Core Binding Factor Research Articles

RAS Mutations in Adult Acute Myeloid Leukemia (AML). Frequency, Mutational Spectrum, and Identification of a Comutation Bias for KRASK117 (TET2/ASXL1)

Introduction RAS mutations are subclonal events associated with both good and bad prognosis AML categories (Papaemmanuil E et al.N Engl J Med 2016; 374). An adverse prognostic meaning of RAS mutations in treated AML with myelodysplastic-related changes has also been reported.In solid tumors, it has been shown that patients with KRASQ61K do not confer resistance to therapy, unlike other mutations: KRASQ61K introduces a cryptic splice donor site and causes a short inactive isoform (Kobayashi Y et al. Nature 2022 ;603). However, most KRASQ61K patients acquired the silent mutation G60G, which removes the new splice donor site and increases therapy resistance. This splicing mechanism was not present in cases with NRASQ61K and HRASQ61K. We investigated the frequency of RAS mutations, their molecular associations, and the prevalence of splicing mechanisms involving KRASQ61K and NRASQ61K in AML. Methods We included a consecutive series of adult de novo AML enrolled in the CETLAM trials, a total of 620 patients who were diagnosed between 2017 to 2022. DNA and RNA were extracted from bone marrow or peripheral blood. Next Generation Sequencing (NGS) was carried out with three panels containing at least 42 AML-related genes, including KRAS and NRAS. Only variants with a VAF (variant allele frequency) >4% and ranked as pathogenic or likely pathogenic were used for the analysis. Conventional karyotype was routinely assessed. cDNA was obtained from 7 patients with NRASQ61K, and was amplified through a conventional PCR. cDNA products were observed in gel electrophoresis and sequenced by the Sanger method. IBM SPSS Statistics was used to perform Chi-Square tests. In statistical tests regarding molecular associations, patients with co-mutated NRAS and KRAS are excluded. Results Our study found RAS mutations in 16.9% (105 patients) of adult AML cases. 11.9% of AML patients had mutated NRAS, while 6.1% mutated KRAS (74 and 38 patients, respectively). In our study, Q61 was the third most common mutation in NRAS with an incidence of 26%, while it was the fourth most common in KRAS with a frequency of 12%. The distribution of mutations in KRAS and NRAS is shown in figure 1. Regarding the Q61 codon, the distribution of each amino acid change in our cohort is shown in Figure 1b. Nine NRASQ61K were found, and cDNA could be obtained from 7 patients. NRASQ61K and non-mutated NRAS samples presented the standard large transcript in the gel electrophoresis. These findings ruled out the presence of exon skipping in AML with NRASQ61K mutations. No single case of KRASQ61K was observed in this extensive series of AML, so we could not identify alternative transcripts described in solid tumors nor compensatory KRASG60G mutations.1.1% of patients (7 cases) presented co-mutated NRAS and KRAS. In 9 subjects, more than one NRAS mutation was found, whereas, in 3 patients we detected two KRAS mutations. Lastly, we focused on molecular associations of RAS mutated patients (Figure 2). Of the total RAS-mutated cohort (105 patients), DNMT3A and NPM1 had the highest mutation frequency. Notably, 40.3% of NRAS-mutated patients had NPM1 mutations, significantly higher (p=0.018) than KRAS (16.1%). Another interesting finding was that KRASK117N mutations were mutually exclusive with DNMT3A and NPM1. All KRASK117N (6 cases) were associated with TET2 and or ASXL1 mutations. Cytogenetics information was available for 87 patients with RAS mutations (59 NRAS and 35 KRAS). NRAS-mutated patients showed a higher frequency of core binding factor (CBF) alterations than KRAS (18.1% vs. 3.6%, respectively) (p=0.063). Complex and miscellaneous karyotypes had higher rates of KRAS mutations than NRAS mutations (57.1% vs. 32.7%, respectively, p=0.032).In 26 cases, RAS mutations were the single mutation or the one with the highest VAF; notably, 42.3% had an NRASQ61 mutation. We had cytogenetic information of 20 patients, and twelve had inv(16) (60%). Conclusions In this cohort of adult AML cases, RAS mutations were present in 16.9%. Mutations at codon G12 were the most prevalent lesions in AML. NRAS mutations were more frequent than those of KRAS. Up to 17% of RAS mutated AML cases showed more than one mutation. CBF chromosomal abnormalities were commonly associated with NRAS mutations. Complex and miscellaneous karyotypes showed KRAS mutations more often. KRASK117N mutations were consistently associated with TET2 and ASXL1 mutations and mutually exclusive with NPM1 and DNMT3A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MYCN Regulates Cell Survival Via EIF4G1 in Inv(16) Acute Myeloid Leukemia

Transcription factors are frequently deregulated in leukemia and are pivotal to its pathogenesis. The core-binding factor (CBF) transcription factor is a heterodimer of CBFβ and RUNX proteins, is critical to hematopoiesis, and dysregulated in AML. The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells by sequestering RUNX1 by protein-protein interaction to induce AML. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in genome-wide RUNX1 binding, apoptosis induction, and enhanced survival in inv(16) mouse model [Illendula* and Pulikkan* et al, Science 2015]. While previous studies have demonstrated that AI-10-49 induced cell death is partly mediated by repression of MYC expression in inv(16) AML cells [Pulikkan et al, Cell 2018], the function of other MYC family members, in inv(16) cell survival, and overall in AML, remains poorly understood. By conducting Real Time RT-PCR in CD34+ bone marrow cells from human AML samples, we identified MYCN upregulation in inv(16) AML. Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 revealed that MYCN expression is downregulated (7-fold). MYCN silencing via CRISPR/Cas9 induced apoptosis in inv(16) AML cells. Furthermore, MYCN genetic targeting by CRISPR/Cas9-RNP approach in human primary inv(16) AML cells transplanted in humanized NSGS xenograft mouse model displayed significant delay in leukemia development, underlining the role of MYCN in inv(16) AML maintenance. ChIP-seq analysis conducted in inv(16) AML cells treated with AI-10-49 demonstrated increased RUNX1 occupancy at MYCN distal genomic element located at +26 Kb (named 'MYCN-e26') downstream from MYCN TSS without any RUNX1 binding at MYCN TSS. ChIP-seq analysis for histone marks such as H3K27ac and H3K4Me1, and chromatin accessibility by ATAC-seq suggested MYCN-e26 acts as a transcriptionally active enhancer element. Analysis of DNase I hypersensitive sites (DHSs) representing accessible chromatin regions in primary AML samples revealed that MYCN-e26 is a bona fide AML enhancer. Finally, deletion of a genomic element in MYCN-e26 surrounding the RUNX1 binding site (200 bp) by CRISPR/Cas9 genome editing downregulated MYCN transcript levels and inhibited inv(16) AML cell survival. Taken together, MYCN-e26 is instrumental in MYCN transcription and cell survival in inv(16) AML cells as a RUNX1 target. To identify the mechanism(s) by which MYCN regulates inv(16) AML cell survival, we performed CUT&Tag sequencing for MYCN and H3K27ac histone mark in inv(16) AML cells. Gene ontology analysis for MYCN binding peaks in transcriptionally active regions revealed mRNA metabolism and stability as the major pathways orchestrated by MYCN in inv(16) AML cells. By comparing the mRNA metabolism/ stability signature genes with transcripts significantly deregulated in human primary inv(16) AML samples, we identified eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) as one of the major genes regulated by MYCN in inv(16) AML cell survival. Our CUT&Tag sequencing analysis demonstrated MYCN binds to EIF4G1 promoter and enhances its transcription. MYCN silencing by CRISPR/Cas9 approach in inv(16) AML cells showed significant downregulation of EIF4G1, suggesting MYCN is required for EIF4G1 expression in inv(16) AML cells. EIF4G1 genetic silencing via CRISPR/Cas9 approach induced apoptosis in inv(16) AML cells in vitro, and significantly delayed leukemia development in NSGS xenograft mouse model, suggesting EIF4G1 is required for the survival of inv(16) leukemia-initiating cells. Our studies demonstrate that CBFβ-SMMHC modulates MYCN levels by inhibition of RUNX1 binding to MYCN distal enhancer in inv(16) AML cells. In addition, our studies bring novel function of MYCN in inv(16) leukemic cell survival by regulating EIF4G1. Future therapeutic approaches targeting MYCN/EIF4G1 axis in combination with AI-10-49 warrant better clinical outcome for the treatment of inv(16) AML.

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.

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

A Prospective Phase 2 Study of Venetoclax and Low Dose Ara-C (VALDAC) to Target Rising Molecular Measurable Residual Disease and Early Relapse in Acute Myeloid Leukemia

Introduction The 2021 European LeukemiaNet (ELN) recognizes MRD relapse as ≥1 log rise confirmed on repeat testing (Heuser Blood 2021). Molecular MRD relapse (e.g. NPM1mut) precedes clinical relapse by ~3-4 months (Ivey NEJM 2016) and provides a window of opportunity for pre-emptive therapy to be delivered prior to clinical relapse. Venetoclax (VEN)-based low intensity therapy is associated with rapid achievement of clinical response in older patients (pts) with AML (DiNardo NEJM 2020 and Wei Blood 2020) but only has modest activity in relapsed/refractory AML. We hypothesized that VEN-based therapy initiated at the time of MRD relapse could be associated with improved clinical response and longer clinical benefit. We therefore conducted a prospective study to explore the efficacy of low dose ara-C (LDAC) + VEN in pts with AML and MRD relapse (ACTRN12619000746134). Methods This multicenter phase 2 study stratified pts into either oligoblastic relapse (marrow blasts 5-15%; Group A), or molecular MRD relapse (Group B). Pts received VEN 600 mg (days 1-28, without dose ramp-up for Group B) and LDAC 20 mg/m2 sc (days 1-10). Primary objectives were morphologic or MRD response (≥1 log reduction) in groups A and B, respectively. Key secondary objectives were realization of allogeneic hematopoietic cell transplantation (HCT), relapse-free (RFS) and overall survival (OS). The study was approved by Alfred Health ethics committee (196/19). NPM1mut and other fusions (per 100 ABL1) from bone marrow were analyzed by RT-qPCR, and IDH1/2mut by Bio-RadTM droplet digital PCR. Results A total of 48 pts were recruited (median age 67; range 18-80). Cytogenetic risk was intermediate in 91%, favorable in 4.5% and adverse in 4.5%. Most (94%) had received prior intensive chemotherapy and only 2 (4%) had received prior HCT in first remission. The median interval from AML diagnosis to study entry was 13.7 months (IQR 9, 23). At the time of study screening, 22 (46%) pts had oligoblastic disease and 26 (54%) MRD relapse. Oligoblastic relapse included pts with early progression not captured by interval MRD surveillance. Pts received a median of 3 cycles (range 1-21+) of VEN-LDAC (ongoing in 11). Reasons for treatment cessation in 37 pts were: HCT (n=19, 51%), treatment failure (n=11, 30%), adverse events (n=3), donor lymphocyte infusion (DLI, n=2) or death before response assessment (n=2). One patient (Group B, NPM1mut) was deemed not evaluable for response assessment after only receiving 9 days of therapy but included in the survival analysis. Among 22 pts with oligoblastic disease, CR/CRi was achieved in 17 (77%). Two additional pts achieved morphologic leukemia-free state and 1 had stable disease. MRD response (≥1 log reduction) among those with an evaluable marker (N=8; all NPM1 mutated) was achieved in 8/8 (100%), with MRD negative in 6/8 (75%) (Figure). Among 25 evaluable pts with MRD relapse, MRD response (≥1 log reduction) was observed in 68% and MRD negative in 52%. Of patients with NPM1mut AML treated at MRD relapse, 13/19 (68%) achieved an MRD response and 10/19 (53%) an MRD negative response (Figure). An MRD response was observed in 2/3 with IDH1/2mut, 1/2 with core-binding factor and 1/1 with KMT2A::MLLT3. Bridging to HCT or DLI (median time 4 months) was successful in 18/27 (67%) pts ≤70 years. With a median follow up time of 14.4 months, the median RFS was 21.5 months and not reached for OS. The estimated 18-month RFS was 63.3% (95% CI, 48.0-83.5) and OS 71.1% (95% CI, 56.5-89.4). Similar outcomes were observed for Groups A and B. Subsequent HCT (as a time-dependent covariate) was the only significant factor for RFS (HR 0.13, 95% CI 0.03-0.61) but not OS (HR 0.48, 95% CI 0.13-1.75) in a multivariate Cox regression model that included age, features at original diagnosis (NPM1, FLT3-ITD and ELN 2017 risk), time since AML diagnosis, and study cohort (A vs B). FLT3-ITD was detected in 3/7 (43%) of relapsed pts. Conclusions In this prospective study of pts with early relapsing AML (oligoblastic or MRD relapse), VEN in combination with LDAC induced a high rate of molecular MRD response, resulting in excellent RFS and OS outcomes. Follow-up is ongoing to determine the durability of response. This novel MRD-targeting approach is being further investigated in a multi-arm, precision-based platform trial ( INTERCEPT: Investigating Novel Therapy to Target Early Relapse and Clonal Evolution as Pre-emptive Therapy in AML; ACTRN12621001265864). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase II Study of PD-1 Blockade Following Fludarabine/Melphalan-Based Autologous Transplantation for Acute Myeloid Leukemia Patients Ineligible for Allogeneic Transplant

Allogeneic transplant (allo) remains the best post-remission therapy for non-favorable risk acute myeloid leukemia (AML) patients, secondary to the potent graft-versus-leukemia (GVL) effect of donor immune cells. Unfortunately, some patients may be ineligible for allogeneic transplant due to psychosocial barriers (e.g. lack of appropriate caregiver support) or unavailability of a suitable donor. Inhibition of the programmed cell death protein-1 (PD-1) pathway is an effective immunotherapeutic anti-cancer strategy for a number of malignancies. We hypothesized that PD-1 blockade following autologous transplant might simulate an allogeneic GVL effect and represent an alternative post-remission strategy in patients with non-favorable risk AML. To this end, we initiated a phase II study whereby AML patients deemed ineligible for allogeneic transplant received a high dose preparative regimen consisting of fludarabine on day -4, -3, -2 (30mg/m2/d) and melphalan on day -1 (180mg/m2 for age ≤60, 140mg/m2 for age >60), followed by autologous stem cell transplantation and post-transplant pembrolizumab starting day +1 (200mg/dose q3weeks x 8 doses) (auto-pembro). Eligible patients were AML patients beyond CR1 or non-favorable CR1 patients, including c-kit-mutated core-binding factor and FLT3-ITD+/NPM1+ AML. Of 32 patients consented/screened for the study, 20 received protocol-directed therapy from January 2017 to July 2021. Reasons for not receiving transplant were mobilization failure [6], failure to achieve CR [2], patient refusal [2], compliance issues [1] and insurance issues [1]. Baseline patient characteristics included a median (range) age of 64 (26-75) years, KPS <80 in 30%, HCT-CI ≥3 in 85% and non-Hispanic white in 45%. Disease characteristics included ELN 2017 favorable, intermediate and adverse risk in 20%, 40% and 40%, disease-risk index low, intermediate and high in 10%, 75% and 15%, secondary/treatment-related in 20%, CR1 status in 80%, and pre-BMT measurable residual disease (MRD) in 30%. Treatment was well tolerated in only 1 non-relapse death. Infections included 5 patients with bacterial infections, 2 with CMV viremia, 1 with localized VZV and 1 patient with HHV6 reactivation. Grade 3 non-hematologic toxicities included febrile neutropenia (30%), nausea/vomiting (25%), diarrhea (25%), hypertension (20%) and stomatitis/esophagitis (15%). There were no study-related grade 4 or 5 toxicities. Possible immune-related adverse events included 4 cases of hypothyroidism, 2 cases of colitis and 1 case each of pneumonitis, erythema multiforme and seronegative arthritis. After a median follow-up of 80 months (range, 15-134), 14 patients remain alive with 10 in continuous remission. Estimated 3-yr OS, DFS, NRM and CIR was 66%, 48%, 5% and 47% respectively. Transplant outcomes in auto-pembro recipients were compared to a contemporaneous cohort of consecutive AML patients receiving allo transplant in first or later CR (n=112) by Cox proportional hazards model, adjusting for significant demographic and risk variables. Compared with allo recipients, auto-pembro recipients were less likely to be non-Hispanic white (45% vs. 69%) and male (35% vs. 59%) but were otherwise similar in regard to baseline patient and disease characteristics. In multivariable analysis, OS and DFS were significantly impacted by age, ELN risk and year of transplant. In contrast, transplant type (auto-pembro vs. allo) had no effect on OS (HR 0.58, p=0.24) or DFS (HR 1.19, p=0.65). Adjusted 3-yr OS and DFS was 76% vs. 66% and 54% vs. 62% respectively (see figure). Although relapse was significant higher following auto-pembro (HR 2.38, p=0.031), it was offset by a corresponding decrease in NRM risk. In conclusion, auto-pembro is a safe and effective consolidation therapy for non-favorable AML patients who are ineligible for allo transplant, with comparable adjusted survival outcomes. Correlative studies are planned to analyze anti-leukemic effect of post-transplant PD-1 blockade following autologous transplant in AML patients. Given these promising results in non-favorable-risk AML patients lacking an allogeneic transplant consolidation option, confirmation in a larger multicenter prospective study is warranted. 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

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

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

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

HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.

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

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

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

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

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

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.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia

BackgroundCleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or...

Clinical application of molecular genetics in acute myeloid leukemia

With the increasing application of genetic testing, we have gradually understood the heterogeneous molecular nature of acute myeloid leukemia (AML). We have been able to identify a large number of translocations and single gene mutations to go with the previously known karyotypic abnormalities associated with this disease. This has led to a refinement in the way we diagnose, prognosticate, and treat this disease. The newer classification systems proposed by the World Health Organization and the International Consensus Classification systems show an increasing reliance on molecular genetics for the diagnosis and classification of AML. This has also generated situations where molecular genetics assume preference over morphologic assessment, namely, in the case of recurring genetic abnormalities where the standard 20% of blast count is not required, and in AML with underlying myelodysplasia where the disease shall be defined not by morphologic dysplasia but by the presence of certain cytogenetic and molecular abnormalities. The new prognostic system proposed by the European LeukemiaNet also places an emphasis on genetic mutations along with the previously known cytogenetic abnormalities. Similarly, the assessment of measurable residual disease (MRD) is recommended to be done by quantitative polymerase chain reaction (PCR) for core-binding factor and NPM1 mutated AML. In the future, next-generation sequencing is also expected to play a prominent role in the assessment of MRD alongside flow cytometry and PCR. Finally, precision oncology is supplementing conventional treatment regimens and patients with FLT3 and isocitrate dehydrogenase mutations are recommended to be offered the respective targeted agents. This review shall discuss the clinical implications of molecular genetics in the current management of AML.