FLT3-ITD Patients Research Articles

Do NPM1 and FLT3-ITD mutations modify prognosis in patients treated with non-intensive regimens?

FLT3-ITD and NPM1 mutations are key to defining the genetic risk profile of acute myeloid leukemia (AML). We aimed to assess the prognostic features of the FLT3-ITD and NPM1 mutations in old and/or unfit individuals with AML treated with non-intensive therapies in the era before azacitidine-venetoclax approbation. The results of various non-intensive regimens were also compared. We conducted a retrospective analysis that included patients treated with different non-intensive regimens, between 2007 and 2020 from PETHEMA AML registry. We compiled 707 patients with a median age of 74years and median follow-up time of 37.7months. FLT3-ITD patients (N = 98) showed a non-significant difference in overall survival (OS) compared to FLT3-ITD negative-patients (N = 608) (P = 0.17, median OS was 5 vs 7.3months respectively). NPM1-mutated patients (N = 144) also showed a non-significant difference with NPM1 wild type (N = 519) patients (P = 0.25, median OS 7.2 vs 6.8 respectively). In the Cox regression analysis neither NPM1 nor FLT3-ITD nor age were significant prognostic variables for OS prediction. Abnormal karyotype and a high leukocyte count showed a statistically significant deleterious effect. Azacitidine also showed better survival compared to FLUGA (low dose cytarabine plus fludarabine). NPM1 and FLT3-ITD seem to lack prognostic value in older/unfit AML patients treated with non-intensive regimens other than azacitidine-venetoclax combination.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Wilms' Tumor 1 Functions As a Tumor Suppressor to Suppress FLT3-STAT Signaling and Epigenetic Remodeling in Acute Myeloid Leukemia (CALGB 8461, 9665 and 20202; Alliance)

The Wilms' Tumor 1 ( WT1) gene is a transcription factor that is recurrently mutated or commonly overexpressed in several cancer types. In acute myeloid leukemia (AML), frequent overexpression of WT1 and poor patient outcomes associated with WT1 mutations highlight its importance in the disease; however, there are no tailored treatments for these patients. Furthermore, WT1's fundamental role as either an oncogene or tumor-suppressor remains unresolved. Here we examine the roles of both wild-type and mutant WT1 in AML through epigenetic and mechanistic studies. Using our findings we propose a personalized treatment strategy. WT1 mutations are enriched in the NPM1 mut subset of AML; thus we first focused our study on a cohort of 581 patients with de novo AML and NPM1 mutations enrolled on Alliance for Clinical Trials in Oncology studies. Transcriptomic analyses revealed that WT1 mut patientsphenocopied a distinct, aberrant gene expression signature associated with FLT3 internal tandem duplications (ITD), a mutation known to activate STAT signaling and associated with poor outcome in AML. We observed that WT1 expression levels were remarkably elevated in FLT3-ITD patients and were driven by STAT5A binding to the WT1 promoter. STAT5A binding and subsequent WT1 upregulation were blocked by small molecule FLT3 inhibitors. These findings linking FLT3 activity with WT1 expression raise two possibilities: either WT1 is an oncogene cooperating with the FLT3-STAT pathway, or WT1 naturally functions to suppress FLT3 signaling in a negative feedback loop subverted by WT1 mutations. As WT1 interacts with TET2 to facilitate epigenetic remodeling and DNA binding, we performed genome-wide DNA methylation analysis on WT1 mut AML patients and observed selective hypermethylation of WT1 binding motifs consistent with loss of function. We confirmed that WT1 mutations in AML commonly cause truncation of the C-terminal DNA binding domain in our patient cohort. Using co-immunoprecipitation, ChIP-sequencing and luciferase reporter assays, we found that mutant WT1 functions as a dominant-negative, inhibiting WT1 derived from the wild-type allele. Investigation of WT1 target genes by intersecting transcriptomic and DNA methylation profiles identified hypermethylation and downregulation of miR-193a, leading to a significant downregulation of miR-193a-3p in WT1 mut AML patients. Overexpression of wild-type, but not mutated, WT1 rescued miR-193a expression in AML cell lines. Enforced expression of miR-193a significantly delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its role as potent tumor suppressor. Therapeutic modulation of miRNA levels in cancer patients has been limited by inefficient delivery and tissue enrichment. To overcome this, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), currently being investigated in a phase I clinical trial (NCT04675996). Biweekly i.v. treatments of INT-1B3 in the immunocompetent Hoxa9/Meis1 (H9M)-transduced model system prevented AML formation, highlighting the potent anti-leukemic activity of this miRNA based therapeutic. Overexpression of miR-193a-loaded LNPs downregulated FLT3 expression and suppressed STAT signaling in primary AML samples. Finally, treatment of primary AML cells with FLT3 inhibitors revealed enhanced sensitivity of WT1 mut cells in the absence of FLT3-ITD, highlighting the role of wild-type FLT3 in WT1 mut cells. In summary, we uncovered a critical negative-feedback loop maintained by WT1 to suppress FLT3 activity. Loss-of-function WT1 mutations subvert the tumor suppressor function of WT1 via failure to maintain miR-193a expression, leading to increased FLT3 expression and STAT5 signaling, subsequently impairing differentiation, increasing proliferation and disease aggressiveness in AML (Figure 1). Our findings advocate for use of FLT3 inhibition and miR-193a supplementation for treatment of WT1 mut patients, a subgroup with poor outcomes and no targeted treatment options.

Characteristics of FLT3-ITD and Efficacy of Sorafenib Treatment in Pediatric AML: A Report from AML-BFM Study Group

Introduction Internal tandem duplication (ITD) of FMS-related tyrosine kinase 3 ( FLT3) gene is one of the most common somatic mutations in acute myeloid leukemia (AML). We and others have previously shown that the prognostic significance of FLT3-ITD in children and young adults relies on its characteristics e.g., co-occurrence with WT1 mutationsand NUP98::NSD1 fusions (Niktoreh et al. 2019) or higher allelic ratio (AR) (Tarlock et al. 2018) lead to worse survival outcomes. Also, recent studies suggested that treatment with FLT3 inhibitors, including sorafenib, might be effective in children with FLT3-ITD positive AML. In this report, we summarize the characteristics of FLT3-ITD and outcomes of treatment with sorafenib in patients recruited in AML-BFM consecutive trials. Methods Patients aged 0-21 years old, diagnosed with FLT3-ITD positive de novo AML between Jan. 2004 and Dec. 2021 in Germany and recruited in AML-BFM 2004 and 2012 studies or the 2012 and 2017 registries were retrospectively analyzed. All patients with FLT3-ITD with high- or intermediate risk stratification (HR or IR) were recommended to receive 100 mg/m² sorafenib taken orally twice daily on days 7 to 21 of their induction and consolidation chemotherapy blocks, the final decision however was made by the treating physicians. Patient samples were examined in AML-BFM reference laboratory in Essen, Germany using standard methods. Patients with Down syndrome and AML-M3 were excluded from the current analysis and survival rates were calculated using Kaplan-Meier method and Log-Rank test (significance threshold p<0.05). Results In total, 137 patients (51% male) with median age of 7.78 (1.3-19, IQR=7.27) years at diagnosis were included. Most of the patients were initially stratified in IR group (70%, N=95) followed by HR (26%, N=35). FABs M2, M4 and M1 consisted 74% of all morphologic subtypes with 26%, 25% and 23% of patients, respectively and with a median of 64,000 (500 -700,400, IQR=53,178) x10 9/L, the majority of patients showed a leukocytosis at diagnosis. The median ITD length in the 107 patients with available data was 51 (9-111, IQR=39) bps and 54% of the 91 patients with available data had an AR above 0.5. WT1 mutations and NUP98::NSD1 were detected in 25% (N=34) and 7% (N=10) of patients, respectively and 4% (N=6), harbored both mutations simultaneously. The three-year probability of overall and event-free survival (3y-OS and 3y-EFS, respectively) in all FLT3-ITD patients were 68.8% (±4.3) and 47.6% (±4.5), respectively and although harboring ITDs longer than the median length had a tendency to worsen the 3y-OS (54.4% in ITD>51 bps vs. 73.3% in ITDs<51 bps, p=0.071), it did not influence the 3-y EFS (41.7% vs. 54.2%, p=0.204). Similarly, an AR higher than 0.5 did not affect the survival outcomes (3y-OS: 67.4% vs. 60.4%, p=0.744, 3y-EFS: 46.8% vs. 47%, p=0.618). In total, 36 patients (27%) were treated with sorafenib of which, 23 (63%) received a consecutive HSCT. We observed no survival advantage between sorafenib-treated and sorafenib-naive groups of patients (3y-OS: 73.7% vs. 67.3%, p=0.408, 3y-EFS: 48.3% vs. 47.5%, p=0.929). Patients with longer ITDs did not benefit from sorafenib treatment (3y-OS: 68% vs. 54.3%, p=0.279, 3y-EFS: 48.9% vs. 38.4 %, p=0.649) but higher AR, showed a tendency of improved survival outcomes when sorafenib treatment was implicated (3y-OS: 80.8 % vs. 61.9 %, p=0.159, 3y-EFS: 67.3 % vs. 39.9 %, p=0.131). Although the number of patients were limited, we observed a possible survival advantage in double or triple FLT3-ITD, NUP98::NSD1 and WT1 mutated patients who received Sorafenib: from 28 patients with WT1 mutations only, four patients received sorafenib three of which, are currently alive and in CR but contrarily, from the 24 untreated patients, 14 (58%) are deceased (78% due to relapse). Additionally, from 10 patients with NUP98::NSD1 (w/o WT1 mutations), two who were treated with sorafenib are alive and in CR, whereas from the remaining 8 patients, 6 (75%) experienced relapse and unfortunately did not survive. Conclusions Our preliminary analysis implicates that although addition of sorafenib to the intensive chemotherapy of children and adolescence with FLT3-ITD positive AML, did not improve the survival outcomes in allpatients; specific genetic subgroups might benefit from this treatment. These results must be validated in bigger cohorts of patients with longer follow-ups.

Comparison of the Prognostic Performance of European Leukemianet 2017 and 2022 Classifications in a Minority-Rich Population of Adult Patients with Acute Myeloid Leukemia

Introduction: Improved understanding of disease biology and treatment has transformed acute myeloid leukemia (AML) prognostication, as reflected in the most recent revision to the 2017 ELN risk classification in 2022. The key modifications include categorizing all FLT3-ITD mutated patients in the intermediate risk group, irrespective of allele frequency (AF), and classifying myelodysplasia-related mutations (MDSmut; ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2) as adverse risk. Several cooperative groups have conducted studies to validate this new classification system. In this study, we compare the performance of the ELN 2017 and 2022 classifications in predicting the outcomes of a predominantly minority AML population. Methods: We conducted a retrospective review of patients >18 years old with newly diagnosed AML treated between July 2014 and February 2022 at Montefiore Medical Center, Bronx, NY. Patients' records were reviewed for demographics, mutations, cytogenetic abnormalities, therapy, and outcomes. Patients were risk-stratified according to both the ELN 2017 and 2022 criteria. Subjects with FLT3-ITD mutation with unknown allele frequency or who did not receive standard of care midostaurin were excluded. Rates of complete remission were assessed in both groups with ordinal regression and compared using Mann-Whitney U tests. Overall survival (OS) and event-free survival (EFS) were compared using Kaplan-Meier logistic regression. Results: We identified 72 newly diagnosed AML patients treated with intensive induction chemotherapy. Thirty-two (44.4%) were male, with a median age at diagnosis of 57 years old (24.4-79.6). Seventeen (23.6%) were White, 18 (25.0%) were Black, and 5 (6.9%) were Asian. Twenty-eight (38.9%) identified as Hispanic. Fifty-five (76.4%) patients had ECOG/PS < 2. The median WBC on presentation was 9.75 (95% CL 5.7-20.8) cells/dL; median blast percentage on bone marrow biopsy was 52% (95% CL 35%-65%). Updating risk classification from ELN17 to ELN22 decreased the favorable risk group from 22 (30.6%) to 16 (22.2%) with 5 patients re-classified due to FLT3-ITD mutations and 1 due to TP53 heterozygosity. The intermediate risk group decreased from 22 to 20 patients, with 4 FLT3-ITD patients reclassified from the favorable group, 1 patient with high FLT3-ITD AF from adverse group and 8 patients upclassified to adverse group due to MDSmut (STAG2 n=4, SF3B1 n=2, SRSF2 n=1, U2AF1 n=1) and cytogenetics (t(8;16) n=1). The adverse group increased from 28 (38.9%) to 36 (50%), predominantly due to myelodysplasia-related mutations. CR rates in patients assigned by ELN17 were 95.5%, 72.7%, and 53.6% compared to 100.0%, 70.0%, 58.3% in the ELN22 (fav, int, adv) risk groups, respectively. Both scores were significant predictors of CR rates as measured via ordinal regression, p=<0.001 for both ELN17 and ELN22, respectively. There were no significant differences in the CR rates between ELN17 and ELN22 risk groups (p=0.862). Reclassification according to ELN22 did not result in statistically significant differences in OS and EFS (p=0.42 and 0.64 respectively). Both risk models had statistically significant differences between risk categories. Risk stratification by ELN22 trended towards improved prediction of both OS and EFS with each increase in risk category, compared to ELN17. The median OS for the ELN17 groups was 2.2, 2.3, and 0.97 years respectively, while it was 4.0, 1.9, and 1.2 years for ELN22 (fav, int, adv) respectively. Similarly, EFS for patients classified by ELN22 followed a more predicted pattern than ELN17, with 1.9, 1.5, and 0.53 years for the three ELN17 groups and 2.8, 1.6 and 0.56 years for ELN22 groups (fav, int, adv) respectively. Combined outcome concordance values (c-index) were in the expected range for AML prediction scores at 0.76 and 0.77. Conclusion: Our study compares the performance of the ELN 2017 and 2022 risk classifications in a predominantly minority AML patient population. Limited by small numbers, we showed that, compared to ELN17, the ELN22 classification provided more predictable trends for CR, OS and EFS, but we were unable to detect statistically significant differences between the 2 scores' prediction. Continuous study of larger, inclusive patient cohorts will be important to validate the prognostication schemas in ethnically and racially diverse populations.

Autologous stem cell transplantation in adult patients with intermediate-risk acute myeloid leukemia in first complete remission and no detectable minimal residual disease. A comparative retrospective study with haploidentical transplants of the global committee and the ALWP of the EBMT

In patients with acute myeloid leukemia (AML) of intermediate-risk (IR) in first remission (CR1) with no measurable residual disease (MRD negative), the choice of the best consolidation is questionable. 1122 adult patients from 196 centers, transplanted in 2010-21 were analyzed: 547 received an autologous stem cell transplantation (ASCT) and 575 a Haploidentical donor transplant. Because of a significant interaction, comparisons were done separately for patients with wild-type FLT3 (FLT3-wt) and FLT3-ITD mutation (FLT3-ITD). In FLT3-wt patients, haploidentical transplants had two year lower relapse incidence (RI) (16.9% versus 32.6%; HR = 0.40, p < 0.001), higher NRM higher (17.2% vs 3.5%; HR = 7.02, p < 0.001), similar LFS (65.9% vs 63.8%; p = 0.37) and lower OS (73.2% vs 80.6%; HR = 1.69, p = 0.018). In FLT3-ITD patients, haploidentical transplants had two year lower RI (8.2% vs 47.8%; HR = 0.14, p < 0.001) higher NRM (20.2% vs 5.6%; HR = 3.43, p = 0.002), better LFS (71.5% vs 46.6%; HR = 0.53, p = 0.007) and similar OS (73.5% vs 61.9%; p = 0.44). In IR AML patients with FLT3-wt in MRD negative CR1, autologous stem cell transplantation is a valid option, while in patients with FLT3-ITD, haploidentical transplant is better. Whether autologous transplantation is superior to chemotherapy in FLT3-wt patients and the role of maintenance therapy with FLT3 inhibitors remain to be studied.

Targeting C/EBPα overcomes primary resistance and improves the efficacy of FLT3 inhibitors in acute myeloid leukaemia

The outcomes of FLT3-ITD acute myeloid leukaemia (AML) have been improved since the approval of FLT3 inhibitors (FLT3i). However, approximately 30-50% of patients exhibit primary resistance (PR) to FLT3i with poorly defined mechanisms, posing a pressing clinical unmet need. Here, we identify C/EBPα activation as a top PR feature by analyzing data from primary AML patient samples in Vizome. C/EBPα activation limit FLT3i efficacy, while its inactivation synergistically enhances FLT3i action in cellular and female animal models. We then perform an in silico screen and identify that guanfacine, an antihypertensive medication, mimics C/EBPα inactivation. Furthermore, guanfacine exerts a synergistic effect with FLT3i in vitro and in vivo. Finally, we ascertain the role of C/EBPα activation in PR in an independent cohort of FLT3-ITD patients. These findings highlight C/EBPα activation as a targetable PR mechanism and support clinical studies aimed at testing the combination of guanfacine with FLT3i in overcoming PR and enhancing the efficacy of FLT3i therapy.

Clinical study of induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation in the treatment of FLT3-ITD(+) acute myeloid leukemia with normal karyotype

Objective: To assess the efficacy of induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of FLT3-ITD(+) acute myeloid leukemia (AML) with normal karyotype. Methods: The clinical data of FLT3-ITD(+) AML patients with normal karyotype in the First Affiliated Hospital of Nanjing Medical University from Jan 2018 to March 2021 were retrospectively analyzed. Results: The study included 49 patients with FLT3-ITD(+)AML, 31 males, and 18 females, with a median age of 46 (16-59) years old. All patients received induction chemotherapy, and 24 patients received sequential allo-HSCT (transplantation group) . The median follow-up time was 465 days, the one-year overall survival (OS) from diagnosis was (70.0 ± 7.4) %, and one-year disease-free survival (DFS) was (70.3±7.4) %. The one-year OS was significantly different between the transplantation group and the non-transplantation group [ (85.2 ± 7.9) % vs (52.6 ± 12.3) %, P=0.049]. but one-year DFS [ (84.7 ± 8.1) % vs (55.2 ± 11.9) %, P=0.061] was not. No significance was found in one-year OS between patients with low-frequency and high-frequency FLT3-ITD(+) (P>0.05) . There were 12 patients with high-frequency FLT3-ITD(+) in the transplantation and the non-transplantation groups, respectively. The one-year OS [ (68.8 ± 15.7) % in the transplantation group vs (26.2 ± 15.3) % in the non-transplantation group, P=0.027] and one-year DFS [ (45.5 ± 21.3) % in the transplantation group vs (27.8±15.8) % in the non-transplantation group, P=0.032] were significantly different between the two groups. Conclusion: Induction chemotherapy followed by allo-HSCT can enhance the prognosis of FLT3-ITD(+) patients, particularly those with FLT3-ITD high-frequency mutation.

Clinical Feature and Effect of Demethylation Therapy in Pediatric Patients with Acute Myeloid Leukemia with Myelodysplasia-Related Changes

Objective:To better understanding of the clinical and biological features of childhood AML-MRC, in this study we retrospectively investigated 70 children with AML-MRC admitted to our hospital and compared with AML-NOS. We also analyzed the effect of demethylation drugs in introduction therapy among these patients. Study design: This retrospective cohort study was conducted at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (IHCAMS). Newly diagnosed AML patients at our hospital between January 2009 and December 2021 with complete data regarding baseline characteristics and treatment outcomes were reevaluated as having AML-NOS or AML-MRC according to the 2016 WHO classification of myeloid neoplasms and acute leukemia, strictly excluding cases of therapy-related myeloid neoplasms and AML with recurrent genetic abnormalities. Patients with acute megakaryocytic leukemia were also excluded. Clinical and laboratory data were searched in electronic medical records. Follow-up information was obtained from electronic records or by contacting family members and was initialized from the day of diagnosis to May 31, 2022, or the day of death. Results: Among the 779 newly diagnosed AML patients in our center, 70 patients met the diagnostic criteria of AML-MRC and 73 patients were AML-NOS. Patients with AML-MRC had lower white blood cell count (9.1 (IQR 3.0~30.0) ×109/L vs.17.6 (IQR 5.9~86.8) ×109/L, p=0.002), lower hemoglobin level (74.5 (IQR 63.5~ 86.0) g/L vs. 80.0 (71.0, 94.0) g/L, p=0.025) and lower bone marrow proliferation level (p=0.001) compared with patients with AML-NOS. More FLT3-ITD positive patients were found in AML-NOS patients than AML-MRC patients (33.8% vs. 7.1%, p<0.001). Among the 70 AML-MRC patients, 41(58.6%) were diagnosed by myelodysplasia-related cytogenetics, 10(14.2%) from MDS history, and 19 (27.1%) from a multilineage dysplasia. Monosomy 7 or deletion 7q(-7/7q-) is the most frequent cytogenetic abnormality (21.6%) followed by complex karyotype (18.8%) in AML-MRC patients. Totally 56 AML-MRC patients and 71 AML-NOS patients accepted chemotherapy in out center. Induction treatment for 69 AML-NOS patients were idarubicin/ cytarabine/ etoposide (IAE) or mitoxantrone/cytarabine/ etoposide (MAE) therapy. For AML-MRC patients, demethylation drugs alone or demethylation drugs combined above regime were also used (15 case) besides IAE/MEA therapy (35 case). Six AML-MRC patients and 2 AML-NOS patients accepted homoharringtonine/cytarabine/ granulocyte colony-stimulating factor (HAG) and 3 patients received hematopoietic stem cell transplantation (HSCT) without induction chemotherapy. Overall complete remission (CR) rate of 1 course were 51.0% in AML-MRC and 68.2% in AML-NOS patients. In IAE/MAE group, higher CR rate were seen in AML-NOS patients (70.3%) than AML-MRC patients (48.4%) p= 0.06). AML-MRC patients treated with demethylation drugs seemed to have higher CR rate than introduced by IAE/MAE (64.2 % vs. 48.3%, p= 0.39). HSCT in CR1 were conducted in 29 AML-MRC patients and 19 AML-NOS patients. Median follow-up periods were 2.17 in AML-MRC group and 2.43 years in AML-NOS. Three-year overall survival (OS) and event-free survival (EFS) were 54.4% (95%CI 42.4%~69.8%) and 75.7% (95%CI 62.8%~91.1%) for AML-MRC, 42.3% (95%CI 30.2%~59.2%) and 45.8% (95%CI 32.8%~63.9%) for AML-NOS patients. Cox analysis revealed that HSCT were associated with higher OS (HR 0.21,95%CI 0.1~0.41, p< 0.001). In AML-MRC patients, 3-year OS in HSCT group were significantly higher than that in non-HSCT group [83.4% (95%CI,69.6%~99.8%) vs. 28.0% (95%CI,14.6%~53.9%), p<0.001]. AML-MRC patients with myelodysplasia-related cytogenetics exhibited lower 3-year OS (41.9% ,95%CI,27.5%~63.8%) compared with MDS history group (64.8% (95%CI,39.3%~100.0%) and multilineage dysplasia (77.1% (95%CI,57.3%~100.0%). complex karyotype (41.9% ,95%CI,27.5%~63.8%) and -7(7q-) (40.0% ,95%CI,21.5%~74.3%) AML-MRC patients had lower 3-year OS than normal karyotype (42.3%, 95%CI,21.8%~82.0%) and the other abnormal karyotype (63.5% ,95%CI,37.3%~100.0%) but without statistical difference. Conclusions: Childhood AML-MRC exhibited a comparable adverse outcome with AML-NOS which can be significantly improved by HSCT therapy. Demethylation may help improve the CR rate of AML-MRC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real-World Outcomes and Cardiac Implications of FLT3 Inhibitors in Acute Myeloid Leukemia

Background:FLT3 is a member of the class III receptor tyrosine kinase family and constitutively activating mutations of FLT3 are found in ∼30% of adult patients with acute myeloid leukemia (AML). Clinically, FLT3-ITD mutations are associated with earlier time to relapse and poorer overall survival (OS). FLT3 inhibitors are divided into first generation multi-kinase inhibitors (such as sorafenib, lestaurtinib, midostaurin) and next generation inhibitors (such as quizartinib, crenolanib, gilteritinib) or based on their specificity of FLT3 inhibition [type I inhibitors (midostaurin, gilteritinib) inhibit FLT3 ITD and TKD mutations whereas type II inhibitors (sorafenib, quizartinib) have only FLT3 ITD activity]. Though FLT3 inhibition has showed marginal survival benefit in the front line setting for AML, some of the benefits may be offset by cardiovascular toxicities such as cardiomyopathies, QT prolongation and arrythmias. Therefore, outcomes and cardiac safety profile of FLT3 inhibitors in AML at our institution was investigated in the real-life scenario. Methods: In this retrospective study, we included all FLT3MTAML patient with ECOG status of ≤ 2 at our institute who either received FLT3 inhibitors (1) at diagnosis, (2) in a study after being refractory to ≥1 treatment, or (3) relapsing after achieving prior remission. We analyzed the safety and tolerability of type I and type II FLT3 inhibitors in terms of dose-limiting toxicities (DLT) and treatment-emergent adverse events (TEAE) from a cardiac standpoint. Baseline 2D Echocardiograms, EKGs pre- and post-treatment, metabolic profile through each hospitalization, major cardiac events in correlation to prior cardiac history and electrolyte abnormalities were included in the final analysis. Results: From June 2016 to April 2021, 63 FLT3MT AML patients were treated at our institution, 41 (65%) with primary AML and 22 with secondary AML (35%). 52 patients (83%) had ITD and 11 (17%) had FLT3 TKD mutations. Furthermore, 29 patients (56%) with FLT3 ITD and 3 (27%) with FLT3 TKD were treated with FLT3 inhibitors. Of the patients who received FLT3 inhibitors, 21 (66%) received type-2, and 11 (34%) got type I FLT3 inhibitors. 17 patients received FLT3 inhibitors as mono-therapy, and 15 in combination with chemotherapy. Median age of patients on FLT3 inhibitor treatment was 77 years (range 40- 83), and median duration of treatment was 96 days (range 45-180). CRi and CRp were seen as the best response in 12 (38%) and 5 (17%) of the 32 FLT3MT patients respectively, and the median time to CRi/CRp was 33 days (range 22 to 36). Time to relapse in patients with CRi/CRp was 4.5 months (range 2 to 7.5 months). Median overall survival of FLT3 ITD patients treated with type II inhibitors was 18 months, versus 14.5 months for type I inhibitors (p = 0.782), and 5.6 months in patients who did not receive FLT3 inhibitor therapy. The most common non-cardiac treatment emergent adverse events (TEAEs) in patients on FLT3 inhibitors were fatigue, muscle weakness, anemia, and decreased appetite. In the total FLT3MT AML cohort, cardiac adverse events were observed in 16 patients (16/63), 12 of which were on FLT3 inhibitors and 4 were not on FLT3 inhibitors (p = 0.0006). All cardiac TEAEs with FLT3 inhibitor therapy were observed only with type II inhibitors (p=0.0018). Notably, 12 (12/32) patients were classified to have serious cardiac TEAEs, and all of them were receiving type II FLT3-inhibitors. Serious cardiac TEAEs included Torsades in 5 (41%) patients, recurrent ventricular fibrillation in 2 (17%), third-degree AV block necessitating emergent pacemaker in 2 (17%), permanent atrial fibrillation in 2 (17%), and asystole culminating in death in 1 patient (8%) [Fig.1]. 12 patients who developed cardiac TEAEs with FLT3 inhibitors had hyperkalemia, 5 had a preceding history of atrial fibrillation and PVC, suggesting a decreased threshold of development of cardiac TEAEs in the presence of known risk factors. Conclusions: Type II FLT3 TKIs had worse cardiac safety profile in our cohort and an increased risk of cardiac toxicity, particularly in patients with prior reduced ejection fraction or A-fib. A careful cardiac history and management of predisposing conditions to serious cardiac events could help improve drug tolerance, morbidity, and mortality. Further studies are required to elucidate the precise mechanisms of increased incidence of cardiac arrythmias and ways to mitigate them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Machine Learning Allows the Identification of New Co-Mutational Patterns with Prognostic Implications in NPM1 Mutated AML - Results of the European Harmony Alliance

Background: Mutations of NPM1 are generally considered as a favorable prognostic marker in acute myeloid leukemia (AML), although patients present with distinct co-mutations that might influence outcomes. The recently updated European LeukemiaNet classification (ELN2022) does now group NPM1mut AML patients with FLT3-ITD (regardless of the allelic ratio) as intermediate prognosis, while NPM1mut without FLT3-ITD is part of the favorable group. However, the mutational landscape of NPM1mut AML is more complex and analysis of a large cohort of patients might allow further dissection to identify less frequent co-mutational patterns of prognostic and predictive importance. Aims: To identify clinically significant co-mutational patterns in NPM1mut AML to further refine risk stratification models for this subset of patients. Methods: From the HARMONY Alliance database, a total of 1093 intensively treated NPM1mut patients were selected. Patients treated with targeted therapies (e.g. FLT3 or IDH1/2 inhibitors) were not included and NPM1 MRD information was not available at this stage of the analysis. A machine learning algorithm was developed in order to identify combinations of up to 4 co-mutated genes with potential impact on overall survival (OS). A heuristic search algorithm was implemented and bootstrap sampling was applied to estimate the impact of all possible gene combinations on OS. In addition, a global dashboard was developed where it was possible to compare mutational combinations with Kaplan Meier and Cox regression models. Finally, clinically significant co-mutational patterns were summarized in a novel risk stratification model for NPM1mut AML, which was validated using a publicly available external patient cohort (Awada et al, Blood, 2021). Results: The study population of 1093 NPM1mut AML patients included 57% females and median age was 53 years. Regarding ELN2022 classification, 57% of patients were classified into the favorable, 42% into intermediate and only 1% into adverse risk groups. The most frequent co-mutations were DNMT3A (54%) and FLT3-ITD (42%), followed by NRAS, FLT3-TKD and TET2 (20% each). Mutations on IDH1 (13%) and IDH2 (15%) showed a similar behavior for all the analyses performed, so for simplification purposes we will refer to IDHmut when any of them was mutated and to IDHwt when both were wildtype. The triple combination of NPM1mut + FLT3-ITD + DNMT3Amut identified a subgroup of patients with adverse prognosis (2-year OS of 33%), similar to patients with TP53mut. Of note, not all FLT3-ITD patients carried an intermediate or adverse prognosis, as we were able to identify a subgroup (FLT3-ITD + IDHmut + DNMT3Awt) with excellent prognosis (2-year OS of 80%), which represented 4% of NPM1mut AML. However, in the absence of FLT3-ITD and the presence of DNMT3Amut, the addition of IDHmut decreased OS towards the intermediate risk (2-year OS 59%). Notably, not all DNMT3Amut patients carried an intermediate or adverse prognosis. In the absence of FLT3-ITD and with IDHwt, mutations on either NRAS, KRAS, PTPN11 or RAD21 revealed a subgroup with favorable prognosis even when DNMT3A was mutated (2-year OS 80%) and this group represented 11% of NPM1mut AML. This information is summarized in a 4-category risk stratification model (figure 1). The revised NPM1mut favorable group presented with a 3-year OS of 78%, which was 63%, 48% and 29% for intermediate-1, intermediate-2 and adverse risk groups respectively (p<0.001). Regarding 3-year relapse free survival (RFS), it was 71%, 59%, 39% and 26% accordingly (p<0.001). In the validation cohort, 3-year OS of was 73% for NPM1mut favorable group, being 59%, 40% and 22% for intermediate-1, intermediate-2 and adverse groups respectively (p<0.001). Multivariate OS analysis in NPM1mut AML identified the following independent prognostic factors: revised NPM1mut model (taking favorable group as reference, HR 1.77 for intermediate-1, HR 2.92 for intermediate-2 and HR 5.13 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.93, p<0.001), age >60 years (HR 1.84, p<0.001), WBC at diagnosis >100x103/uL (HR 1.59, p<0.001), and male gender (HR 1.21, p=0.04). Conclusion/summary: Analysis of large NPM1mut AML cohorts allows the identification of clinically significant co-mutational patterns. We propose a new genetic stratification model for NPM1mut AML that identifies 4 groups with different OS and RFS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Prognostic Impact of FLT3 in NPM1-Mutated AML: Co-Occurrence of FLT3-ITD and FLT3-TKD Confers Poor Outcomes

Background:NPM1 and FLT3 are commonly mutated in patients with acute myeloid leukemia (AML). While FLT3 internal tandem duplication (ITD) is known to confer worse prognosis even in the setting of NPM1 according to the recent European LeukemiaNet (ELN) 2022 criteria, the prognostic impact of FLT3 tyrosine kinase domain (TKD) in this population remains unclear. One recent study suggests FLT3-TKD and NPM1 co-mutation defines a highly favorable prognostic group (Boddu et al.). Furthermore, NPM1-mutated AML with both FLT3-ITD and FLT-TKD mutations are rare and not well described. Objective: In this study, we explored the prognostic impact of FLT3-ITD and/or FLT3-TKD in patients with NPM1-mutated AML. Methods: This was a multicenter, retrospective study (Moffitt Cancer Center, Weill Cornell, and Memorial Healthcare System) of NPM1-mutated AML patients who were diagnosed and treated from 2013 to June 2022. Inclusion was restricted to NPM1-mutated AML patients who also harbored FLT3-ITD, FLT-TKD, or both (n=107). All patients have next generation sequencing (NGS) performed at diagnosis. Kaplan-Meier, univariate (log-rank), and multivariate (Cox regression) analyses were performed. Results: Among 107 patients (47M/60F, median age 62 at diagnosis, range 23-79), 85% (91/107) had de novo AML. By ELN 2017 criteria, 37.4% (40/107) had favorable risk, 50.5% (54/107) had intermediate risk, and 9.3% (10/107) had adverse risk disease. Over 90% had intermediate risk cytogenetics at the time of diagnosis. Common co-mutations included DNMT3A (53.3%), TET2 (24.3%), WT1 (12.1%), IDH2 (8.4%), and IDH1 (6.5%) (Table 1). There were 66.4% (71/107) of patients with NPM1+FLT3-ITD, 23.4% (25/107) with NPM1+FLT3-TKD, and 10.3% (11/107) with NPM1 and both FLT3-ITD and FLT-TKD (NPM1+FLT3). Patients with NPM1+FLT3-ITD achieved CR/CRi in 84.5% (60/71) similar to patients with NPM1+FLT3-TKD, 88% (22/25), p=1.0. Most patients in both groups received intensive frontline therapy (80.3% vs. 72%, p=0.408). Forty-two (59.2%, 42/71) NPM1+FLT3-ITD patients received allogeneic hematopoietic cell transplant (HCT) compared to 36% (9/25) of NPM1+FLT3-TKD patients, p=0.193. The median overall survival (mOS) was not significantly different between NPM1+FLT3-ITD (42.4 months) and NPM1+FLT3-TKD (not reach), p=0.51. The NPM1+FLT3 cohort had dismal mOS of 8.4 months, which is significantly worse compared to those with NPM1+FLT3-ITD (p<0.005) and NPM1+FLT3-TKD (p=0.01) (Figure 1). Univariate analysis showed co-occurrence of FLT3-ITD and FLT-TKD significantly impacted OS (HR 2.94, 95% CI: 1.42-6.08, p=0.004). Multivariate analysis using covariates including age over 60, AML type, mutation burden (4 or more), and co-occurrence of FLT3-ITD and FLT-TKD confirmed its prognostic significance on survival (HR 2.96, 95% CI: 1.41-6.20, p=0.004). Conclusions: Our findings suggest very poor outcomes in patients with NPM1+FLT3 and no significant difference observed in survival between NPM1+FLT3-ITD and NPM1+FLT3-TKD. The prognosis of NPM1-mutated AML is increasingly being refined as more knowledge is gained in the molecular level. More studies are needed to understand the different gene-gene interactions that underlie this unique subset. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting of SFK and G6PD Contributes to Anti-Leukemic Activity of Novel FLT3 Trisubstituted Purine Inhibitors: Effective Therapeutic Approach for AML with FLT3 Mutations

We have recently developed compound LGR3922, a potent inhibitor of FLT3 receptor tyrosine kinase, which displays selectivity and potency in in vitro and in vivo AML models (compound 7d, Gucky T et al, J Med Chem. 2018;61(9):3855). In addition, LGR3922 showed strong inhibitory activity against Src family kinases (SFK) in the in vitro settings (Fig. 1A; ASH meeting abstract, Radosova Muchova et al, Blood. 2016;128(22):1569). Here, we present additional characterization of the lead compound LGR3922 and demonstrate its potent antileukemic activity in MLL/FLT3-ITD cell lines, murine xenografts and ex vivo primary AML samples. LGR3922 exhibited strong anti-proliferative effect against MV4-11 and MOLM-13 cells. Contrary to quizartinib (quiz), LGR3922 simultaneously reduced phosphorylation levels of FLT3-SFK and their downstream molecules STAT5 and ERK in both AML cell lines. Interestingly, the inhibitory effect of LGR3922 on SFK, STAT5 and ERK increased over time, especially in the MOLM-13 cells. Similar results were only seen for the combination treatment with quiz and dasatinib (das). Dual-targeting of FLT3-SFK by LGR3922 was further confirmed on MV4-11 xenografts. In the long-term in vivo experiment with repeated doses of the tested inhibitors, LGR3922 showed similar trends in initial treatment response as the combination treatment with quiz and das; quiz exhibited less efficient inhibitory effects at this stage. The tumor reduction was most significant (p ≤ 0.0001) in LGR3922 group; 3 out of 6 tumors were completely eradicated during the treatment period (Fig. 1B). Complete tumor eradication (in 1 out of 5 animals) over the treatment period was then seen only in combination quiz and das (Fig. 1B). No adverse effects on blood count parameters or body weight were associated with LGR3922 compared to combination treatment with quiz and das, where temporary body weight loss and only limited weight gain were observed. Next, we searched for additional target of the FLT3/SFK inhibitor that would have a synthetic lethal effect for the kinase pair. Besides the kinases that belong to other druggable nodes of AML-associated networks (Fig. 1A), we also tested antioxidant defense enzymes, previously validated as being synthetic lethal with FLT3 inhibitor therapy (Gregory MA et al. Proc Natl Acad Sci U S A. 2016;113(43):E6669). Indeed, we observed that LGR3922 inhibited G6PD activity, already after one-hour exposure (with a dose response over the concentration range), in contrast to quiz and das. We hypothesize that this activity modulating leukemia cell redox metabolism, cooperating with FLT3/SFK inhibition, may elicit more effective eradication of AML cells than quiz and das or quiz/das combination. We then evaluated toxicity of LGR3922 on normal hematopoiesis using colony forming unit (CFU) assays with progenitor cells from several healthy donor controls. LGR3922 inhibited colony formation in a dose-dependent manner; maximal tolerated concentration (MTC) for all healthy samples was 100 nM, in some samples the reduced granulocyte-macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) colony forming ability was not observed up to a concentration of 200 nM. Quiz and das exposure showed MTCs at 20 nM and 5 nM, respectively; in quiz/das combination at 10 nM/0.5 nM; 10 nM/5 nM was already toxic. Then, we performed CFU assays on 4 primary human AML samples with FLT3-ITD mutations (n=3) or FLT3-TKD+KMT2A::MLLT3 (n=1). Cultures with LGR3922 at 100 nM reduced the number of colonies by ∼70% in all FLT3-ITD patient samples. The inhibitory effects were comparable to or a little more potent than MTCs of quiz or quiz/das combination. Das culture had significantly lower effect on colony formation for the tested concentrations. In the FLT3-TKD+KMT2A::MLLT3 positive sample, fusion transcript was quantified in harvested colonies. While LGR3922 significantly reduced KMT2A::MLLT3 fusion transcript, no effects were observed for MTCs of single agent cultures with quiz or das. Only a combination of quiz/das exceeding MTC (10 nM/5 nM) resulted in a slight decrease of KMT2A::MLLT3 fusion transcript levels. Taken together, these data suggest effectiveness of our new LGR3922 compound and provide a rationale for further development and testing of this molecule for MLL/FLT3-ITD AMLs, and, potentially, other types of AMLs. Grant support: Program EXCELES, ID Project No. LX22NPO5102; GA19-09086S; MH CZ-DRO (UHKT, 00023736). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AML-247 Specific Features of the FLT3+ Acute Myeloid Leukemia Course

Preliminary stratification into prognostic risk groups is a mandatory component of the AML patient's treatment, which allows for optimizing the intensity of chemotherapy (CT), as well as planning and justifying allogeneic hematopoietic stem cell transplantation (allo-HSCT). To evaluate the effects of the FLT3 gene mutational status and the allele burden (AB) magnitude in FLT3-ITD patients on the course of AML. Retrospective and prospective study. Study of the FLT3 gene mutational status effect on the course of the disease in 280 patients was carried out. The dynamics of the magnitude of the AB in the FLT3-ITD cases were analyzed in the context of CT, allo-HSCT, and the impact of the AB on survival rates in 91 patients. The adverse effects of FLT3 gene mutations on patients' survival rates were assessed. In the study group, the frequency of FLT3 accounted for 30.5% (FLT3-ITD-24% (48/199), the frequency of FLT3-TKD was 5.5% (11/199), the combination of FLT3-ITD and FLT3-TKD was found in 1.0% (2/199)); the rest of the patients, 69.5% (138/199), had no mutations in the FLT3 gene. Mutations in NPM1 gene were detected significantly more frequently in the FLT3-ITD-in 37 (32.4%) out of 114 patients (p=0.0001). Two groups were identified according to the magnitude of AB in the FLT3-ITD mutations: low AB (<0.5)-n=50 (55%) and high AB (≥0.5)-n=41 (45%). Patients with high AB FLT3 gene, compared with low AB, had a high level of leukocytes (p<0.001) and bone marrow blast cells (p=0.04). Patients FLT3-ITDlow/NPM1+ achieved CR (complete clinical and hematological remission) more often compared with FLT3-ITDhigh/NPM1+, with CR rate of 71.4% and 16.0%, respectively (%2=11.2; p=0.04). The efficacy of allo-HSCT in FLT3 + was significantly higher only in terms of OS; it was not achieved in the allo-HSCT(+) (n=24), compared with the median of 8 months recorded in the allo-HSCT(-) (n=119)(p<0.00001). The application of CT reduced AB by 1.5 (p<0.001). Sequential use of CT and allo-HSCT showed higher efficacy (p<0.001). AML patients with mutations in the FLT3 gene require an individualized approach with inclusion of targeted drugs into the scheme of treatment, which allows survival rates to improve.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Adverse impact of a high allelic burden FLT3-ITD mutation on allogeneic hematopoietic stem cell transplantation in patients with cytogenetically normal AML.

Risks associated with the FLT3-ITD mutation in patients receiving chemotherapy alone for cytogenetic normal acute myeloid leukemia (CN-AML) depend on the allelic ratio (AR) and concomitant NPM1 mutation. Nevertheless, their prognostic ability after allogeneic hematopoietic cell transplantation (allo-HCT) remains undetermined. Moreover, previous studies have revealed that haploidentical transplantation improves outcomes of FLT3-ITD patients. To elucidate whether this alteration also impacts prognosis of myeloablative allo-HCT upon first remission, we retrospectively reviewed the prognostic ability of FLT3-ITD mutations in 205 CN-AML patients. Our analysis demonstrated that FLT3-ITD AR was closely related to pretransplant MRD and induction response. Multivariate analysis showed that high-AR FLT3-ITD, pretransplant MRD and induction response were independent risk factors for CN-AML. In addition, we presented evidence that the high-AR FLT3-ITD patient prognosis was not overcome by haploidentical transplantation, but was markedly improved by cGVHD. More importantly, among patients with negative pretransplant MRD, high-AR FLT3-ITD patients did not have increased relapse risk, compared to low-AR FLT3-ITD and wild-type FLT3 patients. Our findings will aid in accurate prognostic stratification of FLT3-ITD patients. We also recommend further targeted and coordinated approaches to sustain durable remission following induction chemotherapy and allo-HCT in this high-risk patient population.

P565: IMPROVED OUTCOME OF PATIENS WITH ACUTE MYELOID LEUKEMIA HARBORING FLT3 MUTATION IN THE ERA OF TARGETED THERAPY

Background: In recent years, the treatment of AML is rapidly evolving due to the advances in targeted therapy, risk-adapted protocols and MRD-guided decisions. The prognosis of patients harbouring FLT3-ITD differs according to its allelic ratio and the presence of NPM1 co-mutation (NPM1mut). Nonetheless, FLT3-ITD prognostic impact and allogeneic hematopoietic cell transplantation (alloHCT) indication in this setting might be redefined by the widespread use of FLT3 inhibitors. Since 2012, chemotherapy eligible patients are treated following a risk-adapted and MRD-guided protocol in the CETLAM group (AML-12). Since 2016, targeted therapy with midostaurin was progressively incorporated in the protocol for FLT3-mutated (FLT3mut) patients. Aims: To analyse the outcome of FLT3mut AML treated with a risk-adapted protocol in two different time periods, mainly differentiated by the advent of midostaurin. Methods: All adult patients with de novo AML and available FLT3 status treated with the intensive protocol AML-12 (clinicaltrials.gov #NCT04687098) in the Spanish CETLAM centres from 2012 to 2020 were retrospectively analysed. Molecular testing of NPM1, and FLT3-ITD and its allelic ratio were studied as previously described. Patients were divided into two study cohorts: the early (2012-2015) and the late cohort (2016-2020). All patients received the same chemotherapy protocol except for the addition of midostaurin since 2016 in most FLT3mut patients. Complete remission (CR), overall survival (OS), event-free survival (EFS) and risk of relapse (RR) followed ELN criteria. OS and EFS were studied with the Kaplan-Meier method and log-rank test, cumulative incidence and Grey test were used to estimate relapse risk (CIR) and non-relapse mortality (NRM). Results: A total of 906 patients were selected, 390 from the early and 516 from the late cohort. Median follow-up was 38 months; FLT3mut was present in 227 cases (25%) and constitute the focus of this study. There were not differences on main variables between cohorts (age, cytogenetic risk, ELN category, frequency of NPM1 mutation or FLT3-ITD allelic ratio). TKD mutations were only available since 2015. Midostaurin was administered to 62% of FLT3mut patients from the late cohort: 63 with ITDs (20 low and 43 high ratio) and 20 TKDs. CR rates were similar between cohorts. AlloHCT in first CR (CR1) was performed in 60% (early) and 67% (late) patients (p=ns). A higher RR was observed in the early group in comparison to the late cohort (2-year RR 42±11% vs 28±10%; p=0.014), without differences in NRM (Figure 1B), translating into an improved EFS (median EFS 9.4 vs. 26 months, p=0.014) and OS in the late cohort (median OS: 15 months vs. non-reached, p=ns, Figure 1A). Among NPM1mut AML patients, FLT3-ITD and ELN 2017 categories retained its prognostic value in the early cohort, with a worse OS, EFS, and higher RR among FLT3-ITD vs wt patients (HR for OS 1.9 95% CI 1.15-3.2, p=.011; figure 1C) and FLT3mut ELN-intermediate vs favourable patients (OS p<0.001; figure 1D). On the contrary, in the late cohort, FLT3-ITD and FLT3 high allelic ratio lost their adverse prognostic impact, with comparable RR, EFS, and OS regardless of FLT3-ITD status (Figure 1E and F). Image:Summary/Conclusion: This real-life study demonstrated an improved outcome among FLT3mut AML patients treated in the most recent period, probably reflecting the beneficial effect of midostaurin on relapse prevention in this context. Future analyses should revise the role of alloHCT in CR1 for non-favourable FLT3mut AML patients treated with FLT3 inhibitors who achieve MRD eradication.

Abstract 3479: UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome

Abstract Childhood AML is an aggressive disease with high rates of failures and poor survival. We have demonstrated that the molecular landscape of AML in children is distinct, and co-occurrence of variants modulate outcomes. Recent discovery of tandem duplication (TD) of the UBTF gene in AML, with enrichment in FLT3-ITD has implicated yet another mutation whose cooperation with FLT3-ITD may modify outcome. Here, we provide a comprehensive evaluation of UBTF-TD in de novo AML and define its clinical implications within FLT3-ITD patients. Initial interrogation of transcriptome data from 1,158 children enrolled on COG AAML1031 identified 50 cases of UBTF-TD (4.3%). Overwhelming majority of UBTF-TD cases were observed in FLT3-ITD cases (77%), vs. that of 1.2% in those without FLT3-ITD (p&amp;lt;0.001). Given extreme enrichment of UBTF-TD in FLT3-ITD, we inquired whether cooperation of UBTF-TD and FLT3-ITD creates a distinct clinical entity. To this end we screened diagnostic DNA from 400 FLT3-ITD patients treated on three consecutive CCG/COG trials (COG AAML1031, COG AAML0531, and CCG-2961) by PCR and fragment analysis. UBTF-TD was identified in 61 FLT3-ITD cases (15.3%). The data presented here forth focuses on evaluation of implications of UBTF-TD in FLT3-ITD positive patients only. Within the FLT3-ITD patients, initial correlation of UBTF-TD with demographics, disease characteristics, and associated genomic variants was conducted. Patients with and without UBTF-TD had a similar median age at diagnosis (p=0.322), lower diagnostic WBC (p=0.010) and higher marrow blast % (p&amp;lt;0.001). There was a stark paucity of cooperating variants that commonly co-occur with FLT3-ITD, with a single NPM1 mutation (1.6% vs. 29%, p&amp;lt;0.001) and no NUP98 fusions (0% vs. 23%, p&amp;lt;0.001). There was a significant enrichment of WT1 mutations, with 45% UBTF-TD patients with a WT1 mutation (FLT3-ITD/UBTF-TD/WT1), vs. 11% in UBTF-WT (p&amp;lt;0.001). Trisomy 8 (Tri8) was seen in 15% of UBTF-TD. Patients with UBTF-TD had a lower CR rate (44% vs. 60%, p = 0.018), and Higher MRD rate (38% vs. 21%, p&amp;lt;0.001). Patients with and without UBTF-TD had an EFS of 28% vs. 42% (p=0.047) with a corresponding OS of 40% and 57% (p=0.019). Given enrichment of WT1 mutations and Tri8 in patients with UBTF-TD, we studied the outcome UBTF-TD patients in the context of these two variants. FLT3-ITD/UBTF-TD/WT1 patients had a 5-year EFS of 17% vs. 38% for similar patients without WT1 mutations (p=0.0062). Patients with UBTF-TD with additional Tri8 had a similarly poor outcome with an EFS of 23% with a corresponding OS of 33%, providing a distinct high risk UBTF-TD cohort (+WT1 or Tri8), whereas the remaining UBTF-TD patients had a more favorable outcome with EFS and OS of 64% and 86%, respectively (p&amp;lt;0.0001, and p&amp;lt;0.0001). UBTF-TD is a novel genomic entity with high enrichment in patients with FLT3-ITD and a distinct clinical outcome driven by cooperating WT1 mutation and Tri8. Citation Format: Leila Robinson, Amanda Leonti, Todd A. Alonzo, Yi-Cheng Wang, Michele S. Redell, Rhonda E. Ries, Jenny L. Smith, Tiffany A. Hylkema, Quy Le, E Anders Kolb, Richard Aplenc, Xiaotu Ma, Jeffrey Klco, Katherine Tarlock, Soheil Meshinchi. UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3479.

Impact of FLT3 internal tandem duplication and NPM1 mutations in acute myeloid leukemia treated with allogeneic hematopoietic cell transplantation

Background aimsThe internal tandem duplication of FLT3 (FLT3ITD) and NPM1 mutations (NPM1mut) are well-established prognostic factors in cytogenetically intermediate-risk acute myeloid leukemia (AML) when treated with chemotherapy alone. However, their prognostic value in the setting of allogeneic hematopoietic cell transplantation (HCT) is controversial. MethodsFLT3 and NPM1 mutational status was determined at diagnosis using single-gene polymerase chain reaction or next-generation sequencing in 247 adult patients with cytogenetically intermediate-risk AML who underwent myeloablative HCT. Multivariate Fine–Gray and Cox regression was used to analyze the cumulative incidence of relapse (CIR), relapse-free survival (RFS) and overall survival (OS). ResultsFLT3ITD and NPM1mut were present in 74 of 247 (30%) and 79 of 247 (32%) patients, respectively. There was no significant difference between patients without a FLT3ITD or NPM1mut (FLT3NONITD/NPM1WT) and patients with a FLT3ITD mutation alone (FLT3ITD/NPM1WT) with regard to CIR (P = 0.60), RFS (P = 0.91) or OS (P = 0.66). Similarly, there was no significant difference between FLT3NONITD/NPM1WT and FLT3NONITD/NPM1mut patients with regard to CIR (P = 0.70), RFS (P = 0.75) or OS (P = 0.95). The presence of a concurrent mutation in NPM1 did not appear to modify the impact of having a FLT3ITD mutation. ConclusionsIn contrast to chemotherapy-only treatment, FLT3 and NPM1 mutational status does not appear to predict outcomes in patients with cytogenetically intermediate-risk AML following HCT. These results suggest that HCT may ameliorate the poor prognostic effect of FLT3ITD mutation and that HCT should be considered over chemotherapy-only treatment in FLT3ITD-mutated AML.