.15; CR, P=.39). In multivariate analysis, when considering MSI2 high versus low expression, DNMT3A mutations, age, platelet count, secondary versus de novo AML, cytogenetic risk group, NPM1/FLT3-ITD high vs. low risk group, and MLL5 expression levels, MSI2 expression did not remain an independent prognostic marker for OS (HR 1.31, 95%CI.98–1.76, P=.07). In patients with cytogenetically normal AML (CN-AML), MSI2 also presented a negative prognostic marker for OS (HR 1.59, 95%CI 1.08–2.33, P=.019) but did not influence RFS (HR 1.10, 95%CI 0.69–1.76, P=.68) and CR (P=.39). In multivariate analyses for CN-AML, when considering age, platelet count, DNMT3A mutations, NPM1FLT3-ITD high vs. low risk group, CEBPA mutation status and WT1 SNPrs16754, MSI2 did not independently predict OS (HR 144, 95%CI 0.94–2.19, P=.096). In summary, our data suggest that MSI2 expression might contribute to the transformation from MDS to AML. Whether MSI2 expression is dysregulated through other associated prognostic markers, or whether it is an independent event in AML pathogenesis remains to be determined. However, it may represent a valuable therapeutic target both in MDS and AML patients. Disclosures:Ottmann:Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Hofmann:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Incidence and Prognostic Influence of DNMT3A Mutations in Acute Myeloid Leukemia

To study the incidence and prognostic impact of mutations in DNA methyltransferase 3A (DNMT3A) in patients with acute myeloid leukemia. A total of 489 patients with AML were examined for mutations in DNMT3A by direct sequencing. The prognostic impact of DNMT3A mutations was evaluated in the context of other clinical prognostic markers and genetic risk factors (cytogenetic risk group; mutations in NPM1, FLT3, CEBPA, IDH1, IDH2, MLL1, NRAS, WT1, and WT1 SNPrs16754; expression levels of BAALC, ERG, EVI1, MLL5, MN1, and WT1). DNMT3A mutations were found in 87 (17.8%) of 489 patients with AML who were younger than 60 years of age. Patients with DNMT3A mutations were older, had higher WBC and platelet counts, more often had a normal karyotype and mutations in NPM1, FLT3, and IDH1 genes, and had higher MLL5 expression levels as compared with patients with wild-type DNMT3A. Mutations in DNMT3A independently predicted a shorter overall survival (OS; hazard ratio [HR], 1.59; 95% CI, 1.15 to 2.21; P = .005) by multivariate analysis, but were not associated with relapse-free survival (RFS) or complete remission (CR) rate when the entire patient cohort was considered. In cytogenetically normal (CN) AML, 27.2% harbored DNMT3A mutations that independently predicted shorter OS (HR = 2.46; 95% CI, 1.58 to 3.83; P < .001) and lower CR rate (OR, 0.42; 95% CI, 0.21 to 0.84; P = .015), but not RFS (P = .32). Within patients with CN-AML, DNMT3A mutations had an unfavorable effect on OS, RFS, and CR rate in NPM1/FLT3-ITD high-risk but not in low-risk patients. DNMT3A mutations are frequent in younger patients with AML and are associated with an unfavorable prognosis.

The WT1 synonymous SNP rs16754 Is Associated with Higher mRNA Expression and Predicts Significantly Improved Outcome In Favorable-Risk Pediatric AML: a Report From the Children's Oncology Group

AbstractAbstract 950SNP rs16754, a synonymous single nucleotide polymorphism of the WT1 gene, has recently been reported to have prognostic implications in adult normal-karyotype acute myeloid leukemia (AML). We sought to determine the prevalence and clinical implications of WT1 SNP rs16754 in a large cohort of unselected pediatric AML patients. Available diagnostic bone marrow specimens (N=790) from patients treated on 3 consecutive CCG / COG trials (CCG-2941, CCG-2961, and COG AAML03P1) were analyzed for the presence of SNP rs16754 via direct sequencing of exon 7 of the WT1 gene. SNP status was correlated with disease characteristics, WT1 quantitative mRNA expression level, and clinical outcome. At least 1 copy of the minor SNP allele was detected in 229 (29.0%) patients, 38 (16.6%) of whom were homozygous for the SNP. SNP rs16754 was significantly more common in Asian and Hispanic patients than in Caucasian patients (P<0.001). The SNP was also less common in patients with inv(16) (P=0.043) and more common in patients with -5/del(5q) (P=0.047). Although FLT3/ITD, NPMc, and CEBPA mutations occurred at comparable frequencies in patients with or without the WT1 SNP, WT1 mutations were significantly less common in SNP-positive patients (3.2% vs. 10.4%, P=0.002). WT1 expression levels were evaluated by quantitative RT-PCR in 114 unselected patient samples with known WT1 SNP and mutation status (SNP-positive, n=31; WT1-mutation positive, n=13; one patient harbored both the SNP and a WT1 mutation). Median WT1 expression in WT1 wild-type patients (without SNP rs16754 and negative for a WT1 mutation, n=71) was 40.61 times that of normal marrow controls (range 0.00–2949.42). Median WT1 expression levels were significantly higher in both patients with SNP rs16754 (215.76 fold normal marrow, range 0.00–1399.23, P=0.0214) and in patients with a WT1 mutation (327.14 fold normal marrow, range 54.17–902.3, P=0.0005). Five-year overall survival (OS) for patients with or without the SNP was 60% ± 7% and 50% ± 5%, respectively (P=0.031). In multivariate analysis, the presence of the SNP was an independent predictor of improved OS (HR 0.64, 95% CI 0.47–0.86, P=0.004). Relapse rates were comparable between the 2 groups, but OS after relapse was significantly higher in SNP-positive patients (31% ± 11% vs. 24% ± 6% at 5 years, P=0.034) suggesting a higher rate of salvage success. The prognostic impact was most pronounced in patients with low-risk disease (those with CBF-AML, CEBPA mutation, or NPMc mutation), where five-year OS for those with or without the SNP was 90% ± 8% vs. 64% ± 9% (P=0.001) with a corresponding disease-free survival of 72% ± 13% vs. 53% ± 10% (P=0.041). The high prevalence of WT1 SNP rs16754, and its association with improved outcome, identifies WT1 SNP rs16754 as a potentially important molecular marker of prognosis in pediatric AML. WT1 SNP status, WT1 mutation status, and WT1 expression levels should be evaluated together prospectively in future pediatric AML clinical trials. Disclosures:No relevant conflicts of interest to declare.