Core Binding Factor Leukemia Research Articles

Molecular relapse after first-line intensive therapy in patients with CBF or NPM1-mutated acute myeloid leukemia – a FILO study

Patients with Core-Binding Factor (CBF) and NPM1-mutated acute myeloid leukemia (AML) can be monitored by quantitative PCR after having achieved first complete remission (CR) to detect morphologic relapse and drive preemptive therapy. How to best manage these patients is unknown. We retrospectively analyzed 303 patients with CBF and NPM1-mutated AML, aged 18–60 years, without allogeneic hematopoietic cell transplantation (HCT) in first CR, with molecular monitoring after first-line intensive therapy. Among these patients, 153 (51%) never relapsed, 95 (31%) had molecular relapse (53 received preemptive therapy and 42 progressed to morphologic relapse at salvage therapy), and 55 (18%) had upfront morphologic relapse. Patients who received preemptive therapy had higher OS than those who received salvage therapy after having progressed from molecular to morphologic relapse and those with upfront morphologic relapse (three-year OS: 78% vs. 51% vs. 51%, respectively, P = 0.01). Preemptive therapy included upfront allogeneic HCT (n = 19), intensive chemotherapy (n = 21), and non-intensive therapy (n = 13; three-year OS: 92% vs. 79% vs. 58%, respectively, P = 0.09). Although not definitive due to the non-randomized allocation of patients to different treatment strategies at relapse, our study suggests that molecular monitoring should be considered during follow-up to start preemptive therapy before overt morphologic relapse.

Intensification of Therapy and Pharmacogenetic Personalization Mitigate Racial Disparities in Pediatric Acute Myeloid Leukemia Outcomes

Introduction Recent reports have highlighted disparities in outcomes for black children, adolescents, and young adults with acute myeloid leukemia (AML) compared to their non-Hispanic white counterparts. An analysis of pediatric AML patients treated from 1996-2013 revealed that black race was associated with inferior event-free survival (EFS) and overall survival (OS) (Conneely 2021). In a study of adolescent and young adult patients with AML, black race was a predictor of poor outcome, particularly among patients 18-29 years old (Larkin 2022). Addressing these outcome disparities and developing treatment strategies to overcome them is crucial. In this study, we evaluated the interactions between outcomes by race and cytarabine pharmacogenomics in pediatric AML patients treated on two multi-institutional clinical trials. Methods Pediatric patients with newly diagnosed AML treated on AML02 (NCT00136084) or AML08 (NCT00703820) were included. Patients received high-dose cytarabine, daunorubicin, and etoposide (HDAC), low-dose cytarabine, daunorubicin, and etoposide (LDAC), or clofarabine and cytarabine (Clo/AraC) as initial therapy. The pharmacogenomics ACS10 scores, derived from combination of 10 single nucleotide polymorphisms, were calculated as reported (Elsayed 2022). Results Overall, 86 patients were black and 359 were white; 68 had other or unknown racial backgrounds and were not included in this analysis. Complete remission rates after two courses of induction therapy did not differ between black and white patients (92.6% vs 95.0%, p=0.63), nor did the rates of MRD negativity after one course of therapy (55.8% vs 55.4%, p=0.85). The EFS rates of black and white patients were nearly identical, with 5-year estimates of 58.3% (95% CI = 48.8%-69.8%) and 58.2% (95% CI = 53.2%-63.6%; p=0.89, Fig 1). Likewise, OS did not differ between black and white patients (63.8%; 95% CI = 54.3%-74.8% vs 69.4%; 95% CI = 64.7%-74.5%; p=0.24). The cumulative incidence (CI) of relapse did not differ by race (26.0% vs 26.1%, p=0.99), nor did the CI of treatment-related mortality (7.0% vs 6.8%, p=0.78). The prevalence of core binding factor (CBF) leukemia was higher in black patients as compared with white patients (30% vs 19%, p=0.04), but we did not find differences in EFS between black and white patients with or without CBF AML. We recently showed that low ACS10 scores were associated with worse outcomes in patients treated with low-dose cytarabine-based induction. In the present study, there was a significant difference in the distribution of ACS10 scores according to race, with low scores occurring in 73% of black patients compared with 30% of white patients (p<0.001). When patients on all treatment regimens were considered, there were no differences in EFS between black and white patients with low (p=0.88) or with high ACS10 scores (p=0.72). However, black patients with low ACS10 scores had significantly better outcomes after treatment with Clo/Ara-C induction compared with LDAC induction (p=0.01 for EFS and p=0.04 for OS, Fig 2). In multivariable model adjusting for age, risk group, and leukocyte count, Clo/Ara-C induction was identified as the best treatment option for black patients with low ACS10 scores (EFS: HR = 0.2, p=0.03). Discussion Our findings contrast with recent reports, as we did not observe significant outcome differences between black and white patients in terms of complete remission rates, EFS, OS, CI of relapse, or CI of treatment-related mortality. The higher prevalence of low ACS10 scores in black patients compared with white patients likely contributes to outcome disparities observed among patients treated with LDAC in the present study and those in the literature, which have included patients who almost exclusively received low-dose cytarabine-based induction therapy. In the present study, black patients with low ACS10 scores had significantly worse outcomes compared to those with high scores when treated with LDAC. However, these differences in outcome were eliminated by the use of HDAC or Clo/Ara-C induction. By contrast, black patients with high ACS10 scores did not appear to benefit from HDAC or Clo/Ara-C induction. Our results suggest that pharmacogenomics differences between black and white patients should be considered when tailoring induction regimens to improve outcomes of black patients and bridge the racial disparity gap in AML treatment.

Multiparametric Flow Cytometry-MRD Assay: Lesson from Phase II Trail REL AML 001

Background: Core-binding factor (CBF) AML with RUNX1/RUNX1T1 (AML1/ETO) and CBFB/MYH11 fusion genes is known as a heterogenous group, accounting for about 10% to 15% of AML. In the Phase II Trial REL AML 001 (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345) adult CBF leukemia patients treated with a continuation therapy with Midostaurin were included, and the measurable residual disease (MRD) was performed by molecular MRD assessment (qPCR) and multiparametric flow cytometric-MRD (MCF-MRD) assay. The findings of MCF-MRD assay were compared to qPCR. Methods: 31 patients with AML1/ETO (n=15) and CBFB/MYH11 (n=16) were evaluated between December 2018 to July 2022 with both techniques. A total of 145 determinations (n. AML1/ETO = 56 and n. CBFB/MYH11= 89) were performed. MCF panel included 2 10-color tubes: CD15 FITC/ CD56 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD7 APC/ CD13 APC-R700/ CD19 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for RUNX1/RUNX1T1 cases and CD64 FITC/ CD2 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD200 APC/ CD13 APC-R700/ CD14 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for CBFB/MYH11 cases. A mean of 1,733,240 CD45 positive events were acquired, with a mean lower limit of detection (LLOD) of 0.001% (ranging 0.01-0.0003%). In 10 cases (6 RUNX1 and 4 CBFB/MYH11) the molecular and phenotypical features at onset were not available. MCF analyses were performed by InfinicytTM software and reference image tool was used wherever possible. Results: We found a concordance of 76% between MCF cases and RUNX1/RUNX1T1 qPCR, and a concordance of 83% between MCF cases and CBFB/MYH11 qPCR (Tables 1a and 1b). In relapsed cases we observed a dynamic and continuous increase of abnormal MCF events, that was not found in cases without clinical relapse although with positive qPCR (Table 2). The HLADR expression evaluated as the geo mean intensity at presentation and/or at relapse resulted higher in RUNX1/RUNX1T1 patients than in CBFB/MYH11 cases (29845 vs 8979, respectively, p=0.0039 Figure 1). Interestingly, we found in a single case with atypical CBFB/MYH11 fusion gene a high value of HLADR geo mean (44522). The down-regulated expression of HLADR in CBFB/MYH11 blasts may be a useful clue to distinguish AML blasts from the scarcely represented normal CD2 positive myeloid precursors, and from the strongly HLADR positive elements that are found in a regenerating bone marrow background. Conclusion: Although MCF-MRD assay showed lower sensitivity than CBF qPCR, MCF offered interesting informations about the dynamics of the abnormal clone size with time, evaluated as the increasing number of MRD clustering events, which may predict an impending relapse.

Fever with Significant Procalcitonin Increase Following Gemtuzumab Ozogamicin Infusion in Hematologic Malignancy Patients without Evidence of Infection

In patients diagnosed with hematologic malignancies, including acute myeloid leukemia (AML), infections remain a major cause of non-relapse mortality. The accurate detection and management of infections during the treatment of these diseases are crucial for successful outcomes. Procalcitonin (PCT) has been proposed as a valuable tool for identifying severe bacterial infections, even in patients with hematologic malignancies. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody conjugated to calicheamicin, is used in certain subsets of AML patients, such as core binding factor (CBF) leukemia and AML with favorable-risk, as well as in cases of refractory AML. However, GO administration comes with its own set of risks, including liver dysfunction, veno-occlusive disease (VOD), bone marrow suppression, bleeding, and infusion reactions. Among these reactions, those causing high fever and shivering can be particularly challenging to differentiate from sepsis or severe infections. Nevertheless, there have been no reports evaluating the utility of PCT in discriminating infections during GO-induced infusion reactions. To shed light on the impact of GO administration on PCT levels, we undertook a retrospective analysis. Over the period between 2014 and 2022, we administered GO (3.0 mg/sqm per dose) to 42 patients with hematologic malignancies, a total of 97 times (median 2 doses per patient, range 1-6 doses per patient). Of the recipients, 23 had undergone allogeneic stem cell transplantation, while 19 were non-transplant patients. Prior to GO administration, all patients received premedication comprising corticosteroids, antihistamines, and acetaminophen. Among the 97 instances of GO administration, 40 (41.2%) resulted in fever (≥37.5 ℃) within 24 hours. For 19 cases, we conducted a complete set of PCT measurements and blood cultures within 24 hours of GO administration and analyzed the PCT levels and clinical parameters. These 19 cases corresponded to 19 individual patients, of whom nine had CBF leukemia, including t(8;21) AML or inv(16) AML, and ten had non-CBF leukemia. Out of these patients, 12 experienced fever (≥37.5 ℃), while seven did not. The findings indicated that patients with fever (≥37.5 ℃) exhibited significantly higher PCT levels compared to afebrile patients (p=0.018). Furthermore, PCT levels showed a significant positive correlation with C-reactive protein levels (p=0.003). However, among the 19 patients, only one patient with a positive PCT result had a documented infection identified through blood cultures or imaging studies.Other factors, such as patient gender (p=0.467), underlying disease (p=0.224), disease remission status at GO administration (p=0.492), transplant status (p=0.556), and the number of GO administrations (p=0.906), did not significantly influence PCT levels. It is essential to acknowledge that this analysis is based on a limited number of cases from a single institution. Nevertheless, approximately 40% of patients experienced fever within 24 hours of GO administration, despite receiving premedication.Furthermore, the presence of fever after GO administration was associated with a significant increase in PCT levels, even in the absence of identifiable infection. This raises the possibility of false positives for PCT in the context of GO-induced infusion reactions, underscoring the need for cautious interpretation of PCT values for infection differentiation in patients undergoing regimens that include GO. Further research with larger patient cohorts is warranted to validate these findings and improve our understanding of the implications of PCT measurements in this clinical setting.

AML1-ETO and CCND2 Overexpression Cooperate to Drive AML Initiation and Progression

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

Evolutionary Portrait of Adult Core-Binding Factor Leukemia Patients Treated with a Continuation Therapy with Midostaurin: Preliminary Results

Genetic and Clinical Background The clinical outcome of Core Binding Factor Leukemia (CBFL) has been reported to be influenced by KIT mutations, that was frequent in CBFL, and by co-occurring different spectra of cooperating mutations. In addition, gene expression studies found KIT to be highly expressed in CBFL regardless of its mutational status. In vitro experiments revealed that Midostaurin (Mido) is effective in inhibiting both wild type (WT) and a range of KIT mutants. In vivo studies demonstrate the efficacy in KIT-positive malignancies such as Aggressive Systemic Mastocytosis, Mast Cell Leukemia, and SM with Associated Hematological Neoplasm. Therefore, we designed a Phase II trial to evaluate the safety and efficacy of Mido, in association with Intensive Chemotherapy (IC), in CBFL regardless of KIT mutational status. (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345). 34 adult patients with t(8;21) (n=16) and inv(16) (n=18) were enrolled between December 2018 to July 2022. Method Patients with de-novo CBFL received standard induction therapy with an anthracycline containing regimen ("7+3"-like) + Mido, three cycles of post-remission consolidation chemotherapy with high-dose cytarabine + Mido, and 12 months of Mido as Maintenance. At diagnosis, 32 out of 34 patients were studied by a comprehensive NGS panel targeting 40 DNA genes and 29 RNA fusion driver genes associated with major myeloid disorders (Oncomine Myeloid Research Assay). Aim To study the clonal evolution of CBFLs, NGS and MRD status (performed by qPCR and high-resolution multicolor flow cytometry) were assessed at established check-points during consolidation, maintenance therapy, and at relapse. ResultsKIT was the most frequently mutated gene (28%) and there was an enrichment of FLT3 and NRAS (Figure 1). Additionally, the spectrum of recurrent mutations was different, as codon 816 KIT mutations and FLT3-ITD were common only in RUNX1::RUNX1T1 cases. Overall, the CR rate was 97%. At a median follow-up of 18 months (range 2-43 months), we recorded a RI of 37.5 %, an OS of 85 %, and a DFS of 60%. We performed targeted next generation sequencing for six out of 12 relapsed samples in the cohort t(8;21) (n=5) and inv(16) (n=1), showing loss, retention or gain of somatic mutations (Figure 2). Three out of six relapsed patients showed at diagnosis a CEBPA mutation (Gly104del) co-occurring with KIT in RUNX1::RUNX1T1 cases and with NRAS in CBFB::MYH11. Conclusion In patients with CBFL, we found FLT3 and/or KIT mutations actionable by a regimen consisting of intensive chemotherapy and Mido in 47% of cases. In two out of three relapsed cases the KIT mutation is maintained, although frequency of the mutant allele is lower at relapse, while all FLT3 and NRAS mutated patients lose the mutation upon disease relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Protein Interactomes of CBFB-MYH11 and RUNX1-RUNX1T1 Suggest Different Mechanisms of Action for Initiating Acute Myeloid Leukemia

Core Binding Factor (CBF) leukemias are a subset of Acute Myeloid Leukemia (AML) initiated by translocation of genes encoding the heterodimeric core-binding factor (RUNX1 and CBFB) via generation of the oncogenic fusion genes, CBFB-MYH11 and RUNX1-RUNX1T1. While both are classified as favorable risk, relapse occurs in 40-50% of patients with conventional chemotherapy. To better understand the mechanisms by which these fusions initiate AML, we used an MSCV-based, GFP-tagged retroviral vector to introduce a highly active biotin ligase ("TurboID", or "TID") fused to either CBFB-MYH11 ("CM") or RUNX1-RUNX1T1 (AML1-ETO/"AE") at both the N-and C-termini of the proteins into primary murine hematopoietic progenitor cells. With this system, proteins within 10 angstroms of TurboID-fusion proteins are efficiently labeled with biotin, enriched with a streptavidin bead pulldown, and identified using mass spectrometry. We first defined top protein interactors with CBFB-MYH11-TID compared to TurboID protein only ("TID"), and plotted the strength of interactions in the heat map shown in Figure 1A, where each row represents an independent biological replicate (n=11 for TID, n=6 for CM-TID, and n=6 for AE-TID), and each column represents an interacting protein. N- and C-terminal TurboID fusions behaved similarly and are shown together in the figure. AML1-ETO-TID interactions with the same interacting proteins are "passively" plotted, revealing that 11 proteins interacted with both fusions (including RUNX1, RUNX2, and CBFB itself - as expected). AML1-ETO had a unique set of interacting proteins (data not shown) that were primarily nuclear localized, as defined by the Human Protein Atlas (e.g. BCOR, NCOR1, SIN3A, etc.). In contrast, most CBFB-MYH11-interacting proteins were unique for that fusion (including multiple myosin proteins; see Figure 1A), and most were annotated as non-nuclear in the Protein Atlas (denoted with an asterisk in the figure). The predicted cytoplasmic localization of CBFB-MYH11-TID was confirmed by immunofluorescence using an antibody directed against the TID protein. We independently confirmed these results using a CBFB-MYH11-GFP fusion protein, which likewise localized in cytoplasmic aggregates, while a CBFB-GFP fusion was found exclusively in the nucleus. GFP-MYH11 was localized to the cytoplasm, indicating that the MYH11 portion of CBFB-MYH11 directs cytoplasmic localization in a dominant fashion, perhaps by interacting with other cytoplasmic myosin heavy chains. Immunofluorescent staining for endogenous RUNX1 in cells expressing CBFB-MYH11-GFP showed that RUNX1 co-localized with the cytoplasmic CBFB-MYH11, strongly suggesting that the fusion protein acts as a "trap" to sequester RUNX1 in the cytoplasm. To test this hypothesis, we generated a CBFB-MYH11 variant with an N104A mutation in CBFB that is known to disrupt RUNX1 binding. We confirmed complete loss of the CBFB-MYH11 interaction with RUNX1 and RUNX2 using the TurboID system (Figure 1B), and documented restoration of nuclear localization of RUNX1 by immunofluorescence. Using bulk RNA-seq on days 4 and 7 after transduction, we found that WT CBFB-MYH11 overexpression dysregulated many genes (562 up, 352 down, >2-fold change, FDR<0.05), including Runx1 and Runx2, both of which were upregulated. Importantly, the expression phenotype was completely eliminated in cells transduced with the N104A mutated CBFB-MYH11, suggesting that it was dependent on the cytoplasmic sequestration of RUNX1 and/or RUNX2. Finally, analysis of the existing human AML TCGA and Bloodspot transcriptional datasets confirmed upregulation of RUNX1 (p<0.0002 in both datasets) and RUNX2 (p<0.001 in TCGA) in CBFB-MYH11 AMLs relative to non-CBFB-MYH11 AMLs, suggesting that these genes may be activated by a feedback loop that is regulated by functional RUNX1/2 protein levels in the nucleus. In sum, these data suggest that CBFB-MYH11 alters the function of RUNX1 and RUNX2 by sequestering them in the cytoplasm, rather than by directly altering their activity as transcription factors. Thus, novel approaches designed to release mislocalized RUNX1/2 from CBFB-MYH11 cytoplasmic complexes should be explored as a novel therapeutic approach for CBFB-MYH11 AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splicing Gene Mutations Do Not Affect Outcomes in Newly Diagnosed Patients with Acute Myeloid Leukemia

Background: Recent studies have shown that splicing factor (SF) gene mutations in AML implicate a negative prognostic significance associated with secondary AML and are now included as adverse in the 2022 ELN guidelines (Dohner et.al., Blood 2022). However, the contribution of these mutations to the prognosis of patients (pts) with AML, relative to their age, prior history of myeloid disorders, cytogenetics (CTG), concurrent mutations and frontline (FL) therapy requires further characterization. Methods: We retrospectively analyzed all pts with newly diagnosed (ND) AML treated at our center over the last 5 years (yrs) and compared outcomes for patients with and without SF mutations (Smut= SF3B1, SRSF2, U2AF1 and ZRSR2). FL therapy was classified as intensive (INT) and low intensity (L-I), with/without venetoclax (VEN). Relapse free survival (RFS) was calculated from the time of response to relapse or death and overall survival (OS) was calculated from the time of therapy initiation to death; both were censored at last follow up (F/U) Results: From January 2017 to April 2022, 994 ND pts with AML were reviewed; 266 (27%) had Smut in ≥ 1 of the SF genes while the rest were wild type (SWT). The most common Smut was SRSF2 (53%). Only 3% pts had >1 Smut (Table 1). The median age of Smut group was 72 yrs compared to 65 yrs in the SWT group, and significantly more pts in the former had secondary/therapy related AML (s/t AML) (51% vs 41%). The Smut group had less complex karyotype (CK), non CK adverse risk CTG and did not include any pts with core-binding factor (CBF) leukemias. Fewer pts in the Smut had a pathogenic TP53mut but had higher frequencies of RUNX1 and ASXL1 mutations. Overall, 71% Smut pts were classified as ELN 2017 adverse risk compared to 54% SWT pts (p<0.0001). 20% pts in the Smut group vs. 44% SWT pts received INT therapy (p<0.0001). Of the Smut pts who received L-I therapy, 62% received a hypomethylating agent (HMA) and 66% received a VEN- based combination, frequencies similar to the SWT group. At a median F/U of 26 months (mos), the median RFS and OS was 9.6 vs. 12.4 and 13.1 vs 13.3 mos for the Smut and SWT groups respectively. Given the significant age heterogeneity between the 2 groups we analyzed by young (<60 yrs) and older (≥60 yrs) pts with non-CBF AML and compared survival outcomes (Fig.1). Both Smut and SWT pts had similar RFS and OS in their respective age groups. In a small subgroup of older pts with de novo AML with CK treated with L-I regimens, the RFS and OS were especially dismal for those with Smut (RFS= 1.5 vs 5.7 mos, p=0.14 and OS= 2.6 vs 9.3 mos, p=0.003). For other subgroups (stratified based on ELN risk stratification, de novo or s/t AML, CK, TP53mut) outcomes of Smut and SWT were similar in both young and older pt groups treated with INT or L-I therapies. Smut pts who received VEN in the L-I arm had better RFS and OS, especially if they had denovo AML (RFS= 19 vs 5.5 mos and OS 25 vs 4 mos). On univariate (UV) analysis using age, CTG [adverse risk (AR) /intermediate risk (IR)], therapy arm (INT/L-I), Smut/SWT status and concurrent mutations (TP53, ASXL1, RAS, RUNX1, NPM1, FLT3-ITD) as covariates for overall response (OR= CR/CRi/MLFS): lower age, IR-CTG, TP53wt, RASwt ,FLT3mut, NPM1mut and use of INT therapy were favorable while all the factors other than FLT3mut , age and therapy arm remained significant on the multivariate (MV) model. Within the Smut cohort, the presence of SF3B1mut alone negatively affected the odds of OR (0.65, 95%CI=0.3-0.98) on UV but not on MV analysis. Cox regression analysis demonstrated adverse risk CTG, TP53mut and s/t AML had higher hazards of relapse, while the above factors along with the presence of RASmut increased the hazards of death. On stratifying by specific Smut, no individual mutation impacted OS. Cox regression analysis of the above covariates affecting RFS and OS in Smut pts who received L-I therapy showed VEN based combinations significantly reduced the hazards for relapse (0.5, 95% CI=0.3-0.8) and death (0.5, 95%CI=0.3-0.7). Overall, the presence of Smut did not affect response rates to therapy, RFS or OS in both young and older pts treated with L-I (majority VEN based) or intensive therapy. Conclusion: Smut are more common in elderly pts with AML and in s/t AML. Smut does not necessarily affect response rates and survival outcomes compared to SWT pts when controlled for these covariates, and with current therapeutic options including HMA + VEN based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Autologous stem cell transplantation in favorable-risk acute myeloid leukemia: single-center experience and current challenges.

Autologous stem cell transplantation (ASCT) has gained growing consideration as a treatment option for favorable-risk acute myeloid leukemia (FR-AML) in first complete remission (CR1), compared with chemotherapy. We report the long-term outcomes of 117 consecutive patients with FR-AML fit for intensive chemotherapy diagnosed in our center between 1999 and 2020, who underwent ASCT. Sixty-five of the 117 were eligible for intensive post-remission treatment, and 42 of those 65 received ASCT. Median follow up was 132 months. Overall survival (OS) and disease-free survival (DFS) were 75% and 76%. Higher doses of CD34 + stem cell infusions negatively impacted DFS in multivariate analysis. Core-binding factor (CBF) leukemia was an independent prognostic factor for improved DFS. No differences based on pre-transplant measurable residual disease (MRD) were observed. In CBF leukemia, 10-year DFS is 72% for MRD-positive patients versus 100% for MRD negative patients. ASCT is effective and safe in FR-AML patients. In CBF leukemia, ASCT provides excellent results regardless of achievement of bone marrow MRD negativity. In NPM1-mutated/FLT3-wild type (mNPM1) AML, early molecular response seems to have more impact on prognosis. Prospective investigation of the role of gemtuzumab ozogamicin in this setting is ongoing.

Increased Survival Disparities Among Children and Adolescents & Young Adults with Acute Myeloid Leukemia: A Dutch Population-Based Study

Introduction: For many cancers, adolescent and young adult (AYA) patients have a poorer prognosis than pediatric patients. This study compares long-term survival outcomes of children and AYAs diagnosed with acute myeloid leukemia (AML) in the Netherlands. This has not been done before despite the availability of high-quality data collected by the nationwide, population-based Netherlands Cancer Registry (NCR).Methods: Data on all AML patients aged 0-39 years, diagnosed between 1990-2015, were obtained from the NCR (N=2058). Relative (disease-specific) survival was estimated for all patients, children (0-17 years), and AYAs (18-39 years) using the traditional cohort approach. Early mortality was defined as death within 30 days of diagnosis. Multivariable analyses were performed using generalized linear models (excess mortality) and logistic regression (early mortality).Results: The prognosis of Dutch AML patients below the age of 40 years has improved considerably during the past decades with 5-year relative survival rates increasing from 39% in 1990-1999 to 61% in 2010-2015 (p trend<0.01). AYAs had a substantially lower 5-year relative survival than children (43% vs. 57%, Figure). Although survival increased over time in both age groups, the improvement was more pronounced among children. Therefore, the survival gap was largest in the most recent period. In 2010-2015, 5-year relative survival was 74% for children compared to only 54% for AYAs. More detailed analyses showed that 1-9 year old children had the best prognosis with a 5-year relative survival of 84% in 2010-2015. With respect to the AML subtypes, core-binding factor (CBF) leukemia was more structurally tested and registered as from 2001. Within the group of patients diagnosed with CBF leukemia since 2001 (children: N=68, AYAs: N=68), children also had a better 5-year relative survival than AYAs (84% vs. 75%, not statistically significant).When compared to children, the excess risk of dying due to AML was 70% higher in AYAs [95% confidence interval (95% CI), 47%-97%] after adjustment for follow-up time, sex, period of diagnosis, and stem cell transplantation. The excess hazard ratios (HRs) of mortality were 2.0 (95% CI, 1.6-2.4) for 18-29 year olds and 2.1 (95% CI, 1.7-2.6) for 30-39 year olds, using 1-9 year olds as reference.Early mortality declined over time from 9% to 4% (p trend<0.01), and did not differ between children and AYAs. However, multivariable-adjusted odds ratios (ORs) of early death were doubled when comparing older age groups to 1-9 year olds.Conclusions: AYAs diagnosed with AML in the Netherlands between 1990 and 2015 had a worse prognosis than pediatric patients. The survival disparity seemed most evident in recent years, suggesting that improvements in care resulting in better outcome for children have not led to equal benefits for AYAs yet. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CSF3R T618I Collaborates with RUNX1-RUNX1T1 to Expand Human Haematopoietic Stem and Progenitor Cells

The cytokine receptor colony stimulating factor 3 (CSF3R) controls differentiation, proliferation and function of granulocytes as well as mobilization of hematopoietic stem cells upon activation via G-CSF. An oncogenic role of CSF3R mutations has been mainly described in chronic neutrophilic leukemia (CNL) and, more rarely, in atypical chronic myeloid leukemia and pediatric acute myeloid leukemia (AML) (Maxson et al., 2013, N Engl J Med; Maxson et al.,2016, Blood).CSF3R mutations are classified in two distinct categories: truncating mutations and proximal-membrane domain mutations. While truncating mutations lead to a higher sensitivity toward the ligand, the more common proximal-membrane mutations are ligand-independent in activating downstream signaling.Recently, we found CSF3R recurrently mutated in 5% (13/292) of patients with adult core-binding factor (CBF) leukemia (Opatz et al., 2020, Leukemia). Both CBF leukemia subgroups, characterized by either an inversion of chromosome 16 (inv(16)) or translocation t(8;21), were affected. The most prevalent mutation hotspot was T618 in the t(8;21) subgroup, localizing to the proximal-membrane region, recurrently affected also in CNL.We set out to study the role of CSF3R mutations in AML t(8;21). Using CD34+ hematopoietic stem cells from adult healthy donors, we observed that co-expression of the CSF3R mutant T618I (CSF3R T618I) but not CSF3R wildtype (WT)together with the t(8;21) related RUNX1-RUNX1T1 fusion led to a competitive outgrowth of cells (Figure 1A,B). In contrast, over-expression of either CSF3R WT or CSF3R T618Ialone did not lead to a competitive advantage. Furthermore, in a culture composed of >95% RUNX1-RUNX1T1 positive expanded cells, super-infection with the retroviral CSF3R T618I construct led to complete outgrowth of double positive cells over a period of 60 days. These data suggest an oncogenic cooperation between RUNX1-RUNX1T1 and CSF3R T618I. Comparing the cytomorphology of RUNX1-RUNX1T1 single positive cells and RUNX1-RUNX1T1/CSF3R T618I double positive cells, the single positive cells were heterogeneous with a mixture of mature and immature cells (Figure 1C), whereas double positive cells remained mostly undifferentiated, featuring a homogeneous blast-like morphology (Figure 1D). In accordance with these findings, limiting and serial dilution experiments clearly showed an increased self-renewal potential for double oncogene expressing cells compared to cells expressing RUNX1-RUNX1T1 only. This further supports a transforming potential of CSF3R T618I in combination with RUNX1-RUNX1T1.RNA sequencing of outgrown double oncogene expressing cells revealed upregulation of distinct pathways related to myeloid differentiation, in particular neutrophil cell activation and migration. In addition, we found GLI2 as potential downstream effector and putative pharmacological target in AML t(8;21) with CSF3R T618I (Figure 1E). GLI2 expression is a negative prognostic marker in AML, associated with significantly shorter event free and overall survival (Wellbrock et al., 2015, Clin Cancer Res). Acting in the hedgehog-signaling pathway, GLI2 is not expressed in healthy adult tissues. Of note, patient data and mouse models support a specific collaboration of upregulated GLI2 and FLT3-ITD in AML (Wellbrock et al., 2015, Clin Cancer Res; Lim et al., 2015, Sci Transl Med).Conducting further experiments in the granulocyte-macrophage colony stimulating factor (GM-CSF) dependent t(8;21)-positive cell line SKNO-1, we observed cytokine independent growth in the presence of CSF3R T618I but not CSF3R WT. GM-CSF withdrawal of SKNO-1 native cells, vector control or CSF3R WTexpressing cells led to cell cycle arrest that could be overcome by expression of CSF3R T618I.Subsequently, we investigated pharmacological counteraction of CSF3R T618I through inhibition of putative effectors. CSF3R T618I expressing SKNO-1 cells were significantly more sensitive towards treatment with the GLI inhibitor GANT61, as compared to vector or CSF3R WT controls (Figure 1F).Taken together, our results suggest a specific oncogenic collaboration between RUNX1-RUNX1T1 and CSF3R T618I with potential therapeutic implications. [Display omitted] DisclosuresRedondo Monte: Minaris Regenerative Medicine: Current Employment. Greif: AstraZeneca: Honoraria.

CYTOGENETIC AND CLINICAL DATA OF VARIANT THREE-WAY TRANSLOCATIONS T(8;V;21) IN CHILDHOOD ACUTE MYELOID LEUKEMIA

Core binding factor (CBF) leukemias represent an important prognostic group in acute myeloid leukemia (AML). The t(8;21)(q22;q22)/ RUNX1 - RUNX1T1 is one of the most frequent abnormalities in childhood AML (7–16%) and is related to a good prognosis. However, on rare occasions, an additional chromosome may be involved in this translocation, which may result in a poor clinical outcome for the patient. In this sense, the prognostic value of the RUNX1 - RUNX1T1 fusion has been widely discussed in the literature. Approximately 3–4% of AML cases with t(8;21)/ RUNX1 - RUNX1T1 occur in the context of complex rearrangements, with only a few cases reported in children (0.05–1.1%). Our aim in this work is to demonstrate the clinical and molecular data from four AML pediatric patients with variant three-way translocations t(8;V;21), characterizing in detail the extra chromosomes and fusion products. In our cohort of 146 children diagnosed with AML, we observed 22 with complex karyotypes (>3 abnormalities). To date, we detected the t(8;21) in 5/22 cases, among which 4 patients presented the involvement of a third chromosome in t(8;21), characterizing variant three-way translocations t(8;V;21). To characterize the patient's karyotypes, G-banding, FISH, and high-resolution molecular cytogenetics experiments (PCP, WCP, M-FISH, and MCB) were performed on bone marrow and/or peripheral blood cells under standard protocols. Regarding the mechanism of this type of translocation, Downing et al. (1993) and Tanaka et al. (2012) reported that there are at least two stages for the formation of such complex variants. The region 21(q22) (harboring the RUNX1 gene) translocates to the long arm of chromosome 8, while region 8q22 (harboring the RUNX1T1 gene) translocates to a third chromosome. Interestingly, in our 4 cases, t(8;V;21) demonstrated the same biological behavior in the rearrangement as the other cases reported in the literature. Although, unlike the cases previously reported in the literature, the karyotypes characterized in our study were cryptic, reaffirming the importance of an accurate diagnosis of RUNX1 - RUNX1T1 fusion products. With our approach by molecular cytogenetic techniques, we were able to precisely define the breakpoints of the three-way translocations, which will be of importance for future studies, investigating putative genes with impact on the genesis of pediatric CBF leukemias.

Prospective Study of Acute Myeloid Leukemia (AML) Clinical Epidemiology and Cytogenetic Risk Group in Older Adults in the ECOG-ACRIN E2906 Clinical Trial: The Acute Leukemia Epidemiology, Survival and Endpoints (ALESE) Study

Prospective Study of Acute Myeloid Leukemia (AML) Clinical Epidemiology and Cytogenetic Risk Group in Older Adults in the ECOG-ACRIN E2906 Clinical Trial: The Acute Leukemia Epidemiology, Survival and Endpoints (ALESE) Study

Response Kinetics and Factors Predicting Survival Among Patients with Core Binding Factor (CBF) Acute Myeloid Leukemia (AML)

Background: Core Binding Factor (CBF) leukemia, characterized by inversion (16) or t(8;21) cytogenetic abnormalities, is associated with a relatively favorable prognosis and highly responsive to treatment with fludarabine, cytarabine, and granulocyte colony stimulating factor combination (FLAG) along with either gemtuzumab ozogamicin (FLAG-GO) (Borthakur, Am J Hematol, 2014) or idarubicin (FLAG-IDA) (Borthakur ASH 2013). Response monitoring is facilitated by the use of real time polymerase chain reaction (qPCR) assessment for fusion gene transcripts. In this study, we assess factors underlying disease response kinetics and their predictive utility (including the role of quantitative fusion gene transcript ratio)in determining survival outcomes in CBF AML.Methods: One hundred and four patients (pts) with CBF AML were treated with frontline sequential regimens of FLAG-GO or FLAG-IDA at MDACC from 2007 to 2014. Baseline and periodic qPCR values were collected for analysis; logarithmic transformations were applied on qPCR data and depth (quality) of response expressed as quantitative changes in log reductions/increases (log (qPCR)). In order to determine the rate of response, random regression was applied on repeated measures of the log (qPCR) for all 104 pts. Regression coefficient for the each individual's PCR response over time was extracted from the model and measured as the slope [rate of log-reduction over time] (or rate of response). Stepwise cox regression models were applied to evaluate factors predicting for survival in the multivariate analysis. Maximally selected log rank statistics (MSLRS) methods were used to determine optimum cut-offs for continuous covariates, wherever applicable.Results: Baseline pt and disease characteristics are outlined in table 1. Median log (qPCR) reduction (∆qPCRind) over first FLAG induction cycle was 2.8 (0.0-7.0). Median number of FLAG consolidation cycles received were 6 (range, 1-6). Median log alteration from post-induction to post-consolidation phase (log (qPCR)end-induction - log (qPCR)end-consolidation = ∆qPCRcons) was 1.4 (-2.9-7.0). Rates of response (slopes) for the duration of treatment course for all cases are plotted in figure 1A. ∆qPCRind correlated strongly with the overall regression slope (p = 0.0012) indicating that depth of initial response predicts for an overall steep slope (i.e., faster rate of response). Also, there was a trend toward significance by regimen type (p = 0.07) and cytogenetics (p = 0.05), with FLAG-IDA and t(8;21) associated with slower rates of response. At a median follow up of 22 (1-76) months among survivors, 92 (88%) pts are alive; 18 (17%) have relapsed (5 pts during consolidation). Depth of initial response did not, however correlate with rate of log (qPCR) response during the consolidation phase of treatment. Among patients in continued remission at the end of consolidation phase, and not treated with further maintenance, RFS did not differ by the presence or absence of a detectable qPCR transcript-at-end-consolidation (p = 0.87). Among analyzed factors, ∆qPCRind, ∆qPCRcons predicted for a statistically significant improved relapse free survival (RFS); ∆qPCRind, ∆qPCRcons, age, and antecedent hematological disorder were significantly associated with overall survival (OS) (table 2). Importantly, rate of response did not predict either for RFS or OS. Patients with a ∆qPCRind of > 2.8 (by MSLRS on RFS) (n = 47, 45%) and a positive ∆qPCRcons (i.e., ∆qPCRcons > 0) (n = 84, 81%) had a significantly improved RFS compared to those without (Figures 1B & 1C, respectively). When accounting for both variables, pts who failed to achieve either of the two qPCR depth-of-response measures had the lowest RFS compared to those achieved at least one; pts who achieved both had the best RFS (Figure 1D).Conclusions: ∆qPCRind, ∆qPCRcons (both, measures of depth of response) are important predictors of RFS and OS, and quantitative cut-offs derived from our study serve as clinically useful estimates to identify pts at an increased risk of relapse. Patients with inv(16) and those treated with FLAG-GO have a faster rate of response (as measured by qPCR) to treatment. However, neither regimen, cytogenetics, nor rate of response to treatment impact RFS or OS. [Display omitted] DisclosuresCortes:Sun Pharma: Research Funding; Teva: Research Funding; ARIAD: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Kantarjian:Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Delta-Fly Pharma: Research Funding; Pfizer: Research Funding.

Association between Nuclear SET Domain (NSD) Protein Lysine Methyltransferases (KMT) Expression and Poor Prognosis in Acute Promyelocytic Leukemia (APL) Patients Treated with ATRA and Anthracycline Protocol

Introduction: In the last decades, it has been described the potential role of Nuclear SET domain (NSD) Protein Lysine Methyltransferases (KMT) family members (NSD1/KMT3B, NSD2/WHSC1/MMSET, and NSD3/WHSC1L1) in cancer development and progression, mostly associated with overexpression and poor prognosis. However, little is known about their impact on acute leukemia, especially in acute promyelocytic leukemia (APL). Even though the most important advance in APL was the therapy with ATRA, risk stratification remains an important issue. Besides the influence of molecular genetic markers into APL prognosis during the last years, risk stratification is predicted by leukocytes and platelet counts only. We aimed to evaluate the expression levels of NSD genes and their correlation with APL prognosis.Methods: A total of 153 consecutive patients (age, 9-78y) with newly diagnosed APL who were enrolled in the IC-APL study were included. Details about the diagnosis, eligibility criteria and classification of patients are published elsewhere (Rego EM et al., Blood 2013). The treatment protocol was identical to that adopted in the PETHEMA/HOVON LPA2005 trial, except for the replacement of idarubicin by daunorubicin. For comparison purposes, isolated CD34+ cells (n=16), de novo core binding factor-leukemia (CBF-leukemia, 24 patients), cytogenetically normal-AML (CN-AML, 30 patients) and not otherwise specified AML (NOS-AML, 11 patients) were included. Gene expression profile was analyzed by qPCR using the normalized copy number to express the results. Using survival receiver operating characteristic (ROC) curve analysis and the C index, we dichotomized patients into two groups according to NSD1, 2 and3 expression using the median value as cut off (groups “lower” and “higher” expression).Results: NSD expression was lower in APL, CBF -leukemia, CN-AML, and NOS-AML compared to isolated CD34+ from healthy donor samples (P<.001). Restricting our analysis to APL patients, we show that lower NSD genes expression was associated with high-risk group, according to PETHEMA/GIMEMA criteria (NSD1, P=.007; NSD2, P=.022, and NSD3, P=.019). Baseline features were similar between patients with low and high NSD expression levels, except for high frequency of Bcr2 breakpoint in patients with high expression of NSD1 and NSD3 (P=.006 for both) and lower frequency of FLT3-ITD mutation in group with lower expression of NSD1 (OR:0.28; 95% CI: 0.07-0.9; P=.04). With a median of follow up of 32 months (1-101 months), the estimated 5y OS rate was 79% (95% CI: 72-84%). Patients with higher expression of NSD2 gene had higher 5y OS rate (84%, 95% CI: 71-91%) compared to lower expression group (53%, 95% CI: 23-73%) (HR: 0.21; 95% CI: 0.1-0.5; P<.0001) albeit these data were not supported by multivariate logistic regression analysis considering leukocyte count, age and gender as confounders (HR: 0.48; 95% CI: 0.18-1.3; P=.161). Overall, 126/153 (84%) patients achieved complete hematological remission (CR). The estimated 5y DFS and the CIR rates were 87% (95% CI: 80-92) and 12% (95% CI: 7-17%), respectively. According to NSD gene expression, patients with high expression had a higher 5y DFS rate than those with a lower expression (Table 1). In agreement CIR rate was higher in patients with lower NSD expression in comparison to patients with high expression (Table 1). .Conclusion: This is the first study to show that the expression of NSD genes is decreased in APL. Our results show that low NSD transcript levels may predict a short survival and high risk of relapse in APL patients treated with ATRA and anthracycline-based chemotherapy. [Display omitted] DisclosuresBittencourt:Janssen, Takeda: Honoraria. Pagnano:Roche: Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy; Bristol-Meirs Squibb: Consultancy, Speakers Bureau. Lo Coco:Lundbeck: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Prospective Evaluation of a Continuation Therapy with Midostaurin in Adult Patients with Core-Binding Factor Leukemia and Integrated Genetic Analysis: A Multi Center Phase II Study. Preliminary Results

Genetic and Clinical Background: The clinical outcome of Core Binding Factor Leukemia (CBFL) seems influenced by the mutational status of KIT. In fact, several retrospective studies, in addition to our own, as well as a systematic review, indicate that KIT mutations have a negative prognostic impact in AML with t(8;21) or, to a lesser extent, with inv(16)/t(16;16). In addition, gene expression studies found KIT to be highly expressed in CBFL regardless of its mutational status. Furthermore, recent studies have identified novel recurrent somatic mutations co-occurring with KITmut. In-vitro studies revealed that Midostaurin (Mido) is effective in inhibiting both wild type (WT) and a range of KIT mutants. In addition, it is proven to be effective in KIT-positive malignancies such as Aggressive Systemic Mastocytosis (ASM), Mast Cell Leukemia (MCL), and SM with Associated Hematological Neoplasm (SM-AHN). With this background, we designed a Phase II trial to evaluate the safety and efficacy of Mido in association with Intensive Chemotherapy (IC), in CBFL regardless of KIT mutational status. Methods: The inclusion criteria were the following: age 18 to 60 years, diagnosis of de-novo CBFL, adequate organ function, signed informed consent. The exclusion criteria were: central nervous system involvement, uncontrolled infections, other active malignancies, a Qtc value greater than 470 ms (according to Bazett formula) at the electrocardiogram, significant uncontrolled or active cardiovascular diseases. Patients received standard induction therapy with an anthracycline containing regimen ("7+3"-like) + Mido, three cycles of post-remission consolidation chemotherapy with high-dose cytarabine + Mido, and 12 months of Mido as Maintenance. The Mido dosage was: 50 mg orally twice a day, on days 8-21, in association with IC, and 50 mg orally twice a day as single agent maintenance. In order to attain a reduction in 2 years Relapse Incidence (RI), from the historical value of 48% to 28% (Primary Objective of the Study), we plan to enrol 39 patients (power 82%, alpha error 4,6%). At diagnosis all patients were studied by a comprehensive NGS panel targeting 40 DNA genes and 29 RNA fusion driver genes. MRD status was assessed by qPCR and high-resolution multicolor flow cytometry at established check-points during consolidation and maintenance therapy. Results: 17 patients were enrolled between December 2018 to April 2020 (table1). Overall, the CR rate was 94.2%. At a median follow-up of 9 months (range 3-19 months), we recorded a RI of 12.5%, an OS of 93.7%, and a DFS of 81.2%. 16 patients continue on study and 14 patients are in 1st CR, MRD-negative by flow cytometry and qPCR. Six patients (35.2 %) experienced 12 Treatment Emergent Adverse Event (TEAE), 10 out of whom were infections, with grade 3-4 neutropenia (Table 2). We only recorded one death from SARS-Cov2 infection (Interstitial Pneumonia) in a patient in MRD-negative complete remission. There were no treatment-related deaths. Conclusion: In patients with CBFL, the regimen consisting of intensive chemotherapy and consolidation chemotherapy in association with Mido, followed by Mido maintenance, had an acceptable safety profile and excellent response rates with a significant proportion of patients in MRD-negative complete remission. Trial is continuing to accrue (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345). This work was supported by a grant from Fondazione Regionale per la Ricerca Biomedica (FRRB 2015). Disclosures Krampera: Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Todisco:Jannsen, Abbvie, Jazz: Membership on an entity's Board of Directors or advisory committees. Veronese:Novartis: Other: Travel Expenses; Bayer: Honoraria; AstraZeneca: Other: Travel Expenses; Janssen Cilag: Honoraria. OffLabel Disclosure: Midostaurin for treatment of Core Binding Factor Leukemia. The drug has been used as KIT inhibitor.

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p&amp;lt;0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

Baseline Mutations Lack Impact on Clinical Outcomes and Molecular Response in Core Binding Factor Leukemia Treated with Highly Effective Regimen

Baseline Mutations Lack Impact on Clinical Outcomes and Molecular Response in Core Binding Factor Leukemia Treated with Highly Effective Regimen

AML-226: Dasatinib Plus Chemotherapy in Core Binding Factor Leukemia: A Case Study

Core binding factor (CBF) acute myeloid leukemia (AML) patients have t (8:21) and inv(16) or t (16;16). They respond to dose-intensive cytarabine regimens with a complete remission (CR) rate of over 90%. Five-year event-free survival is 44% with a median of 91 weeks. We report a case of a relapsing inv(16) CBF leukemia salvaged by adding a tyrosine kinase inhibitor (TKI) to the chemotherapeutic regimen. Case presentation Ms. J.R is a 21-year-old patient with quadriplegia and inv16 with mutated KIT exon 8 gene diagnosed December 2017. She received induction with FLAG-IDA and six consolidation courses. Four months later, she relapsed with 56,000 WBC/dL and 82% bone marrow blasts. The inv(16) FISH was 87% positive and CBFB MYH11/total ratio was 26.585. Salvage therapy was initiated with decitabine-venetoclax-gentuzumab ozogamicin (GO). There was a marrow CR with a 21% positive inv(16) FISH test and a CBFB-MYH11/total ratio of 32.191. Due to the persistence of the KIT mutation, the TKI dasatinib was added to decitabine-venetoclax-gentuzumab ozogamicin (GO). After the TKI addition, she remained in CR. The CBFB-MYH11/total ratio decreased to 9.046, and the inv(16) FISH test remained positive at 2%. A second similar course given was complicated by a pulmonary fungal infection, acute appendicitis, and CMV colitis. All resolved with antifungals, laparoscopic surgery, and anti-CMV therapy, respectively. Upon recovery, she was in CR with undetectable CBFB-MYH11 transcripts and a negative FISH test. Because a bone marrow transplant is not possible, she has started a modified maintenance regimen with dasatinib, decitabine, and venetoclax. Discussion CBF leukemia chimeric proteins disrupt normal hematopoiesis by recruiting histone deacetylases and DNA methyltransferase 1 to CBF target genes, resulting in increased chromatin deacetylation and promoter hypermethylation. KIT exon 8 and 17 mutations occur in 20–25% of t (8;21) and approximately 30% of inv(16) leukemias. KIT mutations result in constitutively activated promotion of leukemic cells survival. Screening for KIT mutations and adding TKIs during induction or salvage therapy could be important for the treatment of CBF leukemias with an analogous impact, like adding TKIs in Ph-positive diseases.

The clinical mutatome of core binding factor leukemia

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10–15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum (‘mutatome’) in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.