FLT3-ITD Negative Patients Research Articles

Exploring Potential Molecular Mechanisms of Drug Response in FLT3-ITD Negative AML Patients Treated with Quizartinib Vs Placebo Plus Standard Chemotherapy in the Quiwi Trial

Introduction Wild-type FLT3 AML presents significant therapeutic challenges due to limited treatment options. The QUIWI trial investigated quizartinib (Quiz) combined with standard chemotherapy, showing promising results. To understand drug response and refractoriness, we conducted a correlative analysis, exploring differential expression patterns among distinct patient groups. We reasoned that the molecular fingerprints of good responders to Quiz plus chemotherapy would be different with respect to patients who respond only to standard chemotherapy, helping to elucidate critical molecular mechanisms associated with Quiz response. Our study provides a basis for personalized therapeutic strategies in FLT3-ITD negative AML. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced, out of which 55 cases were FLT3-ITD and were discarded for this analysis. For this analysis, only samples with a minimum of 20% blasts were considered. The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Differential expression was assessed with the DESeq2 algorithm, using Blast proportion to adjust for disparities among the samples. P-values were adjusted for multiple testing using the FDR method. OS was defined as time from start of screening to death. Gene ontology and pathways analysis were performed using the WebGestalt portal. Results In this study, we compared the gene expression profiles of Quiz versus placebo-treated patients categorized as good responders, defined arbitrarily as those who continue alive and have a minimum follow-up of 6 months after diagnosis. Out of 82 eligible patients, we identified 100 (q-value < 0.05) and 172 (q-value < 0.1) differentially expressed genes between the Quiz and Placebo groups. Notably, 132 genes were overexpressed in the Quiz group, while 40 genes showed overexpression in the placebo group. Moreover, we observed a highly significant overexpression of ribosomal genes in the Quiz group (17 overlaps, KEGG pathways q-value < 2.2 x 10 -16), and a significant overexpression of genes related to the attenuation of the heat shock protein transcriptional response, which is involved in protein folding, in the placebo group (Reactome q-value, 0.008; gene ids: HSPA1A, HSPA1B and DNAJB1). Additionally, the Quiz group exhibited a significant overexpression of transmembrane receptor protein tyrosine phosphatase-related genes (Gene Ontology Molecular Function q-value, 0.02; gene ids: PTPRB, PTPRG and PTPRU). Conclusion This correlative analysis of the Quiwi trial revealed critical molecular mechanisms associated with Quiz response. The overexpression of ribosomal and transmembrane receptor protein tyrosine phosphatase-related genes in good responders within the Quiz group, along with the overexpression of the heat shock pathway genes in the placebo group, aligns with existing knowledge about the FLT3 pathway biology and the molecular determinants to tyrosine-kinase inhibitors. These findings offer potential biomarkers for personalized therapeutic approaches to improve clinical outcomes in this challenging AML population.

Patient Specific Droplet Digital PCR Targeting FLT3-ITD Allows to Detect Small Clones in Acute Leukemias at Diagnosis and during Follow-up

Approximately 30% of Acute Myeloid Leukemia (AML) patients are characterized by an Internal Tandem Duplication (ITD) in the FLT3 gene that is associated with a worse clinical course, a higher relapse rate, and shorter overall survival. The prognostic significance of measurable residual disease (MRD) positivity after chemotherapy or before hematopoietic stem cell transplantation (HSCT) has been largely demonstrated. Despite this, FLT3-ITD is not used as a marker of MRD because it is reported to be an unstable marker. Moreover, the methods currently used for the detection of FLT3 ITD are characterized by a low Limit of Detection (LoD), therefore small clones (<1%) cannot be identified. For this reason, it is difficult to monitor the persistence or reappearance of a FLT3-positive clone during remission and after HSCT. The possibility of identifying the FLT3-ITD clones as long as they are still very small would allow an early treatment with selective inhibitors. This study aimed 1) to follow up small clones in patients with FLT3 ITD positive at diagnosis and negative after therapy. 2) to verify the presence of small clones at diagnosis in patients who were negative for FLT3-ITD at diagnosis and relapsed as FLT3-ITD positive. We set up a patient-specific approach based on droplet digital PCR (ddPCR). We analyzed 48 samples from 11 AML patients. Seven patients were diagnosed as negative for FLT3-ITD but relapsed with a positive clone (group A), while 4 were identified as FLT3-ITD positive at diagnosis (group B). For each patient, the FLT3-ITD sequence was identified by Sanger sequencing and different patient-specific probes were designed. The LoD of each assay was calculated by mixing the DNA of a wild-type sample with an increasing amount of mutated DNA. Finally, the presence of FLT3-ITD clones was tested in the collected samples by a 2-plex ddPCR, having labeled the patient-specific probes with FAM and the total FLT3 probe with HEX. The target concentration in each sample was expressed as a percentage of FLT3-ITD/FLT3. ddPCR results were compared with the data obtained in the diagnostic routine with LeukoStrat CDx FLT3 mutation assay (Invivoscribe). FLT3-ITD was evaluated at diagnosis and during follow-up in both groups. Regarding group A, ddPCR was able to identify 3 out of 7 patients, evaluated as FLT3 wild-type by the diagnostic test, in which the FLT3-ITD clone was already present at diagnosis, with a percentage of FLT3-ITD/FLT3 of 0.003%, 4.25% and 0.31% for patients 3, 4 and 5. (Fig. 1). In the remaining 4 patients, the absence of an ITD clone at diagnosis was confirmed by ddPCR, but we were able to detect the appearance of a small clone about 9 months before the relapse in 2 of them (pt 1 and 2). In Group B (patients FLT3-ITD positive at diagnosis by routine tests) several time points of follow up were evaluated by ddPCR (Fig.2). In 2 out of 4 patients, FLT3-ITD was negative after treatment and remained negative throughout the follow-up. In patient 8 ddPCR approach was able to identify the presence of a small mutated FLT3-ITD clone (1.43%), which was undetectable by routine test 9 months after diagnosis and 3 months before relapse. In patient 10 ddPCR detected the presence of a small FLT3-ITD clones of 0.004% 0.048% 8 and 3 months before relapse respectively. Our study demonstrates that in FLT3-ITD negative patients a very small clone can be present at diagnosis or can appear soon after the end of chemotherapy. In both cases this clone remains under the limit of detection of 5% for months until the overt relapse. These data are purely cognitive at the moment because the probes are designed on the ITD at relapse, but these data provide the rational for the development of a "universal" ddPCR able to detect the majority of ITD sequences. Finally, our study demonstrates that in patients FLT3-ITD positive at diagnosis, ddPCR allows to detect the persistence or the reappearance of small clones that expand until clinical relapse. The possibility of an early diagnosis of molecular relapse offers a window of time for intervention with selective FLT3 inhibitors in the attempt to prevent or delay relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microdeletion in Chromosome 17 Cytoband q21.31 Identified As Located within the KANSL1 Gene Prevailed in Flt3-ITD Negative Normal Karyotype AML Patients As Opposed to Their AML Counterparts and Influenced the Phenotype of the Disease

Recent advances in genetic tools for leukemic diagnosis have facilitated studies on genetic traits which in concert with a driving mutation if present may influence the phenotype of the disease. Flt3-ITD is present in about 30% of AML patients and tends to be associated with a poor prognosis. Currently the use of inhibitors of mutated and in consequence activated kinases is undergoing clinical trials. The dim prognosis of Flt3-ITD positive AML and the hope associated with kinases inhibitors prompted us to evaluate in some depth the genetics of AML cases in relation to the presence of Flt3-ITD .Ninety-two AML patients (diagnosed according to the FAB/WHO criteria, F/M=49/43, age median: 57, range: 21-81 years) who were seen in our institution were recruited to the present study which was conducted as follows:The patients underwent GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). 52 lacking GTG and/or FISH abnormalities were called as normal karyotype (NK) patients, among those, 20 were Flt3-ITD positive. The other 40 patients had karyotype abnormalities or were FISH positive for AML-associated abnormalities among which 10 had Flt3-ITD .In all 92 patients microarray analysis was performed using Agilent - Catalog Agilent Cancer CGH+SNP 180K or Roche - WG Catalog NimbleGene 12x270K microarrays.We found (Partec Genomics Suite and Biodiscovery Nexus 8 software) that NK Flt3-ITD negative had, when the whole genome was analyzed, a higher number of total copy number variation (CNV) aberrations (amplifications and/or deletions, vs 32.21±8.70 vs 16.15±2.60, p=0.008) including one copy losses, (2.79±0.57 vs 1.20±0.35 p=0.019)as opposed to NK patients having Flt3-ITD mutation .During the study, an interim analysis was performed at the time we switched from Roche to Agilent microarrays which revealed the significant prevalence of CNVs in AML Flt3-ITD negative patients over Flt3-ITD positive ones within the region 17q21.31 specifically in the positions from 44281092 to 4476549, 44702953 to 44745272 and 44745272 to 44765496. The same region appeared again as significantly discriminating Flt3-ITD negative and positive patients at the final analysis. Therefore, this region was analyzed in some depth. Hg19 annotation was used and according to that the region encompasses the KANSL1 gene among two others (NSFP1 and NSF). The NSF gene encodes a protein involved in transport to the Golgi and NSFP1 is a pseudogene. KANSL1, a component of the MLL1 complex (thought to contribute to leukemogenesis), was the focus of our interest, and the gene was further investigated to validate the microarray-based platform results and to localize the CNV area more precisely. In the region 44281092 to 4476549 within the KANSL1 gene Flt3-ITD negative patients had more deletions and amplifications as compared to those Flt3-ITD positive (31/62 vs 7/30, p=0.012). The same was valid when only deletions were considered (19/62 vs 4/30, p=0.058).Then to validate microarray platform results the same 80 samples were examined using the TaqMan Copy Number Assay (ThermoFisher) for the KANSL1 gene (primers and probe designed for the region 44297111-44297201 lying in chr17), and as a reference the RNaseP gene was employed. The results obtained using the microarray platform correlated well (r=0.671, p<0.001) with those obtained using RT-PCR copy number assay for KANSL1 .61 DNA samples obtained from the patients' solid organ biopsies were examined using the Copy Number Assay for KANSL1 gene CNV. In all instances except one the same CNP was observed in the marrow as that in the solid organ biopsies. It lends credit to the technology employed in this study, but still we do not have enough data to exclude the somatic origin of the microdeletions.The microdeletions are of biologic significance, as suggested by the following findings: 1.All three patients with FISH-documented MLL amplification had deletions within the KANSL1 gene (chr:17 44281092 to 4476549).2.Microdeletions within the KANSL1 gene were more frequently observed in the group M0-M2 patients than in other subtypes of AML (18/51 vs 5/35, p=0.026).In conclusion, a lack of Flt3-ITD and normal karyotype was associated with the frequent presence of microdeletions within the chr17 cytoband q21.31. The latter was identified as (i) present within the KANSL1 gene and (ii) influencing the phenotype of AML.Supported by the INNOMED/I/1/NCBR/2014 DisclosuresNo relevant conflicts of interest to declare.

Factors Predicting Response and Resistance to Midostaurin in FLT3 Positive and FLT3 Negative AML in 483 Primary AML Patient Samples

Introduction: Midostaurin is an FDA approved, multi-target tyrosine kinase(TK) inhibitor shown to inhibit the FLT3 kinase and have therapeutic benefit in FLT3 mutated AML especially FLT3-ITD mutations (FITD+). However, midostaurin has also shown therapeutic response in select FLT3-ITD negative (FITD-) AML patients likely due to inhibitory effects on other TKs .We analyzed primary AML patient samples from our ‘Beat AML’ database to look at disease related factors, aside from FLT3 status, that may influence midostaurin drug sensitivity and affect patient outcomes.Methods: We collected peripheral blood and bone marrow samples from 483 newly diagnosed AML patients (348 FITD+, 135 FITD-), extracted mononuclear cells and plated them on a Midostaurin dose curve, without growth media or feeder cells. Metabolic viability was assessed after 72-hours and drug response was summarized with an area under curve (AUC) metric on a 0-100 scale. We annotated these samples for known chromosomal translocations and somatic mutations in leukemia-related genes and performed association-based analyses to identify genomic predictors of midostaurin drug sensitivity.Results: FLT3-ITD was predictably the strongest association with midostaurin-response, so further analysis was subsetted into FITD+ and FITD- samples (Figure A). For our analysis, we included FLT3-TKD mutant samples in the FITD- cohort as we found no significant difference between FLT3-TKD status and non-FLT3 mutated samples.Within FITD- samples, we found inv(16) Core Binding Factor AMLs demonstrated increased sensitivity to midostaurin (p < 0.05) . We investigated the frequency of c-Kit mutations in inv(16) AMLs, given Midostaurin's activity against c-Kit, but did not observe significant differences in drug response between any combination of inv(16) and c-Kit mutational status due to a relatively small sample size.(Fig B) Further, among all FITD- samples evaluated, we found mutations in KRAS and TP53 to be predictive of midostaurin resistance (p < 0.05, Fig B). This relationship was also observed through analysis of publically available cell line drug screens within the Genomics of Drug Sensitivity in Cancer (GDSC), where mutations in NRAS and TP53 suggested Midostaurin resistance.In FITD+ AML samples, we found the FLT3-ITD mutant allele fraction (MAF) to strongly correlate with the extent of Midostaurin sensitivity. FLT3-ITD MAF was significantly higher in Midostaurin-sensitive samples compared to resistant samples (p < 0.001)(Fig C). Additionally, we observed an increase in Midostaurin efficacy (p < 0.05) in FLT3-ITD positive(15;17) APL,a subset of APL with relapse rates 15-times higher than FLT3 WT APL (Breccia et. al. Haematologica 2013) (Figure C). Further, we found that neither NPM1 or FLT3-TKD status conferred additional sensitivity to Midostaurin. There was no significant difference in AUC between FLT3-ITD and NPM1 double-mutant samples and FLT3-ITD alone, and similarly no significant difference between FLT3-TKD and FLT3 WT samples.Discussion: With the approval and clinical use of Midostaurin in FLT3 positive AML patients it is important to find and validate factors influencing Midostaurin efficacy. Data from the RATIFY clinical trial has shown Midostaurin improved overall survival in patients with both high and low FLT3-ITD allelic ratios (Stone et. al. N Eng J Med 2017). We show that the ex vivo Midostaurin response is correlated with FLT3-ITD status and its MAF but not significantly with the FLT3-TKD mutation. Our data suggests that FITD+ APLs are responsive to this FLT3-inhibitor thus offering the potential of combination therapy and improved outcomes with current APL regimens.We also demonstrate that Midostaurin shows efficacy in FLT3-ITD negative patient samples, illustrating the multi-targeted nature of the inhibitor. Within these samples,inv 16 predicted Midostaurin sensitivity regardless of c-Kit mutational status. Additionally, mutations in TP53 and the Ras-pathway predict Midostaurin resistance (these mutations rarely co-occurred with FLT3-ITD in our cohort, thus limiting their impact in FLT3-ITD positive cases). Additional in vitro and clinical data is necessary to determine whether these findings should influence treatment decisions.Newer FLT3 inhibitors in clinical trials need to be characterized in a similar manner so as to efficiently target subsets of FLT3 mutated AML. [Display omitted] DisclosuresTyner:Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Janssen Pharmaceutica: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Syros: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Agios Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; Leap Oncology: Consultancy. Borate:Novartis, Jazz: Consultancy, Speakers Bureau.

High Throughput Molecular Characterization of Normal Karyotype Acute Myeloid Leukemia in the Context of the Prospective Trial 02/06 of the Northern Italy Leukemia Group (NILG).

By way of a Next-Generation Sequencing NGS high throughput approach, we defined the mutational profile in a cohort of 221 normal karyotype acute myeloid leukemia (NK-AML) enrolled into a prospective randomized clinical trial, designed to evaluate an intensified chemotherapy program for remission induction. NPM1, DNMT3A, and FLT3-ITD were the most frequently mutated genes while DNMT3A, FLT3, IDH1, PTPN11, and RAD21 mutations were more common in the NPM1 mutated patients (p < 0.05). IDH1 R132H mutation was strictly associated with NPM1 mutation and mutually exclusive with RUNX1 and ASXL1. In the whole cohort of NK-AML, no matter the induction chemotherapy used, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall and disease-free survival (p < 0.05). FLT3-ITD positive patients who proceeded to alloHSCT had a survival probability similar to FLT3-ITD negative patients and the transplant outcome was no different when comparing high and low-AR-FLT3-ITD subgroups in terms of both OS and DFS. In conclusion, a comprehensive molecular profile for NK-AML allows for the identification of genetic lesions associated to different clinical outcomes and the selection of the most appropriate and effective treatment strategies, including stem cell transplantation and targeted therapies.

Characteristics and Outcome of Patients with Core Binding Factor Acute Myeloid Leukemia and FLT3-ITD: Results from an International Collaboration

Background: Core binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) and is associated with a favorable outcome, particularly if treated with repetitive cycles of high-dose cytarabine as post-remission therapy. Long-time 10-year overall survival (OS) rate was reported of 58% in FLT3-ITD negative patients (pts; Allen et al. Leukemia 2013). Nevertheless, 30-40% CBF-AML pts experience relapse. FLT3-ITD mutations occur in roughly 5-10% of adult CBF-AML. However, their prognostic relevance is still controversial. Aims: To characterize CBF-AML with FLT3-ITD and compare outcomes according to their genetic background. Methods: We retrospectively studied 65 AML pts with CBF-AML and FLT3-ITD (median age at diagnosis, 54 years; range, 22-81 years) diagnosed between 1996 and 2018 within seven study groups/institutions of the US and Europe. Results: Thirty-two (49%) of the 65 pts harbored t(8;21). Median white blood cell and platelet counts at diagnosis of patients with t(8;21) and inv(16) were 18.3/nl (range, 1.8-202/nl) and 31/nl (range, 7-372/nl), respectively. AML diagnoses were de novo in 61 (94%) and therapy-related in 4 (6%) of the pts. Thirty (46%) pts were female. Cytogenetic analysis revealed additional abnormalities (abn) in 38 (58%) pts, most frequently loss of X or Y (n=13; n=12 associated with t(8;21)), complex karyotype (≥3 abn; n=12; n=7 occurring in t(8;21)), trisomy 22 (n=7, all associated with inv(16)) or trisomy 8 (overall n=6, n=5 occurring in inv(16)). Four pts were positive for both mutations, FLT3-ITD as well as FLT3-TKD. Median ITD allelic ratio were 0.44 (range, 0.003-50) and median ITD size 60 bp (range, 3-120 bp). Three older pts (median age, 75.5 years) were treated with either azacitidine + sorafenib, azacitidine + venetoclax or with etoposide + tipifarnib. All three patients receiving non-intensive therapy died within one year and were excluded from further analysis. Complete remission (CR) after anthracycline-based induction therapy was achieved in 98% (n=61/62) of patients fit for intensive treatment including two pts treated with 7+3 ± midostaurin within the RATIFY trial. One patient died during induction. Fifteen (24%) pts underwent allogeneic hematopoietic cell transplantation. Of those, 10 pts were transplanted in 1st and 5 pts in 2nd CR. Median follow-up for the entire cohort was 4.43 years (95%-CI, 3.35-8.97 years). Median and 4-year relapse-free survival (RFS) rates were 3.41 years (95%-CI, 1.26 years - not reached) and 44.9% (95%-CI, 32.9-61.4%). Median and 4-year overall survival rates (OS) were 4.48 years (95%-CI, 2.26 years - not reached) and 51.8% (95%-CI, 39.6.2-67.9%). Neither type of CBF-AML (p=0.60), nor additional chromosomal abn (p=0.80), nor presence of a complex karyotype (p=0.50) had a prognostic impact on OS. Higher age (≥60 years) was an in trend negative prognostic factor on RFS and OS (p=0.07, each). High allelic ratio (≥0.5) had no impact on RFS (p=0.3), but in trend on OS (p=0.10). Conclusions: Despite a high remission rate pts with FLT3-ITD had an inferior outcome as compared to previously published data on CBF-AML without FLT3-ITD. Thus, CBF-AML with FLT3-ITD should not be classified within the low-risk category. CBF pts with FLT3-ITD warrants further study and should be included in FLT3-inhibitor trials. Disclosures Brunner: Astra Zeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Novak:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel,Accommodations; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel,Accommodations; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stoelzel:Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Thiede:Daiichi Sankyo: Honoraria; AgenDix GmbH: Employment, Equity Ownership; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Diaceutics: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Levis:Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

The Presence of Minimal Residual Disease, As Determined By Highly Sensitive Quantitation of NPM1-Mutatation, Provided Powerful Prognostic Information in Acute Myeloid Leukemia

Background: As recurrence of acute myeloid leukemia (AML) is difficult to predict, it is important to detect it by measuring minimal residual disease (MRD). PML-RARA, RUNX-RUNX1T1, CBFB-MYH11 are regarded as the reliable MRD markers. However, in AML with normal karyotype and many other forms, no MRD markers have been established. NPM1 mutations, occurring in approximately 30% of adult AML cases, and 50-60% of AML cases with normal karyotype, represent one of the most frequent mutations in AML. Recently, NPM1 mutation is reported to be useful in assessing MRD. We undertook a retrospective and prospective investigation of the usefulness of NPM1 mutation as an MRD marker in Japanese patients with AML. Methods: The subjects were 38 NPM1-mutated AML patients with first hematological remission at several hospitals related to our institution between 2001 and 2018. This study was approved by the ethics committee of Nippon Medical School and the informed consents were obtained from all patients, according to the Declaration of Helsinki. We analyzed peripheral blood cells or bone marrow cells at diagnoses, and evaluated only bone marrow cells after diagnoses. Detection of NPM1 mutation was carried out using allele-specific real time PCR following creation of a complementary primer. After dilution of the samples, sensitivity to TCTG, CATG, and CCTG was found to be 0.001%. The NPM1 mutant copies were qualified only at successful amplification of internal control. Results: The median age of the patients was 58 years (18-79 years). There were 32 cases with intermediate cytogenetic prognosis and 6 cases with unclear chromosomal profile. Of the 38 cases, 14 cases (37%) were FLT3-ITD-positive and allogeneic hematopoietic stem cell transplantation was carried out in 14 cases (37%). The base sequence was TCTG in 36 cases and CCTG in 2 cases. Persistence of NPM1-mutatation was present in 25 patients with first hematological remission (66%). Compared with patients with MRD negative, patients with MRD positive were associated with DNMT3A mutation (MRD positive 12/25 vs MRD negative 0/13, p=0.003). The rate of relapse in patients with MRD positive was significantly higher than those of in patients with MRD negative (MRD positive 76% vs MRD negative 23%, p=0.004). The rates of relapse free survival (RFS) and overall survival (OS) in patients with MRD positive were significantly lower than those in patients with MRD negative (RFS at 2 years: MRD positive 14% vs MRD negative 86% p=0.003; Figure 1, OS at 2 years: MRD positive 25% vs MRD negative 93%, p&lt;0.001). In FLT3-ITD negative group, the rates of RFS in patients with MRD positive were significantly lower than those in patients with MRD negative. (RFS at 2 years: MRD positive 21% vs MRD negative 92% p=0.001; Figure 1). Conclusion: The presence of MRD with NPM1 mutation is significantly associated with relapse and it is useful to decide their treatment strategy. Especially, there is the usefulness of NPM1 mutation as an MRD marker in NPM1 positive Flt3-ITD negative AML patients who are generally classified as favorable risk. According to previous reports, it is known that NPM1-mutated AML sometimes relapse with losing NPM1 mutations. However, in this study, all NPM1-mutated AML relapse without losing NPM1 mutations. We need to collect more patients and are going to confirm whether there are patients who relapse with losing NPM1 mutations or not. We plan to analyze the genetic background of MRD positive and negative patients with next-generation sequencing. We are going to announce the genetic characteristics in addition to this result at ASH. Disclosures Usuki: Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Pre-Transplant NPM1 Mutant Transcript Level Is Highly Predictive of Outcome after Allograft and Thresholds Are Dependent on FLT3 ITD Status

IntroductionRelapse remains the most common cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (SCT) and carries a grave prognosis. Multiple studies have identified the presence of minimal residual disease (MRD) prior to SCT assessed by flow cytometry (FCM) as a strong predictor of relapse: however, little is known about the impact of molecular MRD pre-SCT. Molecular techniques can provide 100-1000 fold greater sensitivity than FCM in NPM1 mutated AML allowing identification of patients with very low level MRD prior to SCT. Peri-transplant management decisions for patients with positive molecular MRD are highly challenging due to the absence of robust outcome data.MethodsBetween 2009-14 the NCRI AML17 study enrolled 3215 adult non-APML patients aged 16-77 eligible for intensive chemotherapy. Central screening for NPM1 mutations was positive in 861/2949 (29%); 530 patients provided serial samples for MRD monitoring. Paired blood (PB) and bone marrow aspirates (BM) were requested after each chemotherapy cycle and then quarterly; additional samples were requested pre- and post-SCT. Samples were analysed by RT-qPCR using a suite of 27 mutation-specific reverse primers. Results were only fed back to clinicians after June 2012 when patients could be treated for confirmed re-emergent or persistent molecular positivity. Post-remission treatment was determined according to the validated NCRI risk score, with poor-risk patients recommended for SCT during first complete remission (CR1). Survival analysis was performed using the logrank test.ResultsIn total 108/530 patients received SCT (CR1 57 (52%), after molecular relapse or progression (MR) 30 (28%), CR2 21 (19%)). Five-year survival post-SCT (5y-OS) was 65% in CR1, and 54% in MR/CR2 (p=0.3). After MR 26/30 patients received chemotherapy prior to SCT. Evaluable pre-SCT PB and BM samples taken within 60 days of SCT were available for 104 and 78 patients (both available n=74).Considering PB samples, 5y-OS was 73% (median OS (mOS) not reached (NR)) for MRD-ve patients (n=74) vs. 30% (mOS 8.1 m) for any PB positivity (n=30) (p<0.0001). Patients with a negative pre-SCT BM had 5y-OS of 79% (mOS NR) vs 47% (mOS 11.9 m) if the BM was positive (p=0.002).Of the 47 patients who received additional chemotherapy for morphologic or molecular relapse or progression, 26 (55%) converted to MRD negativity accounting for 44% of 59 patients who were MRD negative pre-SCT. Ninety-three per cent of 59 pre SCT-MRD negative patients had been MRD negative in the PB after the second cycle of induction (a previously identified marker of favourable outcome).A threshold of 200 mutant NPM1 transcripts/105 ABL copies in the pre-SCT PB sample split patients into 3 groups with 2y-OS of 81% (negative, n=74), 54% (low, n=13) and 9% (high, n=17; p<0.0001). In the BM a threshold of 1000 copies defined 3 groups with a 2y-OS of 86% (negative, n=36), 56% (low, n=32) and 12.5% (high, n=8; p<0.0001).Thirty four patients were positive for FLT3-ITD at diagnosis (5y-OS 55%) and 74 were negative (5y-OS 62%; p=0.3). For patients without FLT3 ITD, negative, low and high levels of MRD in the pre-SCT PB sample were associated with 2y-OS of 79% (n=53), 88% (n=8) and 0% (n=9) (p<0.0001). For BM samples, 2y-OS was 78% (n=28), 62% (n=24) and 0% (n=5; p=0.0001). For patients with FLT3 ITD, corresponding 2y-OS for PB samples were 89% (n=21), 0% (n=5) and 25% (n=8; p=0.0005); for BM samples 2y-OS was 83% (n=9), 38% (n=8) and 25% (n=4; p=0.02).We assigned 80 patients to high and low risk groups based on FLT3 ITD status and pre-SCT MRD level in PB and BM. High-risk patients (FLT3 ITD and any detectable level of MRD in the PB or BM, or FLT3 ITD negative with > 200 copies in the PB or > 1000 copies in the BM) had 3y-OS of 14% (n=27, mOS 6.5 months) compared with 80% for low-risk patients (n=53, p<0.0001) (fig 1).ConclusionsPatients who test negative for NPM1 mutant transcripts immediately before SCT have a favourable outcome which is also observed in FLT3 ITD negative patients who are MRD positive at levels which do not exceed 200 copies in the PB or 1000 copies in the BM. FLT3 ITD mutated patients with any level of MRD prior to SCT, and FLT3 ITD negative patients with transcript levels above these thresholds, have a very high risk of relapse and may benefit from further chemotherapy or FLT3 inhibition prior to or after SCT; studies to investigate this are urgently needed. [Display omitted] DisclosuresHills:Daiichi Sankyo: Consultancy, Honoraria. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Russell:Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau.

FLT3-ITD As a Prognostic Factor for Chinese Patients with Acute Myeloid Leukemia

Internal tandem duplication of the FLT3 gene (FLT3-ITD) is one of most common genetic mutations found in patients with acute myeloid leukemia (AML). Although FLT3-ITD has been widely studied and used as an important prognostic indicator of AML in the western world, its association with disease features and prognosis in Chinese AML patients to date has rarely been investigated.In this study, 177 Chinese patients with AML were enrolled including a FLT3-ITD mutation positive group (n = 83) and a FLT3-ITD mutation negative group (n = 94).In FLT3-ITD-positive AML patients, the number of patients whose peripheral blood leucocyte count (≥ 100 × 109/L) before treatment was significantly higher than in FLT3-ITD negative patients (32.1% vs 7.6%), but comparing to FLT3-ITD mutation negative patients, 1 year and 2 year event free survival rates (EFSR) of FLT3-ITD-positive AML patients was lower ((37.0% vs 56.0%) and (14.8% vs 38.4%)), and also their 1 year and 2 year OS rates were significantly lower ( (50% vs 100%) and (18.0% vs 36.5%)). However, it was found that the rate of induced remissions in FLT3-ITD-positive AML patients was much lower than in negative patients (39.4% vs 56.0%). The median survival time of the FLT3-ITD+/NPM1- group (n = 39) was superior to the FLT3-ITD+/NPM1+ group (n = 27) (16.1 months vs 5.8 months). Lastly, after comparing chemotherapy regimes, we found that the allogeneic hematopoietic stem cell transplantation treatment method significantly improved the survival rate of FLT-ITD positive AML patients (not reached vs 14.5 months). Therefore, for FLT3-ITD-positive Chinese patients with AML, the relative rate of complication with leucocyte was increased; both the event free and overall survival rates were lower, these characteristics are the same as the FLT3-ITD-positive patients in western countries. The lower induced remission rate and shorter survival time suggested FLT3-ITD+/NPM1+ mutation is inferior to FLT3-ITD+/NPM1- mutation, which is not consistant with most reports. In addition, allogeneic hematopoietic stem cell transplantation was clearly an effective treatment for patients with FLT-ITD positive AML in China. DisclosuresNo relevant conflicts of interest to declare.

The Clinical Features and Prognostic Impact of PRDM16 gene Expression in Adult Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a complex disease caused by various genetic alterations. Some prognosis-associated cytogenetic aberrations or gene mutations such as FLT3-internal tandem duplication (ITD), t(8;21)(q22;q22)/RUNX1-RUNX1T1, and inv(16)(p13q22)/CBFB-MYH11 have been found and used to stratify the risk. Numerous gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT, NPM1, CEBPA and FLT3in the recent development of massively parallel sequencing technologies. However, even after incorporating these molecular markers, the prognosis is unclear in a subset of AML patients.Recently, NUP98-NSD1 fusion gene was identified as a poor prognostic factor for AML. We have reported that all pediatric AML patients with NUP98-NSD1 fusion showed high expression of the PR domain containing 16 (PRDM16; also known as MEL1) gene, which is a zinc finger transcription factor located near the breakpoint at 1p36. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of EVI1. Furthermore, PRDM16 is essential for hematopoietic stem cell maintenance and remarkable as a candidate gene to induce leukemogenesis. Recent reports revealed that high PRDM16 expression was a significant marker to predict poor prognosis in pediatric AML. However, the significance of PRDM16 expression is unclear in adult AML patients.MethodsA total of 151 adult AML patients (136 patients with de novo AML and 15 patients with relapsed AML) were analyzed. They were referred to our institution between 2004 and 2015 and our collaborating center between 1996 and 2013. The median length of follow-up for censored patients was 30.6 months. Quantitative RT-PCR analysis was performed using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between PRDM16 gene expression and other genetic alterations, such as FLT3-ITD, NPM1, and DNMT3A, and clarified the prognostic impact of PRDM16 expression in adult AML patients. Mutation analyses were performed by direct sequence analysis, Mutation Biased PCR, and the next-generation sequencer Ion PGM.ResultsPRDM16 overexpression was identified in 29% (44/151) of adult AML patients. High PRDM16 expression correlated with higher white blood cell counts in peripheral blood and higher blast ratio in bone marrow at diagnosis; higher coincidence of mutation in NPM1 (P = 0.003) and DNMT3A (P = 0.009); and lower coincidence of t(8;21) (P = 0.010), low-risk group (P = 0.008), and mutation in BCOR (P = 0.049). Conversely, there were no significant differences in age at diagnosis and sex distribution. Patients with high PRDM16 expression tended to be low frequency in M2 (P = 0.081) subtype, and the remaining subtype had no significant differences between high and low PRDM16 expression. Remarkably, PRDM16 overexpression patients were frequently observed in non-complete remission (55.8% vs. 26.3%, P = 0.001). Patients with high PRDM16 expression tended to have a cumulative incidence of FLT3-ITD (37% vs. 21%, P = 0.089) and MLL-PTD (15% vs. 5%, P = 0.121).We analyzed the prognosis of 139 patients who were traceable. The overall survival (OS) and median survival time (MST) of patients with high PRDM16 expression were significantly worse than those of patients with low expression (5-year OS, 17% vs. 32%; MST, 287 days vs. 673 days; P = 0.004). This trend was also significant among patients aged <65 years (5-year OS, 25% vs. 48%; MST, 361 days vs. 1565 days, P = 0.013). Moreover, high PRDM16 expression was a significant prognostic factor for FLT3-ITD negative patients aged < 65 years in the intermediate cytogenetic risk group (5-year OS, 29% vs. 58%; MST, 215 days vs. undefined; P = 0.032).ConclusionsWe investigated the correlations among PRDM16 expression, clinical features, and other genetic alterations to reveal clinical and prognostic significance. High PRDM16 expression was independently associated with non-CR and adverse outcomes in adult AML patients, as well as pediatric AML patients. Our finding indicated that the same pathogenesis may exist in both adult and pediatric AML patients with respect to PRDM16 expression, and measuring PRDM16 expression was a powerful tool to predict the prognosis of adult AML patients. DisclosuresInokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Abstract 5042: A CRISPR/Cas9 knockout screen identify novel drug resistance genes in AML

Abstract Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. Despite remarkable progress made in the therapy of AML, the mainstays of treatment have not significantly changed over the last 20 years. Technological advances have greatly accelerated genomic discovery and precision medicine holds promise to better tailor medications to AML with specific mutations. Importantly, one major obstacle to greater success with target therapy of leukemia is drug resistance. But the mechanisms underlying drug resistance in AML are poorly understood. The activating mutations of FLT3 are now recognized as the most common molecular abnormality in AML and FLT3ITD mutations are found in nearly 30% of AML patients. The poor prognosis of patients harboring FLT3 mutations renders FLT3 an obvious target of therapy. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3ITD positive and negative AML patients and as a maintenance therapy. So far, those clinical trials have showed very promising result. However, drug resistance to AC220 has also been reported through the early clinical studies. To understand the underlying mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confer resistance to AC220. In our screen, we identified SPRY3 and GSK3 as positive hits and the related Wnt and FGF-Ras-ERK signaling activation as the major mechanisms to cause resistance to the FLT3 inhibitor AC220. In the next step, we will explore the detailed underlying mechanisms by which SPRY3 and GSK3 mutations lead to drug resistance to AC220. We will also confirm our findings in primary AML patient samples. Taken together, our study will provide new insight into signaling pathways that contribute to the acquired resistance in AML. The knowledge learned from this study may lead to the development of more efficient therapeutic avenues for AML. Citation Format: Jian Huang. A CRISPR/Cas9 knockout screen identify novel drug resistance genes in AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5042.

Decision Analysis of Postremission Therapy in Cytogenetically Intermediate-Risk Acute Myeloid Leukemia: The Impact of FLT3 Internal Tandem Duplication, Nucleophosmin, and CCAAT/Enhancer Binding Protein Alpha

We performed a decision analysis comparing allogeneic hematopoietic cell transplantation (allo-HCT) versus chemotherapy in first complete remission for patients with cytogenetically intermediate-risk acute myeloid leukemia, depending on the presence or absence of FLT3-internal tandem duplication (ITD), nucleophosmin (NPM1), and CCAAT/enhancer binding protein alpha (CEBPA) mutations. Adjusted means of the patient-reported EQ-5D index were used as quality-of-life (QOL) estimates. In 332 patients for which FLT3-ITD status was available, FLT3-ITD was present in 60. In 272 patients without FLT3-ITD, NPM1 mutations were present in 83. CEBPA biallelic mutations were detected in 53 patients. For patients harboring FLT3-ITD, allo-HCT improved life expectancy (LE) (52 versus 32 months during 10-year observation) and QOL-adjusted life expectancy (QALE, 36 versus 21). Monte-Carlo simulation identified allo-HCT as the favored strategy in 100% of simulations. In patients without FLT3-ITD, allo-HCT improved LE/QALE with or without NPM1 mutations. However, sensitivity analyses showed that the results were not robust enough. For patients harboring CEBPA biallelic mutations, chemotherapy was favored (LE, 53 versus 84; QALE, 37 versus 59), whereas, for patients with monoallelic mutations or wild-type CEBPA, allo-HCT was favored (LE, 68 versus 54; QALE, 48 versus 37). Sensitivity analyses did not change the results in either group. In conclusion, based on a Markov decision analysis, allo-HCT was a favored postremission strategy in patients with FLT3-ITD, and chemotherapy was favored in patients with biallelic CEBPA mutations. A prospective study is warranted to determine the value of allo-HCT, especially in FLT3-ITD–negative patients.

Gemtuzumab Ozogamicin Reduces Relapse Risk in FLT3-ITD+ Acute Myeloid Leukemia: A Report from the Children’s Oncology Group

CD33 is variably expressed on most acute myeloid leukemia (AML) blasts and is the target of gemtuzumab ozogamicin (GO), a calicheamicin-conjugated anti-CD33 monoclonal antibody. COG studies AAML03P1 and AAML0531 evaluated the safety and efficacy of GO combined with conventional chemotherapy to determine the impact of GO on treatment outcomes. We have previously demonstrated that those with high CD33 expression are more susceptible to GO. As FLT3-ITD is associated with high levels of CD33 expression, this group of patients represents a subgroup of particular interest for this therapeutic approach. Patients with high-allelic ratio (HAR) FLT3-ITD have poor outcomes with conventional chemotherapy alone and experience improvement with allogeneic hematopoietic stem cell transplant (HCT). Thus, COG AAML0531 allocated HAR FLT3-ITD+ patients enrolled after April 14, 2008 to consolidation allogeneic HCT with the best available donor.In combined evaluation of COG AAML0531 and its preceding pilot study AAML03P1, 479 patients received conventional MRC based induction chemotherapy (0531 Arm A) and 735 patients received conventional chemotherapy + GO (03P1 and 0531 Arm B). A total of 183 FLT3-ITD+ patients were treated on 0531 Arm A (n=71) and on 03P1/0531 Arm B (n=112). Overall, patients with FLT3-ITD had significantly lower rates of complete remission (CR) compared to FLT3-ITD negative patients, 64% v. 77% respectively (p<0.001). Among FLT3-ITD+ patients, CR rates were identical in those with or without induction GO exposure of 64% vs. 64% respectively (p=0.98).Analysis of 5-year outcomes for FLT3-ITD+ patients treated with GO compared to no GO demonstrated no difference in overall survival (OS) (50% v 49% respectively, p=0.74). Importantly, cumulative incidence of relapse (CIR) at 5 years from CR for patients treated with GO was 37% vs. 59% in those who did not receive GO (p=0.018). This GO-associated improvement in relapse was offset by higher treatment related mortality (TRM) among GO compared to no GO recipients (16% v 0% respectively, p=0.008), leading to similar DFS of 47% vs. 41% respectively (p=0.45). The benefit of decreased relpase risk (RR) was most significant for patients receiving GO in addition to HCT. Among FLT3-ITD+ patients who underwent HCT, those who received GO (n=33) had a 5-yr RR of 22% compared to 56% for the no GO cohort (n=25, p=0.003). There was a trend towards increased TRM among patients receiving GO compared to no GO (22% v. 4% respectively, p=0.078), with a corresponding DFS in GO recipients of 56% vs. 40% for the no GO cohort (p=0.09). Evaluation of the 8 GO recipients who died at HCT revealed that 3 (38%) were the result of complications from transplant-associated sinusoidal obstructive syndrome.Patients with HAR FLT3-ITD, who experience poor outcomes with conventional chemotherapy alone, were analyzed separately to evaluate the impact of induction GO on outcomes. Among HAR FLT3-ITD+ patients who underwent HCT, those treated with GO (n=26) had a significantly lower RR of 15% compared to 53% among no GO recipients (n=15, p=0.007). Additionally, patients receiving GO had a trend towards higher DFS of 65% compared to 40% for no GO group, (p=0.079). In this cohort, TRM in GO vs. no GO recipients was 19% vs. 7% respectively (p=0.297). Among HAR FLT3-ITD+ patients who did not receive HCT, there were no significant differences in DFS, RR, and TRM among the GO versus no GO recipients.Data from the two consecutive COG studies AAML03P1 and AAML0531 suggest that FLT3-ITD+ patients may benefit from the addition of GO to intensive chemotherapy. There is further evidence that HCT may augment the therapeutic impact of induction GO by further reducing the risk of relapse. However, clinical impact of GO was tempered by higher incidence of TRM in GO recipients. CD33 targeting represents an attractive approach in FLT3-ITD+ patients as they often have elevated blast CD33 expression. Further understanding of the toxicity profile of GO, especially when used in conjunction with intensive chemotherapy and HCT, is needed to enhance its therapeutic benefit. Additionally, its impact may be most significant in certain biologic subsets of AML. Our findings demonstrate that CD33 targeting is an important treatment strategy in AML that warrants further investigation in FLT3-ITD+ patients. DisclosuresNo relevant conflicts of interest to declare.

Internal tandem duplication of the FLT3 gene confers poor overall survival in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based chemotherapy: an International Consortium on Acute Promyelocytic Leukemia study.

Activating internal tandem duplication (ITD) mutations in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor outcome in acute myeloid leukemia, but their prognostic impact in acute promyelocytic leukemia (APL) remains controversial. Here, we screened for FLT3-ITD mutations in 171 APL patients, treated with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy. We identified FLT3-ITD mutations in 35 patients (20 %). FLT3-ITD mutations were associated with higher white blood cell counts (P < 0.0001), relapse-risk score (P = 0.0007), higher hemoglobin levels (P = 0.0004), higher frequency of the microgranular morphology (M3v) subtype (P = 0.03), and the short PML/RARA (BCR3) isoform (P < 0.0001). After a median follow-up of 38 months, FLT3-ITD(positive) patients had a lower 3-year overall survival rate (62 %) compared with FLT3-ITD(negative) patients (82 %) (P = 0.006). The prognostic impact of FLT3-ITD on survival was retained in multivariable analysis (hazard ratio: 2.39, 95 % confidence interval [CI] 1.17-4.89; P = 0.017). Nevertheless, complete remission (P = 0.07), disease-free survival (P = 0.24), and the cumulative incidence of relapse (P = 0.94) rates were not significantly different between groups. We can conclude that FLT3-ITD mutations are associated with several hematologic features in APL, in particular with high white blood cell counts. In addition, FLT3-ITD may independently predict a shorter survival in patients with APL treated with ATRA and anthracycline-based chemotherapy.

Next-generation sequencing to identify mutations that may predict outcome after allogeneic stem cell transplantation for AML.

7043 Background: Relapse is the most common reason for failure of allogeneic stem cell transplant (SCT). Identifying patients at increased relapse risk is crucial for improving outcomes. Prognostic somatic mutations have been identified in AML but their prognostic value after SCT is unknown. We hypothesize that next generation sequencing (NGS) can predict SCT outcome in AML. Methods: We performed NGS for 33 hematologic malignancy associated genes in 62 adult patients treated with SCT for de novo AML (2003-2013). Relapse free survival (RFS) was compared by Cox regression with adjustment for cytogenetic risk, conditioning intensity, donor type and graft source. Results: At least 1 mutation was identified in 56 (90%) patients (median 2, range 0-6) including 35 of 36 (97%) patients with intermediate cytogenetics (median 3, range 0-6). Among patients with intermediate cytogenetics transplanted in CR, poor prognostic mutations were found in 9 of 15 FLT3ITD negative patients (2 DNMT3A; 3 DNMT3A + IDH; 1 DNMT3A +...

FLT3-ITD Mutation in Relation to FLT3 Expression in Pediatric AML: A Prospective Study from India

There is lack of data with regard to FLT3 expression in FLT3-ITD positive pediatric AML patients. Further, FLT3-ITD has not been systematically analyzed for outcome from Indian subcontinent. Amongst 64 consecutive pediatric AML patients, FLT3-ITD was present in 12 (19%) patients. All patients with FLT3-ITD achieved CR; those with FLT3-ITD mutation had inferior DFS (P = .029). FLT3 expression by flow-cytometry was observed in all FLT3-ITD positive patients, whereas 40/52 (77%) FLT3-ITD negative patients expressed FLT3 (P = .06). FLT3 expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome. In FLT3-ITD negative patients, higher surface expression of FLT3 significantly predicted poor EFS (P = .001) and OS (P = .007).

Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients

BackgroundSomatic mutations in isocitrate dehydrogenase 1 (IDH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the workThe aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. MethodsResidual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDH1gene, codon 132 mutations screening. ResultsThe frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p=0.03). Patients with IDH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p=0.08). ConclusionIDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDH1 mutations in AML.

The Clinical Features and Prognostic Impact of DNMT3A Gene Mutation in Japanese Patients with De Novo AML

Abstract 2537 Background and Aims:Several gene mutations were found in acute myeloid leukemia (AML) and were expected to be used as prognostic factors. The nucleophosmin 1 (NPM1) and CCAAT/enhancer-binding protein alpha (CEBPA) gene mutations are associated with a favorable outcome and lack of a transplant benefit, but FMS-like tyrosine kinase 3 (FLT3) mutations leading to an internal tandem duplication (ITD) are associated with adverse outcome. Recently the DNA methyltransferase 3A (DNMT3A) gene mutation was identified by whole-genome sequencing in patients with AML. DNMT3A encodes for the enzyme DNA (cytosine-5)-methyltransferase 3A and belongs to the family of other methyltransferases like DNMT1 and DNMT3B. These enzymes are involved in adding methyl groups to the cytosine residue of CpG dinucleotides and thus play an important role in epigenetic regulation of genes, but the mechanism of DNMT3A mutation -associated leukemogenesis was still unknown. About clinical impact of DNMT3A mutation, several groups reported that DNMT3A mutation have been associated with adverse outcome. We analyzed clinical features and prognostic impact of DNMT3A mutation in patients with Japanese patients with AML. Methods:We retrospectively analyzed 188 cases of de novo AML treated at Nippon Medical School Hospital and its affiliated facilities from 2000 to 2010. We analyzed 188 samples at initial presentation and 70 samples at relapse. Mutation analyses were performed for FLT3ITD by PCR amplification, and NPM1, CEBPA, and DNMT3A mutations by direct sequence. To validate sequencing results, PCR products were inserted into the pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Recombinant plasmids isolated from 8 to 12 white colonies were sequenced. Results:The median age was 53.0 years (range, 17–87 years) with 62.1% being males. Patients were followed-up for 0.03–126.1 months after initial presentation, with a median of 19.9 months. DNMT3A mutations were detected in 35 cases (18.6%) at initial presentation, and 12 cases (17.1%) at relapse. Most frequently, codon R882 located in exon 23 was mutated, and 32 cases (91.4%) of these mutations were located within methyltrasferase domain.We evaluated the correlation of clinical and genetic characteristics with DNMT3A mutations. Age (p=0.2884), sex (p=0.6964), and platelet count (p=0.9940) were not significantly different in AML patients with and without DNMT3A mutations. However the frequency of higher white blood cell count (p=0.0001), M5 in FAB classification (p=0.0018), and intermediate risk karyotype (p=0.0032) in patients with DNMT3A mutations were significantly higher than those in patients without them. Also, patients with DNMT3A mutations had a mutation in NPM1 (p<0.0001) and FLT3ITD (p=0.009) more frequently.We next assessed the prognostic impact of DNMT3A mutations. For total cohort of patients with AML, complete remission rate and rates of relapse free survival (RFS) at 5 years were not statistically different between AML patients with and without DNMT3A mutations. However the overall survival (OS) at 5 years in patients with DNMT3A mutation (11.0%) was significantly lower than those in patients without them (28.9%) (p=0.0005). Among the intermediate risk karyotype or FLT3ITD negative AML patients, RFS at 5 years were not statistically different between AML patients with and without DNMT3A mutations, but OS at 5 years in patients with DNMT3A mutation (intermediate risk karyotype: 12.6%, Flt3ITD negative: 11.6%) was significantly lower than those in patients without them (intermediate risk karyotype: 23.9%, p=0.0231, FLT3ITD negative: 30.9%, p=0.0046). Conclusions:This study shows that DNMT3A mutation is an important adverse prognostic factor among intermediate risk karyotype or FLT3ITD negative AML patients. Recently TET2 and Isocitrate Dehydrogenase (IDH) 1/2 gene mutations in de novo AML were reported. These genes including DNMT3A play an important role in epigenetic regulation of genes such as methylation etc, and mutations of these genes may be associated with leukemogenesis of AML. Now we are analyzing TET2 and IDH1/2 mutations among same our AML cohort, and we will show the prognostic impact of TET2, IDH1/2 and DNMT3A mutations in patients with de novo AML. Disclosures:No relevant conflicts of interest to declare.

WT1 overexpression at diagnosis may predict favorable outcome in patients with de novo non-M3 acute myeloid leukemia

We reviewed the frequency and prognostic significance of FLT3 (fms-like tyrosine kinase receptor-3) and NPM (nucleophosmin) gene mutations and WT1 (Wilms’ tumor) and BAALC (brain and acute leukemia, cytoplasmic) gene expression in 100 consecutive patients with intermediate and poor cytogenetic risk de novo acute myeloid leukemia (AML) receiving conventional anthracycline–AraC based therapy. We observed a strict relationship between unfavorable karyotype and BAALC >1000 (p = 0.0001). Multivariate analysis of 81 patients with intermediate karyotype revealed that younger age (p = 0.00009), NPM gene mutation (p = 0.002), and WT1 >75th percentile (>2365) (p = 0.003) were independent, positive factors for complete remission (CR). WT1 expression above 2365 was correlated also to longer event-free survival (EFS) and overall survival (OS) in the same subset of patients (p = 0.003 and p = 0.02, respectively); the same finding occurred in younger patients with AML with intermediate karyotype (p = 0.008 and p = 0.01, respectively). In patients with intermediate karyotype, FLT3 internal tandem duplication (ITD) negatively affected EFS (EFS at 30 months: 30% vs. 6% in FLT3-ITD negative and FLT3 positive patients, respectively; p = 0.01) and OS (OS at 30 months: 38% vs. 20%, p = 0.03). The positive prognostic value of high WT1 expression does not have a clear explanation; it may be implicated either with WT1 anti-oncogenic function, or with the stimulating effect of WT1 oncogene on the leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.

The Impact of FLT3-ITD Mutation on the Expression of Multidrug Resistance Genes MDR-1, MRP-1, BCRP and LRP In Adult Acute Myeloid Leukemia

AbstractAbstract 1808 BackgroundMultidrug resistance (MDR) is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). From 20–50% of patients with AML are primarily resistant to induction chemotherapy and in the great majority of those who respond well to initial therapy the disease relapses. While some mechanisms of MDR have been well characterized, this phenomenon still remains incompletely understood. Three ATP-dependent drug transporting proteins: p-glycoprotein (Pgp; MDR-1), multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) and one major vault protein-lung resistance-related protein (LRP) and the internal tandem duplication (ITD) in the receptor tyrosine kinase FLT3 have been found to have a strong correlation with MDR and poor outcome in AML. About 30% of AML cases harbor FLT3-ITD mutation. Mainly MDR-1 protein and FLT3-ITD mutation are targets for inhibition by numerous agents in clinical practice. ObjectivesIn this retrospective study we estimated expression of MDR genes encoding MDR-1, MRP-1, BCRP, LRP proteins and detected FLT3-ITD mutation in patients with AML. We divided all patients into two groups according to FLT3-ITD mutation and we compared expression of MDR genes and other prognostic factors in AML: age (<60 vs ≥60 years), cytogenetic/molecular aberrations (good, intermediate, poor), de novo/secondary AML, white blood cell count (WBC), extramedullary involvement, type of AML according to the FAB classification, CD34 expression on blast cells and myeloperoxidase activity (<50% of blast cells, ≥50%) between groups Moreover the rate of complete remissions (CR) and relapses and disease free survival (DFS) and overall survival (OS) in both groups were estimated. Materials and MethodsA total of 126 adult patients at median age of 53 years (range 21–87) with newly diagnosed between June 2005 and March 2010, previously untreated AML and 40 healthy donors were included in this study. Diagnosis of AML was based on standard morphological and immunophenotypical criteria according to the WHO classification. Most of the patients were treated with anthracycline and cytarabine during the induction regime. There were 39 FLT3-ITD positive (30,4%) and 87 FLT3-ITD negative (69,6%) patients. DNA was extracted from bone marrow (90 patients) or peripheral blood (36 patients) samples and used to detect FLT3-ITD with PCR method. RNA was extracted from the same samples and following reverse transcription RQ-PCR method was performed for assessment of expression of MDR-1, MRP-1, BCRP and LRP genes. Parameters of RQ-PCR reaction were established based on the Europe Against Cancer protocol. Every tested sample was amplified simultaneously for the presence of control ABL gene for standardization of mRNA level for tested genes. Results of the expression of tested genes were presented as coefficients calculated with an intermediate method according to Pfaffl's rule. ResultsTo our surprise, significant higher expression of MDR-1 gene was found either in bone marrow or peripheral blood samples of the patients who do not harbor FLT3-ITD in contrast to the patients harboring this mutation (median 0,3025 vs 0,0756, p=0,003 total; median 0,2690 vs 0,0583, p= 0,002 bone marrow samples; median 0,5775 vs 0,0920, p=0,001 peripheral blood samples). For MRP-1, BCRP and LRP genes we did not find significant differences in their expression between two groups. When adjusted for other prognostic factors, only WBC was significantly higher in FLT3-ITD positive group of patients: 1–294×109/l, median 74 vs 0–286×109/l, median 6,5 in FLT3-ITD negative group of patients (p=0,0001). We did not observe significant differences in CR and relapses rates, DFS and OS between groups. ConclusionsIt seems that FLT3-ITD mutation may in some mechanisms influence MDR-1 gene expression We observed significant inhibition of MDR-1 expression in FLT3-ITD positive patients. Because risk factors for AML and outcome of AML therapy did not differ between groups it seems that FLT3-ITD mutation and high expression of MDR-1 gene are independent high risk factors which negatively influence results of AML therapy. These results might provide a powerful risk classification and useful information for treatment strategies including inhibitors of tyrosine kinase or MDR-1 protein.This project was supported by grant of The Ministry of Science (N N402 208935). Disclosures:No relevant conflicts of interest to declare.