Core-binding Factor Acute Myeloid Leukemia Research Articles

Comparison of efficacy between homoharringtonine, aclarubicin, cytarabine (HAA) and idarubicin, cytarabine (IA) regimens as induction therapy in patients with de novo core binding factor acute myeloid leukemia.

To compare efficacy between homoharringtonine combined with cytarabine and aclarubicin (HAA) and idarubicin and cytarabine (IA) regimens as first induction chemotherapy in patients with core binding factor acute myeloid leukemia (CBF-AML). Cox regression model and propensity score matching (PSM) were used to identify the regimen associated with a better remission rate and outcomes. In total, 374 patients with CBF-AML (243 with RUNX1::RUXN1T1 and 131 with CBFB::MYH11) were included in this study. The patients received the HAA or IA regimen (187 each) as the first induction therapy. For patients with RUNX1::RUXN1T1, multivariate analyses showed that the HAA regimen was significantly associated with a higher CR/CRi rate after the first induction (hazard ratio [HR] = 5.3 [95% CI 2.3, 12.2]; p < 0.001) and more favorable relapse-free survival (RFS) (HR = 0.5 [0.3, 0.8], p = 0.01). In PSM analysis, the HAA regimen also had a higher CR/CRi rate (96% vs. 77%, p < 0.001), especially for those harboring wild-type KIT (KITWT) (96% vs. 83%, p = 0.02) or non-D816 KIT mutation (100% vs. 63%, p = 0.002), as well as more favorable RFS (p = 0.01), compared with the IA regimen. However, there was no difference in the remission rate or outcomes between the two regimens for patients with CBFB::MYH11. The HAA regimen as first induction chemotherapy resulted in a higher CR/CRi rate in AML patients with RUNX1::RUNX1T1, especially those harboring KITWT and non-D816 KIT mutation, and a more favorable RFS compared with the IA regimen. The efficacy between the two regimens did not differ in those with CBFB::MYH11.

Prevalence of Gene Rearrangement by Multiplex PCR in De Novo Acute Myeloid Leukemia in Adult Iraqi Patients.

Gene rearrangements of acute myeloid leukemia (AML) play a significant role in categorizing patients and provide valuable information about prognosis and treatment choices. However, in Iraq, the prevalence and prognostic significance of gene rearrangements in AML have not been previously examined. This study utilized a multiplex reverse transcription real-time PCR (RT-qPCR) system to identify gene rearrangements in a group of 115 adult patients from Iraq who had been diagnosed with De Novo AML. The diagnosis of AML was confirmed through blood film and flow cytometry. The ethical committee of the College of Medicine at the University of Baghdad provided approval for this research study. In this study, 66.1% of the patients diagnosed with acute myeloid leukemia (AML) exhibited distinct genetic abnormalities. Among these abnormalities, the most frequent was the rearrangement involving the KMT2A gene, observed in 19.9% of the patients. The risk stratification analysis revealed that 40% of the patients were classified as having a favorable risk, 4.3% as intermediate risk, and 25.2% as adverse risk. A subtype of AML known as core-binding factor (CBF) AML was identified in 21.7% of the cases, with 84% of these patients achieving complete remission. The NPM-RARA gene rearrangement, found in 43% of acute promyelocytic leukemia (APL) cases, was associated with a 71% complete remission rate. Among patients with KMT2A rearrangement, which accounted for 19.9% of all AML cases, the MLL-AF10 rearrangement was the most common, although only one patient with KMT2A rearrangement achieved complete remission. Furthermore, the analysis of demographic data revealed a significant association between increased risk and advanced age, presence of comorbidities, and FAB classification (M0 subtype). The prevalence of genetic rearrangements in Iraqi De Novo AML patients is higher than the global trend, highlighting the importance of genetic characterization in risk assessment and treatment decisions.

Clinical impact of c-KIT and CEBPA mutations in 33 patients with corebinding factor (Non-M3) acute myeloid leukemia.

Corebinding factor acute myeloid leukemia (CBF-AML) is the most common cytogenetic subtype of pediatric AML. CBF-AML is associated with a relatively favorable outcome, although the relapse rate of approximately 40% indicates a high degree of clinical heterogeneity. The clinical impact of additional cytogenetic aberrations, including c-KIT and CEBPA mutations, in pediatric CBF-AML has not been well characterized, especially in the multi-ethnic region of Yunnan Province in China. In this study, we retrospectively analyzed the clinical features, gene mutations, and prognoses of 72 pediatric patients newly diagnosed with non-M3 AML in Kunming Children's Hospital, China, from January 1, 2015 to May 31, 2020. Of the 72 pediatric patients with AML, 46% (33/72) had CBF-AML. Thirteen patients with CBF-AML (39%) had c-KIT mutations, five (15%) had CEBPA mutations, and eleven (33.3%) had no other cytogenetic aberrations. The c-KIT mutations, resulting from single nucleotide substitutions and small insertions or deletions, occurred in exons 8 and 17. All of the CBF-AML-associated CEBPA mutations were single mutations and occurred in patients with RUNX1-RUNX1T1 fusion. We found no significant differences in the clinical data between CBF-AML patients with c-KIT or CEBPA mutations and CBF-AML patients without other aberrations, and no prognostic significance was established for these mutations. Our study is the first to report the clinical impact of c-KIT and CEBPA mutations in pediatric patients with non-M3 CBF-AML from the multi-ethnic Yunnan Province, China. c-KIT and CEBPA mutations occurred at a higher frequency in CBF-AML cases and were associated with unique clinical characteristics; however, no potential molecular prognostic markers were identified.

An ETV6::NTRK3 fusion transcript in a core-binding factor acute myeloid leukemia.

An ETV6::NTRK3 fusion transcript in a core-binding factor acute myeloid leukemia.

Outcomes of different therapies in acute myeloid leukemia patients with core-binding factor.

e19036 Background: Acute myeloid leukemia (AML) with RUNX1:RUNXT1 fusion or CBFB:MYH11 fusion is defined as core-binding factor AML (CBF-AML) and has been considered to show favorable prognosis. Methods: We conducted a retrospective analysis on previously untreated CBF-AML patients who received standard-dose induction therapies between Jan 2018 and Dec 2020 in our medical center. Patients were classified into 5 groups based on the induction regimens: VA, DA, IA, HAA, and DAV. The dosage of these regimens were determined as follows: VA, venetoclax 100mg d1,200mg d2, 400mg d3-28, Azacytidine 75mg/m2 d1-7; DA, daunorubicin 60mg/m2 d1-3, cytarabine 100mg/m2 d1-7; idarubicin 8-10mg/m2 d1-3, cytarabine 100mg/m2 d1-7; HAA, homoharringtonine 2mg/m2 d1-7, cytarabine 100mg/m2 d1-7, aclarubicin 12mg/m2 d1-7; DAV, daunorubicin 60mg/m2 d1-3, cytarabine 100mg/m2 d1-7, venetoclax 100mg d4, 200mg d5, 400mg d6-11. The response rate and survival data were analysed between these groups. Results: There’s a total of 106 CBF-AML patients involved, 85(80.2%) with RUNX1:RUNXT1 fusion and 21(19.8%) with CBFB:MYH11 fusion. The most common concomitant mutation was KIT (33%), and the most frequent hotspot mutation site was D816V in exon 17. The median age was 46 years ranging from 7 to 78, and 46.2% of the patients were male.Patients were classified into 5 groups based on the induction regimens, and there were 9 in VA group, 21 in HAA group, 23 in DA group, 38 in IA group, and 15 in DAV group. The CR rate of each group was 55.6%(VA), 87.0%(DA), 65.8%(IA), 90.4%(HAA), and 100%(DAV) respectively. Among the patients who received CR, patients in DAV group showed more pronounced decreasing of fusion gene levels (median 1450 fold) than other groups, while VA group had the slightest (median 8.9 fold). As to the survival, the median follow-up time was 15.2 months, and the median overall survival (OS) were not reached in all the groups. In the RUNX1:RUNXT1 fusion subgroup, DAV group showed significantly improved OS and event-free survival (EFS) than other groups, while VA groups was the worst. The median EFS were as follows: 3.5months(VA), not reached(HAA), 10.9months(DA), 7.9months(IA), and not reached(DAV) respectively. Conclusions: Our study demonstrated a promising therapeutic results of DAV regimen, and an unsatisfactory therapeutic results of VA regimen in CBF-AML. CBF-AML patients showed significantly improved CR rate, elimination of fusion genes, OS, and EFS when received intense chemotherapy, especially DAV and HAA regimen, than VA regimen. These results indicated that intense chemotherapy may be an optimal choice for CBF-AML.

CAG (cytarabine, aclarubicin and granulocyte colony-stimulating factor) regimen for core binding factor acute myeloid leukaemia with measurable residual disease.

Acute myeloid leukaemia (AML) with t (8;21) or inv (16), called core binding factor (CBF) AML, has a favourable prognosis. However, some CBF-AML patients have persistent measurable residual disease (MRD) and are more likely to relapse after standard chemotherapy treatment. The CAG regimen, composed of cytarabine, aclarubicin and granulocyte colony-stimulating factor, has been proven to be effective and safe in treating refractory AML patients. We performed a retrospective study to evaluate the efficacy of the CAG regimen to eliminate MRD detected by RUNX1::RUNX1T1 and CBFβ::MYH11 transcript levels by quantitative polymerase chain reaction (Q-PCR) among 23 patients. Molecular response was defined as the ratio of fusion transcript after treatment to that before treatment less than or equal to 0.5. The molecular response rate and median decrease ratio of fusion transcripts at the molecular level of the CAG regimen were 52% and 0.53, respectively. The median fusion transcripts before CAG treatment was 0.25% whereas after CAG was 0.11%. Among the 15 patients who had a poor molecular response to the high/intermediate-dose cytarabine regimen, the median decrease ratios of transcripts at the molecular level of high/intermediate-dose cytarabine and CAG were 1.55 and 0.53 (P = 0.028), respectively, and 6 of 15 patients achieved a molecular response to CAG (40%). The median disease-free survival was 18months, and the overall survival rate at 3years among all patients was 72.7% ± 10.7%. The common grades 3-4 adverse events were nausea (100%), thrombocytopenia (39%) and neutropenia (37.5%). The CAG regimen may have activity in CBF-AML patients and could provide a new option for patients who have a poor molecular response to high/intermediate-dose cytarabine.

Outcome prediction by the 2022 European LeukemiaNet genetic-risk classification for adults with acute myeloid leukemia: an Alliance study

Recently, the European LeukemiaNet (ELN) revised its genetic-risk classification of acute myeloid leukemia (AML). We categorized 1637 adults with AML treated with cytarabine/anthracycline regimens according to the 2022 and 2017 ELN classifications. Compared with the 2017 ELN classification, 2022 favorable group decreased from 40% to 35% and adverse group increased from 37% to 41% of patients. The 2022 genetic-risk groups seemed to accurately reflect treatment outcomes in all patients and patients aged <60 years, but in patients aged ≥60 years, relapse rates, disease-free (DFS) and overall (OS) survival were not significantly different between intermediate and adverse groups. In younger African-American patients, DFS and OS did not differ between intermediate-risk and adverse-risk patients nor did DFS between favorable and intermediate groups. In Hispanic patients, DFS and OS did not differ between favorable and intermediate groups. Outcome prediction abilities of 2022 and 2017 ELN classifications were similar. Among favorable-risk patients, myelodysplasia-related mutations did not affect patients with CEBPAbZIP mutations or core-binding factor AML, but changed risk assignment of NPM1-mutated/FLT3-ITD-negative patients to intermediate. NPM1-mutated patients with adverse-risk cytogenetic abnormalities were closer prognostically to the intermediate than adverse group. Our analyses both confirm and challenge prognostic significance of some of the newly added markers.

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis

AbstractAdenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Therapy-related core binding factor acute myeloid leukemia.

Therapy-related acute myeloid leukemia (t-AML) usually stems from exposure of the bone marrow to cytotoxic chemotherapy and/or radiation therapy. t-AML is usually associated with poor overall survival, but occasionally t-AML can involve favorable-risk cytogenetics, including core binding factor AML (CBF-AML), which shows a recurrent chromosomal rearrangement with t(8;21) (q22;22) and 'inv(16) (p13.1;q22)/t(16;16)(p13.1;q22)', leading to 'RUNX1::RUNX1T1 and CBFB::MYH11' fusion genes, respectively. Therapy-related CBF-AML (t-CBF-AML) accounts for 5-15% of CBF-AML cases and tends to have better outcomes than t-AML with unfavorable cytogenetics. Although CBF-AML is sensitive to high-dose cytarabine, t-CBF-AML has worse overall survival than de novo CBF- AML. The objective of this review is to discuss the available data on the pathogenesis, mutations, and therapeutic options in patients with t-CBF-AML.

Drivers of Deep Molecular Response and Outcomes in Patients with Core Binding Factor Acute Myeloid Leukemia

Introduction: Core-binding factor acute myeloid leukemia (CBF-AML), including t (8;21) and Inv (16), constitutes 15% of all AML cases and generally carries a favorable prognosis. However, relapse rate is ≈ 30% and predictors are not well understood. Although CBF-AML responds to induction chemotherapy, post-remission treatment can be further optimized, particularly with current next generation sequencing (NGS) and measurable residual disease (MRD) detection techniques. Impact of baseline somatic mutations on outcomes remains unclear, as does role of allogeneic stem cell transplant (allo-SCT). In this study, we investigated the potential drivers of treatment response and overall outcomes in CBF-AML. Methods: In this single center retrospective study, clinical, demographic, and molecular data were collected from adult pts with CBF-AML who were treated at Moffitt Cancer Center. We included pts with MRD by polymerase chain reaction (PCR) for CBF abnormality (CBF-MRD) results available from time of induction and consolidation. All statistical analyses were performed in SPSS. Results: Our study included 68 patients (pts), including 26 (38%) t (8;21) AML. The median age at diagnosis was 48 years, and 54% pts were male. Pts with inv (16) AML had significantly more leukocytosis than t (8;21) (10x109/L vs. 23x109/L; p=0.001). Only 5% of pts (n=3) in our cohort had extramedullary disease on presentation. Common mutations identified on baseline NGS (n=60) were KIT (35%), NRAS (27%), KRAS (15%), FLT3-ITD and FLT3-TKD (12% each; 2 pts had both types). Mutation in KIT was more common in t (8;21) AML (50% vs. 25%; p=0.04), whereas inv (16) cases were enriched in NRAS mutations (36% vs. 13%; p=0.04). Overall, 80% of CBF-AML pts had at least one kinase mutation (KIT/RAS/FLT3). Majority of pts were treated with "7+3" (44%) and "7+3+ gemtuzumab ozagamicin (GO)" (40%) as induction regimens (Table 1). Daunorubicin was the predominant anthracycline, used in 80% of pts treated with "7+3" and 48% received dose of 90 mg/m2. Overall, 31 (46%) pts received GO as part of induction treatment, with equal distribution of "day 1 only" and "day 1,4,7" schedules at 3mg/m2. Median number of consolidation cycles was 3, and GO was incorporated in consolidation (commonly with high dose cytarabine) for 29 (43%) pts. After induction, 16% pts experienced CBF-MRD negativity which increased to 73% following consolidation, with no significant difference between t (8;21) and inv (16) AML. Use of GO during induction or consolidation was associated with significantly higher CBF-MRD negativity rate at completion of induction and consolidation (83% vs. 50%; p= 0.021). However, we did not find any significant correlation between groups based on presence of baseline kinase mutation/s and CBF-MRD negativity at end of induction or consolidation. In FLT3-positive cases, 100% of pts (5/5 evaluated;4 ITD and 1 TKD) achieved FLT3-MRD negativity by PCR (sensitivity of 5X10-5) following consolidation. Overall, 35% of pts (n=24) underwent allo-SCT in our cohort, including 15 in first remission (CR1). Kinase mutations were present at baseline in majority of these pts (75%; 87% in CR1 group). Notably, 9 pts (7 in CR1 group) in the allo-SCT cohort had positive CBF-MRD at the end of consolidation. Seven pre-transplant CBF-MRD positive cases turned MRD negative following allo-SCT. Relapse rate was low (n=10; 15%) in our CBF-AML pt population. Among pts with relapse, 6 were CBF-MRD positive post-induction and 3 had MRD even after consolidation. Use of GO in induction was associated with significantly lower relapse rate (0% vs. 28%; p= 0.001). Presence of kinase (KIT/RAS/FLT3) mutation at diagnosis was associated with shorter relapse free survival (RFS) in these pts (median RFS- 7.7 vs. 12.9 months (mo); p= 0.014; Figure 1). After a median follow up of 33.4 mo, median OS was 94.1 mo in our pt cohort. There was no significant OS difference between subgroups based on CBF-AML subtype, baseline mutations, or receipt of GO during treatment. Conclusions: CBF-AML continues to be a favorable risk disease, with a median OS >7 years in our study population. Post-treatment CBF-MRD negativity rate was 73% and was predicted by the incorporation of GO in induction or consolidation. Rate of relapse rate was low at 15%, and all occurred in pts who did not receive GO during induction or consolidation. Baseline kinase mutations (KIT/RAS/FLT3) on NGS was associated with a shorter RFS in our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prospective Evaluation of Sorafenib Combined with Chemotherapy in Newly Diagnosed Adult Core-Binding Factor Acute Myeloid Leukemia: An Open-Label, Randomized Controlled, Multicenter Phase II Trial

Objective Core-binding factor acute myeloid leukemia (CBF-AML) includes AML with t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) chromosomal rearrangements, leading to the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively. Although CBF-AML is considered to have a favorable prognosis relative to other AML subtypes, up to 40% of these patients experience relapse. Recent studies demonstrate achievement of complete molecular remission (CMR) at the end of treatment significantly associated with a reduced risk of relapse. A high frequency of mutations and/or high expression of KIT gene is observed in CBF-AML. Sorafenib is a first-generation type-II multitargeted tyrosine kinase receptor inhibitor (TKI) that suppresses various signaling pathways associated with the development of AML, such as RTK (FLT3, c-KIT). Thus the purpose of this study is to evaluate the safety and efficacy of sorafenib combined with chemotherapy in adult newly diagnosed CBF-AML. Methods Patients were randomly assigned (1:1) to control group (chemotherapy alone) or sorafenib group (sorafenib combined with chemotherapy). For control group, patients received one cycle of induction therapy with idarubicin (12 mg/m² on days 1-3) plus cytarabine (100 mg/m² on days 1-7), followed by one cycle of idarubicin (8 mg/m² on days 1-3) plus cytarabine (2 g/m² q12h on days 1-3) and two cycles of high-dose cytarabine consolidation therapy (2 g/m² q12h on days 1-3). For sorafenib group, patients were treated with chemotherapy plus sorafenib at 400 mg twice daily on days 8-21 for induction cycle, and on days 1-21 for each consolidation, and as maintenance for 12 months. Allogeneic or autologous HSCT were implemented based on the measurable residual disease (MRD) level and donor availability after 4 cycles of therapy. MRD levels were serially monitored for RUNX1-RUNX1T1 or CBFB-MYH11 transcripts by real-time quantitative polymerase chain reaction. Results were expressed as a [fusion gene/ABL1] ×100 transcript ratio. Major molecular response (MMR) and complete molecular remission (CMR) are defined as transcript ratio < 0.1% and 0.001%, respectively. The primary endpoint was CMR after 4 cycles of therapy in bone marrow (BM). Results Between January 2020 and March 2022, 64 patients were enrolled and randomly assigned to sorafenib group (n=32) or control group (n=32). There is no significant difference between the two groups about age, gender, CBF subtype, BM blast percentage, additional chromosome abnormalities and gene mutations at diagnosis. The hematological complete remission in the sorafenib and control group were 96.9% and 94.1%, respectively (p=.592). Following three cycles of therapy, MMR was observed in 79.3% of the sorafenib group and 46.9% of the control group (p =.009). Sorafenib group also had a significantly higher CMR than control group (62.1% vs 28.1%, p =.009). After four cycles of therapy, MMR increased to 100% in the sorafenib group and 70.8% in the control group (p =.019), whileCMR was also more frequently achieved in the sorafenib group than that in the control group (90.9% vs 54.2%, p =.006). There was no significant difference of grades 3-4 adverse events between the two groups. Grade 4 neutropenia (neutrophil count less than 0.5×10⁹ cells per L) and thrombocytopenia (platelet count less than 20×10⁹ per L) were observed in all cases. The median duration of neutropenia was 12d (range, 6-25d) in the sorafenib group and 10d (range, 5-22d) in the control group (p =.063) , while the median duration of thrombocytopenia was 12d (range, 5-33d) and 9d (range, 6-19d) (p =.052), respectively.The most common grade 3-4 non-hematological adverse events in sorafenib and control group were pneumonia (31.3% vs 57.4%, p=.019), mucositis (21.9% vs 24.1%, p =.816), blood stream infection (37.5% vs 31.5%, p =.568). Conclusion Sorafenib combined with chemotherapy significantly increased the CMR after 4 cycles of therapy, however, the incidence of haematological and non-haematological adverse events did not apparently increased. The impact of sorafenib combined with chemotherapy on relapse and survival of CBF-AML need further following-up. Keywords: Sorafenib, CBF-AML, CMR, adverse events

Characterization of Survival Outcomes and Clinical and Molecular Modulators in Adult Patients with Core-Binding Factor Acute Myeloid Leukemia (CBF-AML) Treated with Hidac Consolidation: An Alliance Legacy Study

Introduction CBF-AML is a specific disease entity defined by the presence of either t(8;21)(q22q22)RUNX1-RUNX1T1, or inv(16)(p13.1q22) or t(16;16)(p13.1;q22) creating the CBFB-MYH11 fusion gene, and is associated with a relatively favorable prognosis. Yet, outcomes are variable and relapse occurs in ~50% of patients (pts). Whereas several clinical and molecular features have been suggested to modulate survival, consensus data are sparse. Moreover, there is persistent controversy about the optimal dose intensity of cytarabine consolidation, which complicates interpretation of correlative studies with respect to outcome associations. Thus, we analyzed outcomes of a large cohort of CBF-AML pts treated with high-dose cytarabine (HiDAC) consolidation with respect to cytogenetic subtypes and co-existing molecular features. We analyzed clinical and molecular characteristics and outcomes of 304 pts with CBF-AML aged 18-59 years [y; inv(16)/t(16;16), n=186; t(8;21), n=118] who were similarly treated and achieved a complete remission upon standard cytarabine/anthracycline induction followed by HiDAC on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols between 1986-2016. All pts had centrally reviewed cytogenetics and samples from 136 pts underwent comprehensive molecular profiling of 80 AML-associated genes for association studies. Kaplan-Meier method was used to estimate probabilities of disease-free survival (DFS) and overall survival (OS), and the log-rank test evaluated differences between survival distributions. Cox proportional hazard regression analyses was used for DFS and OS univariable and multivariable modeling. Results Thirty-eight percent of pts relapsed and 69% were alive 5y after diagnosis (median OS): 12.5y with median follow-up of 8.6y. Outcomes of t(8;21) and inv(16)/t(16;16) AML were similar; relapse rates: 35% vs 40%, 5y OS rates, 64% vs 72%, respectively. Although outcome of pts with inv(16) was not significantly different from outcome of t(16;16) pts, the latter had, by trend, a superior DFS (5y rates, 75% vs 47%, P=0.07); their OS was not significantly different (5y rates, 81% vs 64%, P=0.24). As secondary cytogenetic abnormalities are common and impact on survival has yielded controversial results, we next analyzed the impact on outcomes of our pt cohort. None of the frequently observed secondary abnormalities [i.e., loss of X or Y chromosomes, +8, or del(9q) in t(8;21) pts and +22 or +8 in inv(16)/t(16;16) pts] affected the outcomes of CBF-AML pts we analyzed, which contrasts with some previous reports. The presence of a complex karyotype also did not negatively affect the favorable survival of CBF-AML pts. Next, we assessed the impact of recurrent gene mutations on pt outcomes. While KIT mutations have previously been shown to confer inferior survival, our data suggest that presence of a KIT mutation negatively affects DFS of both t(8;21) and inv(16)/t(16;16) AML pts [5y rates, t(8;21) 36% vs 67%, P= 0.03; inv(16)/t(16;16), 21% vs 61%, P<0.001], and also OS in inv(16)/t(16;16) pts (5y rates, 52% vs 74%, P=0.04). Co-existing FLT3-TKD mutations were almost exclusively found in inv(16)/t(16;16) in 16/18 cases. Though a negative prognostic impact of FLT3-TKD has been suggested, this was not found in our cohort (5y rates, DFS, 56% vs 55%, P=0.93; OS, 78% vs 68%, P=0.69). Furthermore, neither the presence of NRAS or KRAS mutations affected survival outcomes. In multivariable analyses for OS, a higher percentage of peripheral blood blasts was the only feature associated with inferior survival [HR, 1.14 (95% CI, 1.04-1.25), P=0.004]. Conclusions Our analysis of a large cohort of younger CBF-AML pts treated on frontline protocols with 7+3-based induction therapy and HiDAC consolidation, showed though these pts are considered favorable risk, they demonstrate varying long-term survival. Clinically, the presence of high blast count at time of diagnosis, was a strong adverse prognosticator for inferior survival. With respect to recurrent co-occurring cytogenetic aberrations or gene mutations, only KIT mutations associated with inferior survival in pts with t(8;21) and in those with inv(16)/t(16;16). Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov: NCT00048958(CALGB 8461), NCT00899223(CALGB 9665), NCT00900224(CALGB 20202) *co-senior authors

Single Institution Observational Study of Risk-Adapted Therapy for Children with Newly Diagnosed Core-Binding Factor Acute Myeloid Leukemia

Background: Pediatric acute myeloid leukemia (AML) with core-binding factor (CBF) rearrangements (RUNX1-RUNX1T1/CBFβ-MYH11) have favorable outcomes. Therefore, hematopoietic stem cell transplantation (HSCT) is usually not offered to these patients during their first remission (CR1). However, more than one third of patients experienced relapse. Objective: To explore whether HSCT in CR1 for pediatric CBF-AML would benefit this subgroup. Design and Methods: We describe the outcomes of 68 cases with RUNX1-RUNX1T1 and 21 with the CBFβ-MYH11 consecutively treated in a single institution between 2012 and 2018. Practice patterns in pediatric CBF-AML treatment varied according to the presumed risk of relapse or treatment abandonment. Remission induction consisted of two courses of a low-intensity or standard-intensity regimen. Post-remission treatment consisted of two or three courses of standard AML therapy. Hematopoietic stem cell transplantation (HSCT) was offered in first complete remission (CR1) for selected patients. Results: Eighty-nine patients received two courses of a low-intensity or standard-intensity regimen for remission induction and two or three courses of standard AML therapy for post-remission. C-KIT mutations were present in 49.4% of the cases. Eight-three of 87 (95.4%) children attained CR, and two patients were not evaluable. Twenty-nine patients at high risk of relapse underwent HSCT in CR1. The 5-year event-free survival (EFS), overall survival (OS), and relapse rates were 74.8%, 85.0%, and 13.9%, respectively. There was a significant difference in 5-year OS (100% vs. 81.5%, P = 0.016), EFS (96.4% vs. 67.1%, P = 0.003), and relapse-free survival (RFS) (96.4% vs. 68.8%, P = 0.005) between patients who underwent HSCT in CR1 and those treated with chemotherapy alone. Conclusion: Our data suggest a potential benefit of HSCT for selected patients with CBF-AML and reveal a need to improve risk stratification for pediatric CBF-AML.

The Adverse Effect of Myelodysplasia-Related Mutations in De Novo Acute Myeloid Leukemia Is Associated with Higher Variant Allelic Frequency (VAF): A Proposal for a Numeric Cutoff for Variant Allelic Frequency

Mutations in myelodysplasia-related genes ASXL1, BCOR, EHZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 (MRmut) are class-defining alterations in acute myeloid leukemia (AML) in the latest World Health Organization (WHO, with the exception of RUNX1) and International Consensus Classification (ICC) schemes and indicate adverse-risk per European Leukemia Net (ELN) 2022 guidelines, unless occurring in favorable-risk AML. The lower limit of detection (LOD) for most clinical-grade next generation sequencing (NGS) is ~1-2% variant allelic frequency (VAF); however, data on the prognostic significance of MRmut relative to VAF is lacking. No guidelines that provide a numeric cutoff for VAF are available for utilizing these mutations to classify AML. Our aim was to define a clinically meaningful VAF cutoff for MR mutations in de novo AML. We included patients (pts) with de novo, untreated AML with MRmut treated at our institution between 2017 to 2022. We excluded pts with acute promyelocytic leukemia, core binding factor AML, and other ELN adverse-risk categories (adverse karyotype and/or TP53 mutation). NGS at baseline was performed using a panel of 81 genes recurrently mutated in myeloid neoplasms (LOD ~1-2%). We performed a best fit classifier on individual MRmut to determine optimal VAF cutoffs using event-free survival (EFS) and overall survival (OS) as endpoints. VAF for MRmut involving X-linked genes was adjusted for men. Pts (n=145) included 92 (63%) men and 53 (37%) women with a median age of 68 years (range, 58-75). 85 (59%) pts had diploid karyotype AML, 51 (35%) had other intermediate-risk non-diploid karyotype AML, and 9(6%) had insufficient metaphases for analysis. 34 (23%) pts had ELN favorable-risk and 111(77%) had ENL adverse-risk AML. Pts were treated with low-intensity+venetoclax (VEN) (72/50%), low-intensity (26/18%), high-intensity (22/15%), and high-intensity+VEN (12/17%) regimens. Median follow-up was 26.9 months (range, 13.9 - 40.8). 72 (49%) pts had 1 MRmut; the remaining had 2-4 concurrent MRmut. MRmut had the following frequencies and VAFs: ASXL1 (28%; median VAF: 26%, [range 2-48%]); BCOR (18%; median VAF 37% [3-100%]); EHZH2 (9%; median VAF 7% [1-55%]), RUNX1 (32%; median VAF 47% [1-96%]), SF3B1 (9%; median VAF: 38% [1-55%]), SRSF2 (46%; median VAF: 37% [1-76%]), STAG2 (20%; median VAF: 44% [15-98%]), U2AF1 (12%; median VAF: 33% [2-43%]) and ZRSR2 (2%; median VAF: 94% [93-95%]). 34 (23%) pts had ELN favorable-risk AML including 28 (23%) with mutated NPM1 without FLT3-ITD and 6 (4%) with CEBPA bZIP mutations, respectively; in 12/34 pts NPM1 or CEBPA bZIP mutations were subclonal compared with clonal MR mutation(s). A summary of baseline genetic and treatment data is provided in Fig 1A. The overall composite remission rate (CR+CRi) was 88% (64% and 24%, respectively) Median OS was 30.3 months and estimated 12/24 month OS were 77%/60%. The median EFS was 24.6 months and estimated 12/24 month EFS were 65%/51%. Pts with ELN favorable-risk AML with MRmut had longer OS and EFS compared with the adverse-risk group (median OS 34.9 vs 27.5 months, median EFS 34.9 vs 22.9 months) but the difference was not significant (p=0.60 and 0.30, respectively). The VAF cutoff for MRmut associated with EFS and OS, respectively, was: ASXL1: 21%/21%, BCOR 25%/25% , EHZH2 15%/15%, RUNX1 25%/27%, SF3B1 31%/31%, SRSF2 32%/35%, STAG2 40%/21%, U2AF1 44%/44% and ZRSR2 47/47%.There was no significant difference in OS or EFS between pts with 1 MRmut vs those involving ≥2 MR genes. Pts treated with high-intensity, high intensity+ VEN and low intensity+VEN regimens had longer OS (Fig 1B) and EFS compared with pts treated with low intensity regimens without VEN (OS: not reached, 31.7 months, 31.4 months vs 8.9 months, respectively, p<0.001; EFS: not reached, 31.7 months, 29.6 months vs 3.7 months, respectively, p<0.001). Our data suggest that the poor prognostic effect of MRmut in de novo AML is associated with higher VAF and not a binary presence or absence of MRmut. The minimum VAFs associated with worse outcome for each MRmut ranged from 15-47%. Additional analysis to create predictive models is in progress to determine the optimal VAF cutoff when using MRmut to assign AML to the WHO/ICC MR categories and to ELN adverse-risk group. Importantly, our results suggest that pts with MRmut and non-adverse karyotype have significantly better outcomes when treated with high-intensity and/or venetoclax-based treatment regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Factors in Children with Acute Myeloid Leukemia Receiving the First Hematopoietic Stem Cell Transplantation in Second Remission

Introduction In children with acute myeloid leukemia (AML), hematopoietic stem cell transplantation (HSCT) during the first complete remission (CR1) is generally restricted to patients with a high risk of relapse. However, 30-40% of children with AML receiving chemotherapy will eventually relapse; these patients often undergo initial HSCT after achieving second CR (CR2) with re-induction chemotherapy. Previous studies have reported favorable prognostic factors in children with relapsed AML: no prior history of HSCT, longer duration of CR1, and achieving CR2 before HSCT. The identification of prognostic factors in children who received initial HSCT in CR2 may facilitate the refinement of risk stratification in first-line therapy, but they remain largely unknown. In this study, we explored the prognostic factors of pediatric patients with AML who received initial HSCT in CR2. Methods Data from 234 patients were collected from the Transplant Registry Unified Management Program sponsored by the Japanese Society for Transplantation and Cellular Therapy and the Japanese Data Center for Hematopoietic Cell Transplantation. The patients were selected according to the following criteria: (1) de novo AML, (2) age < 16 years and CR2 at the time of receiving HSCT, (3) no prior HSCT, and (4) HSCT performed between 2000 and 2019. Patients without information on survival and disease recurrence or those with Down syndrome (DS) were excluded. A myeloablative conditioning (MAC) regimen was defined as total-body irradiation (TBI) at > 8 Gy, melphalan > 140 mg/m2, or busulfan ≥ 9 mg/kg. All other regimens were analyzed as reduced-intensity conditioning regimens. High-risk (HR) cytogenetics/genetics were defined as either 7-/7q-, 5q-, complex karyotype (three or more chromosomal abnormalities in the absence of one of the recurring translocations or inversions), t(6;11)(q27;q23), t(6;9)(p23;q34), t(16;21)(p11;q22), t(9:22)(q34;q11.2), or fms-like tyrosine kinase receptor 3 internal tandem duplication (FLT3-ITD). Results The median age at HSCT was 7 years (range, 1-15), and the median follow-up period for survivors was 7.7 years (range, 0.2-19.2). The 5-year event-free survival (5y-EFS) and 5-year overall survival (5y-OS) rates were 63.5% (95% CI, 56.6-69.5%) and 66.9% (95% CI, 60.0-72.8%), respectively (Figure 1). The 5-year cumulative incidence of relapse was 24.7% (95% CI, 19.2-30.6%), whereas the 5-year cumulative incidence of non-relapse mortality was 11.8% (95% CI, 7.9-16.6%). The 5y-EFS was 72.6% (95% CI, 61.1-81.2%) for patients with core binding factor AML and 100% (95% CI unavailable owing to small patient number) for patients with t(15;17)(q22;q21). Patients with 11q23/KMT2A rearrangement and HR cytogenetics/genetics showed worse outcomes, with 5y-EFS rates of only 43.8% (95% CI, 21.4-64.3%) and 36.4% (95% CI, 17.8-55.3%), respectively. Among the French American British classification subtypes, patients with M7 AML showed inferior outcomes with a 5y-EFS of 18.8% (95% CI, 4.6-40.2%). For the conditioning regimens, aside from "other MAC,” which included only three patients, melphalan-based non-TBI MAC was associated with the most favorable outcomes, with a 5y-EFS of 77.1% (95% CI, 64.2-85.9%). Multivariate analysis identified duration of CR1 > 1 year and melphalan-based non-TBI MAC regimen as independent favorable prognostic factors, while M7 was identified as an independent adverse prognostic factor (hazard ratio 2.62, p = 0.033, Table 1). Conclusion In the present study, approximately two-thirds of children who experienced relapse after chemotherapy remained in remission after HSCT in CR2. Melphalan-based non-TBI MAC is recommended as a conditioning regimen for this select population. Patients with t(15;17)(q22;q21) showed particularly excellent outcomes after initial HSCT in CR2, whereas those with HR cytogenetics/genetics or non-DS M7 were scarcely rescued with HSCT even after attaining CR2. Precise identification of high-risk populations among them with poor early treatment response and/or unfavorable genetic alterations will contribute to refining the risk stratification and indications for HSCT in CR1. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gemtuzumab Ozogamicin Plus Standard Induction Chemotherapy Improves Outcomes in Intermediate Risk Cytogenetic Acute Myeloid Leukemia

Background: Gemtuzumab ozogamicin (GO) is a CD33-directed antibody-drug conjugate approved for the treatment of newly diagnosed (ND) and refractory/relapsed CD33-positive acute myeloid leukemia (AML). Meta-analysis of individual patient (pt) data from 5 randomized controlled trials (n=3325) confirmed a significant survival benefit of GO added to intensive induction chemotherapy for pts with AML without adverse cytogenetics. While favorable-risk ND-AML pts, specifically core-binding factor AML (CBF-AML), particularly benefited with a 5-year overall survival (OS) improvement of 20.7% (log rank p= 0.0006), pts with intermediate-risk AML also showed a significant 5-yr survival improvement of 5.7% (p=0.005) (Hills RK et al Lancet Oncol 2014: 15, 986). However, the smaller difference in these latter pts coupled with apprehension for risk of sinusoidal obstruction syndrome (SOS) following hematopoietic stem cell transplantation (HSCT) has led many clinicians to not use 7+3 plus GO in the intermediate-risk AML population. Aim/Objective: To perform a retrospective analysis of outcomes of 7+3 plus GO vs 7+3 in ND intermediate-risk cytogenetic AML pts treated at a single comprehensive cancer center. Methods: We studied the demographic, clinical and genetic data of intermediate risk ND-AML pts treated at Roswell Park Comprehensive Cancer Center (n=113) between 2015-2022. Conventional cytogenetics and fluorescence in situ hybridization (FISH) were performed. Genomic profiling was performed using next-generation sequencing methods (FoundationOne®CDx). Risk stratification was defined per the European LeukemiaNet (ELN)-2017 classification. 7+3 plus GO pts received at least one dose of GO 3 mg/m2 (up to one 4.5 mg vial) plus cytarabine 100 mg/m² continuous IV infusion on days 1-7, and daunorubicin 60 mg/m² IV on days 1-3 (7+3). Control pts received 7+3 alone. Prophylactic heparin was administered to prevent hepatic SOS based on institutional standards if not thrombocytopenic. Outcomes included response rate to induction, successful bridging to HSCT, relapse rate, and overall survival. Results: 113 intermediate-risk ND-AML pts were studied: 33 received 7+3 plus GO and 80 received 7+3. Median age was 57 vs. 59 years, respectively (range: 19-76 years). Moreover, there was no significant difference in the percentage of pts > 60 yrs (45% vs 48%). Gender was equally distributed. Pts treated with 7+3 plus GO had a significantly higher rate (p=0.019) of complete remission (CR) and complete remission with incomplete count recovery (CRi) (27/33, 82%) than 7+3 (44/80, 55%; Figure 1A). Among pts with available minimal residual disease (MRD) status detected by multiparameter flow cytometry, 100% achieved MRD-negative status after 7+3 plus GO (18/18) compared to 86% with 7+3 (12/14). Non-responders to 7+3 plus GO were enriched for ASXL1, DNMT3A, TET2, and TP53 mutations as compared to responders who harbored more FLT3, IDH1/2, KRAS/NRAS and RUNX1 mutations. Among pts achieving CR/CRi, 48% after 7+3 plus GO vs 30% after 7+3 (p=0.13) were successfully bridged to HSCT. Cumulative incidence of relapse after CR/CRi was significantly lower after 7+3 plus GO (11%) than 7+3 (43%; p=0.007). Finally, 7+3 plus GO pts demonstrated a superior, yet not statistically significant, median overall survival (OS) benefit (median OS not reached) compared to 7+3 alone (median OS = 35 months; Figure 1B). No pts receiving 7+3 plus GO developed SOS. Conclusions: Intermediate risk ND-AML pts treated with 7+3 plus GO at our academic center had significantly higher rates of MRD-negative CR and CRi, lower rates of relapse, and higher percentage of pts receiving HSCT as compared with 7+3 alone. Non-responders in the 7+3 plus GO group harbored adverse risk genetic mutations that likely dictated the failure to response. Although addition of GO was associated with a non-significant trend towards improved overall survival, longer follow-up is warranted to decipher an absolute survival advantage of this regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Functional Implications of MYC Variants As a New Class of Pathogenic Variants in AML

MYC, a well-known oncogene, is implicated in a majority of cancers, but there is little data on its role in myeloid malignancies. We recently described a novel internal tandem duplication of MYC gene (MYC-ITD) in a subset of patients with AML. As the prevalence of MYC aberrations have not been well documented in AML, we interrogated the genome, transcriptome and associated clinical and laboratory data from the largest cohort of pediatric AML patients to define the prevalence, frequency of co-occurring cytomolecular alterations and outcomes associated with MYC variants. Mutation Profiling: Diagnostic specimens from 1,806 patients with AML (age 0-29 years) enrolled on COG Phase III trials were retrospectively interrogated using contemporary NGS techniques or a PCR based "Tandem Repeat" assay for MYC-ITD. Conventional karyotyping and mutational analysis, including NGS, were used to determine additional cytogenetic and molecular abnormalities. Of the 1,806 patients studied, MYC aberrations were identified in 58 patients (3.2%) with 50% (N=29) harboring MYC-ITD, 46.5% (N=27) with a SNV or indel (MYCSNV/indel) and 2 patients (3.5%) with dual MYC-ITD and SNV. For MYC-ITD, tandem duplications occurred in exon 3 (Max binding domain) with ITD lengths ranging from 252-588 bp. The majority of MYCSNV/indel (77%) were identified at the P72-P78 hotspot with 33.3% (N=9) harboring the P74>L mutation and 18.5% (N=5) having the T73>N mutation. Functional Studies: Given the novelty of the ITD variant, we initially characterized the functional properties of MYC-ITD in vitro. Similar to wild-type (MYCWT), MYC-ITD is localized to the nucleus and requires Max to bind to specific DNA sequences (E-boxes). However, in contrast to MYCWT, MYC-ITD fails to recognize sequences containing a single E-box in vitro, but exhibits higher affinity toward sequences with two or more E-boxes. MYC-ITD is consistently more efficient in activating promoters with multiple E-boxes than MYCWT. Thus, MYC-ITD may possess different DNA-binding and protein-interacting properties, potentially leading to altered transcriptional regulation and biological output. Given the alterations in DNA binding properties, we evaluated the functional consequences of MYC-ITD in our cord blood stem cell/endothelial cell (EC) co-culture system. Normal cord blood stem cells were transduced with MYC-ITD vs. MYCWT and grown in an EC co-culture system. MYC-ITD transduced cord blood stem cells showed significantly higher proliferation compared to MYCWT or control GFP-transduced cells with proliferation advantage of 37-and 45-fold after 14 and 21 days in co-culture, respectively (p<0.001). Clinical Impact: Analysis of cooperating cytomolecular lesions revealed a striking difference between MYC-ITD and MYCSNV/indel. MYC-ITD was highly enriched in core-binding factor (CBF) AML found in 86% (N=25)[(N=18, 62%) with inv(16) and (N=7, 24%) with t(8;21)] (Figure 1A), vs. 26% in the MYCWT cohort and had a paucity of somatic mutations. Conversely, MYCSNV/indel was enriched in NUP98-NSD1 fusions (N=7, 27%), FLT3-ITD (N=10, 38%) and NPM1 mutations (N=9, 33%) (Figure 1A), which was significantly higher than the WT cohort. Patients with MYC-ITD had a morphologic CR rate after course 1 of 92.3% vs. 80% (p=0.249) in patients with MYCSNV/indel with a corresponding relapse rate of 24% and 52% (p=0.046). Lower CR and higher relapse reflects the higher proportion of patients with high-risk lesions (FLT3-ITD, NUP98-NSD1 and WT1) in MYCSNV/indel and favorable CBF in MYCITD. Those with MYC-ITD and MYCSNV/indel had a 5-year event free survival (EFS) of 70% vs. 38.5%, respectively (p=0.018) with a corresponding overall survival (OS) of 76% and 47% (p=0.023) similar to patients with favorable or high risk disease (Figure 1B). Conclusion: Here we define MYC aberrations as a new class of pathogenic variants in childhood AML. We demonstrate functional and clinical characterization of a novel MYC-ITD and contrast this novel variant to that of established pathogenic MYCSNV/indel. We show the high prevalence of co-occurring core-binding factor fusions with MYC-ITD, and high frequency of co-occurring NUP98-NSD1, FLT3-ITD and NPM1 mutations in MYCSNV/indel. Patients with MYC-ITD have superior clinical outcomes while patients with MYCSNV/indel have clinical outcomes similar to high-risk patients, likely due to the associations with the disease associated fusions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P62SQSTM1 Affects Interacting Substrates MDM2, MDMX and Casein Kinase 1alpha to Yield Protection of Beta-Catenin Along with p53 Silencing for Refractory AML

Interventions targeting individual driver mutations in AML have not overcome resistant disease. Missing has been definition of a signal hub that integrates multiple oncogenic drivers for a composite pathway to resistance, where hub abundance determines signal(s) strength. We hypothesized that AML signal hub is p62SQSTM1, a nonmutant-active signal scaffolding for multiple pathways, including NFkB and MAPK (Moscat, Cell. 2016). Genetic epistasis in AML cell lines validated origin from P62 of such signals. Oxidative stress resistance pathways (OXPHOS) driven by p62SQSTM1 may also join with pathway(s) for leukemic progenitor replication, involving HOXA and beta-catenin within resistant AML's, but this has not been established. We sought merging pathways to OXPHOS-mediated resistance also serving to guard replicative potential through HOXA and beta-catenin. AMLs from patients were tested prior to treatment, and later failure, of induction chemotherapy, when given along with targeted agent (for cases with Flt3 mutation). Resistant AML blasts were found to have tandem overexpression of HOXAs and OXPHOS effectors p62SQSTM1, NRF2, NQO1 (NADPH oxidoreductase), BCL-2 ranging from 2-5-fold log10 relative quantity (RQ-Taqman) above chemo-sensitive core-binding factor AML, which lacks HOXA expression. We hypothesized that disrupting the downstream pathway from p62SQSTM1 to OXPHOS ending on MDM2 and/or MDMX (Todoric, Cancer Cell, 2017) is an approach to affect broad cell extinction. Indeed, besides destabilization/destruction of wild-type p53 affected by MDM2, the paralogue MDMX exists in an induced tripartite complex with CK1alpha (CK1) and p53, whose activity for restraining p53 and protecting beta-catenin from phosphorylation/inactivation is disrupted upon stress-induced CK1 activation (Huang, EMBOJ, 2020; Ueda, Cancer Cell, 2021), which we affected by using a proteasome inhibitor (PI). We found by immunoblotting AML blast lysates following PI treatment in vitro with Ixazomib, CK1 translocates to the nucleus and phosphorylation (S33,S37,T41,S45)/inactivation and cytoplasmic export of beta-catenin ensued, accompanying cell apoptosis, thus implicating MDMX inactivation resulting from PI. Primary p53-wild-type AML blasts or isogenic cell lines were treated in vitro with graded doses of the MDM2 inhibitors Pevonedistat (PEVO) or Siremadlin, either added alone, or in combination with Ixazomib at pharmacokinetically-achievable dose ranges typical in patients. Among high-risk primary AML blasts with Flt3ITD alone, or ITD along with NUP98-NSD1 or mDNMT3A, or Flt3neg with mutant EP300, or with PRDM16-SKI fusion as drivers, synergy/cooperativity was observed between MDM2 inhibitor and PI Ixazomib for inhibition of 3H-thymidine incorporation and visible apoptosis in vitro. Full characterization within PRDM16-SKI blasts by immunoblotting demonstrated expected posttranslational modification (PTM) on beta-catenin by PI, whereas distinct PTM for beta-catenin followed PEVO application, and the combination demonstrated further heterogeneity. Treatment of these blasts with Ixazomib or the combination with PEVO also led to induction of p53 in tandem with the destruction of MDM2/MDMX PTM isoforms and nuclear translocation of CK1. Strong upregulation/nuclear translocation of NRF2 resulted as part of the apoptotic pathway. To further discriminate the requirement for, and the nature of PTMs in the functional outcomes, FLAG-tagged (S33-A, S37-A,T41-A) mutant beta-catenin isoform, also lacking lysine ubiquitin target residues, was transfected in a lentiviral vector into p53-mutant K562 cells as host, and cells were compared to the same cells transfected with non-mutant FLAG-tagged beta-catenin. PEVO/Ixazomib did not induce PTM in mutant FLAG-beta-catenin transfectants, but the expected PTMs occurred in wild-type FLAG-tagged beta-catenin transfectants. In this context, PEVO became a stimulator of cell proliferation of the cells bearing mutant PTM-resistant beta-catenin, thus implicating beta-catenin disruption involving these residues as a requisite for optimal AML cell inhibition with the combination. In addition, a combination of PEVO/Ixazomib was as effective for apoptosis as was Flt3 inhibitor among certain Flt3mutant primary AML blasts. A clinical test of this strategy is warranted in high-risk relapsed/refractory p53WT/p62SQSTM1-expressing AML.

Identification and in silico analysis of noval alteration Arg420Gly in KIT proto oncogene among acute myeloid leukemia patients

A number of studies have reported frequent incidence of c-kit gene mutations in association with core binding factor acute myeloid leukemia (CBF-AML). These genetic changes have become important prognostic predictors in patients with abnormal karyotype. Aim of this study was the detection of nucleotide alterations in newly diagnosed acute myeloid leukemia patients for three exons of c-kit gene, including cytogenetically normal patients. Thirty-one de novo AML patients were screened for any possible variations in exon 8, 11 and 17 sequences of c-kit proto-oncogene leading to amino acid substitutions or frame shift. Sanger sequencing method was employed followed by sequence analysis. Mutation data was then correlated with clinical and hematological parameters of patients and prognostic significance of genetic changes was assessed as well. The computational tools were then used to further understand the extent of damage caused by these mutations to c-kit protein. Fifteen (48.4%) mutant patients were observed with single, double or multiple mutations in one, two or all three exons studied. The analysis revealed eight new alterations which were not reported previously. Significant variation among mutant and non-mutant group of patients was observed with respect to FAB subtypes (x2 = 12.524, p = 0.029), Spleen size (x2 = 4.288, p = 0.038) and Red blood cell count (x2 = 8.447, p = 0.007). The survival analysis indicates poor overall and event free survival outcomes in mutant individuals. Furthermore, the in silico analysis suggests that changes in nucleotide sequences can possibly damage the protein structure and effect it’s function. This study emphasizes the need to consider screening of c-kit gene alterations not only in CBF-AML but in cytogenetically normal AML patients as well. In current investigation the effect of mutation Arg420Gly on structure and function of c-kit protein was investigated, as this was the most observed substitution in present cohort. Various bioinformatics tools and techniques were employed, which determined that Arg420Gly is possibly non-pathogenic mutation.

AML-488 Impact of Somatic Mutations on Treatment Response and Outcomes in Patients With Core Binding Factor Acute Myeloid Leukemia

Prognostic significance of somatic mutations remains unclear in patients with core binding factor acute myeloid leukemia (CBF-AML), especially with advanced next generation sequencing (NGS) and measurable residual disease (MRD) detection techniques. To estimate the distribution of mutations and to assess their impact on outcomes in CBF-AML. Single-center retrospective study Patients: Adults with newly diagnosed CBF-AML Main Outcome Measures: MRD negativity, relapse free survival, overall survival Results: Our study includes 59 patients (mean age- 48 years) with CBF-AML [t(8;21), n=21 and Inv(16), n=38], with available NGS and CBF-MRD results during primary treatment. Most common mutations at diagnosis included KIT (31%), NRAS (26%), KRAS (15%), FLT3-TKD (11%), and FLT3-ITD (9%). Mutations in NRAS were enriched (32%) in Inv(16) AML, whereas KIT mutations were common (45%) in association with t(8;21). No pathogenic mutation was noted in 17% of patients. Most common induction regimen was "7+3" (48%), followed by "7+3+GO" (37%), with 36% receiving gemtuzumab ozogamicin (GO) in consolidation (median- 3 cycles). CBF-MRD negativity was 15% at the end of induction, which increased to 70% after consolidation, with no significant difference between patients with or without common kinase mutations (KIT, RAS, or FLT3). In the t(8;21) AML, CBF-MRD negativity rate was lower in KIT-mutant vs. wild type (38% vs. 75%) patients, although it was not statistically significant (p = 0.31). Overall, 23 (39%; 14 on CR1) patients underwent allogeneic stem cell transplant (allo-SCT), with no difference in respect to baseline mutations. The rate of relapse (n = 10; 17%) was also similar with or without ≥ 1 of KIT, RAS, or FLT3 mutations. The presence of any of these 3 mutations was associated with worse relapse free survival (7.7 vs. 12.9 months; p= 0.01), with no significant difference in overall survival (median OS- 94.1 months vs. not reached, p=0.44). Overall, patients with CBF-AML did well independent of induction/consolidation regimen, inclusion of GO, achievement of MRD negativity, or allo-SCT status, with a 2-year OS of > 80%. Mutations in KIT, RAS, and FLT3 are enriched in CBF-AML and contribute to earlier relapse although OS remains favorable across molecular and treatment subgroups.