Presence Of ASXL1 Mutations Research Articles

What Is the Optimal Treatment Modality in Molecularly Defined Secondary AML? a Multicenter Cohort Study

Introduction Secondary ontogeny acute myeloid leukemia (AML), defined by the presence of mutations in ASXL1, BCOR, EZH2, SF2B1, SRSF2, STAG2, U2AF1 or ZRSR2 was recently integrated into both the ICC and WHO diagnostic criteria, as well as into the ELN 2022 risk classification as adverse risk. However, it is unknown whether cytarabine + anthracycline (7+3), liposomal cytarabine and daunorubicin (CPX-351) or hypomethylating agent + venetoclax (HMA+VEN) is the optimal frontline treatment for these patients (pts). We performed the largest multicenter retrospective cohort study to date, dedicated to pts with molecular-defined secondary ontogeny AML, comparing treatment modalities and assessing the impact of clinical and molecular characteristics on response and survival. Methods We included pts with newly diagnosed (ND) AML who were treated at 4 large academical centers with 7+3, CPX-351 or HMA+VEN. Secondary ontogeny was defined as absence of TP53 mutation and presence of at least one of the aforementioned mutations. Composite complete response (cCR) was defined as complete response (CR) + CR with incomplete count recovery (CRi). Logistic regression was fitted to evaluate odds ratio (OR) for cCR. Overall survival (OS) was compared between groups by log-rank test. COX regression for OS were fitted to evaluate effect of secondary/co-mutations within each treatment group, as well as the effect of treatment and allogeneic stem cell transplantation (SCT) as a time-varying covariate. Results Out of 1132 ND AML pts in the entire cohort, 401 (35%) were molecularly defined as secondary ontogeny and included in the final analysis. Initial treatment was 7+3 in 173 (43%), HMA+VEN in 162 (40%) and CPX-351 in 66 pts (17%). Pts in the HMA+VEN group were older (median age 74 years[yrs]; CPX351 66 yrs; 7+3 63 yrs, p<0.001), while pts in the CPX351 group were more likely to have prior myeloid neoplasm (n=52 [79%]; HMA+VEN n=72 [44%]; 7+3 n=48 [28%], p<0.001) and prior HMA exposure (n=27 [41%]; HMA+VEN n=23 [14%]; 7+3 n=28 [16%], p<0.001). The cCR rates were 56% (97/173), 56% (90/162) and 44% (29/66) in 7+3, HMA+VEN and CPX-351, respectively (p=0.21). In the 7+3 group, the presence of RUNX1 co-mutation was associated with lower cCR rate (OR 0.8, 95% confidence interval [CI] 0.7-0.95, p=0.01), while PTPN11 was associated with higher cCR rate (OR 1.4, 95% CI 1.1-1.8, p=0.011). In the HMA+VEN group, monosomal karyotype (OR 0.7, 95% CI 0.5-0.9, p=0.002), NRAS co-mutation (OR 0.6, 95% CI 0.5-0.8, p<.001) and SF3B1 mutation (OR 0.76, 95% CI 0.6-0.9, p=0.008) were associated with lower cCR rate and FLT3-TKD with higher cCR rate (OR 1.5, 95% CI 1.1-2.1, p=0.01). In the CPX-351 group, NRAS co-mutation was associated with lower cCR rate (OR 0.65, 95% CI 0.5-0.9, p=0.005). As most younger pts (aged ≤ 60 yrs, n=85) were treated with 7+3 (n=64) or CPX-351 (n=13) and almost exclusively older pts (age >75, n=78) were treated with HMA+VEN (n=74), we performed age-group based analyses. In pts aged > 60 yrs (n=318), the median OS (mOS) was comparable between groups (7+3: 16 months [mo], 95% CI 13-27; HMA+VEN 15 mo, 95% CI 13-19; CPX-351 11 mo, 95% CI 9-28 p=0.57). Similar results were seen in pts 60-75 yrs (n=238): 7+3 mOS 16 mo, 95% CI 13-27; HMA+VEN mOS 16 mo, 95% CI 13-26; CPX-351 mOS 10 mo, 95% CI 8-28, p=0.74. The effect of secondary/co-mutations on survival differ between groups: For 7+3 splicing mutations were associated with worse OS, whereas IDH1/2 were associated with improved OS ( Figure). Conversely, in HMA+VEN and CPX351 groups K/NRAS co-mutations were associated with worse survival. NPM1 co-mutation did not affect survival in any treatment group. In multivariable analysis, age, prior myeloid disease, K/NRAS co-mutations and monosomal karyotype were associated with worse OS ( Table). Conversely, BCOR mutation, IDH1/2 co-mutation and SCT were associated with improved OS. Treatment with HMA+VEN or CPX351 were not statistically different vs 7+3, although there was a trend for better OS with HMA+VEN vs 7+3 (HR 0.68, 95% CI 0.44-1.03, p=0.069). Conclusion In pts with ND secondary ontogeny AML the OS with HMA+VEN was at least comparable to 7+3 or CPX351 when adjusted for multiple covariates. The effect of co-mutations on response and OS differed between treatment modalities and may aid in optimal treatment selection in this population. Prospective trials should evaluate the potential superiority of HMA+VEN and effect of co-mutations in secondary ontogeny AML.

CD123 As a Therapeutic Target in Accelerated and Blast Phase Myeloproliferative Neoplasms (MPNs)

Background: CD123 expression in MPNs has been reported in case series and CD123 has been a therapeutic target in one phase I study of patients with chronic myelomonocytic leukemia or myelofibrosis in chronic phase. CD123 has also been described as a marker of leukemic stem cells and incorporated in chimeric antigen receptor T-cell products for treatment of acute myeloid leukemia in clinical trials. Herein, we describe the clinical phenotype of patients with accelerated (AP) or blast phase (BP) MPNs paired with CD123 co-expression on CD45(dim) myeloid blasts. Methods: This is a retrospective, single-institution study of patients with AP and BP MPNs. Subjects were identified by search for MPN diagnoses in the Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank (OSUCCC-LTB). Relevant clinical data was collected by retrospective chart review with approval of Institutional Review Board. CD123 expression was measured by immunophenotyping of cryopreserved peripheral blood (PB) or marrow (BM) samples. Blasts were identified by CD45(dim) gating of lymphocytes. Lymphocytes were also gated on CD7, CD13, CD33, CD34, and CD117. CD123 expression was stratified by quartile and correlation with disease phase, symptoms, splenomegaly, transfusion-dependence, and molecular mutations measured by Chi-square test. Results: Forty-one PB or BM samples from 24 subjects were identified from the OSUCCC-LTB. Nine subjects had paired PB and BM specimens and 6 subjects had samples obtained at multiple timepoints in the course of their disease. Median age at time of sample collection was 65.8 years (range 35.2-79.7) and median time from initial diagnosis to sampling was 13.1 months (range 0-96.5). Constitutional symptoms were present at the time of collection in 24 specimens (58.5%) and splenomegaly present in 20 specimens (51.3%). At sample collection, 19.5% had never received treatment, 19.5% had been previously treated, and 61% were currently on treatment. Most patients on treatment (n=16, 64%) were receiving JAK inhibitor at sample collection. Nineteen samples were obtained in chronic phase (46.3%), nine in AP (22.0%), and thirteen in BP (31.7%). Median WBC count was 4.37K/uL (range 0.3-174.6), median peripheral blasts were 3.2% (range 0-76%), median hemoglobin was 8.8 g/dL (range 5.6-13.2), and median platelets were 42K/uL (range 5-564). Sixteen samples (39.0%) were from transfusion-dependent patients, of which, 68.8% were both red blood cell (RBC) and platelet transfusion-dependent, 25.0% were RBC transfusion-dependent only, and 6.2% were platelet transfusion-dependent only. Median lactate dehydrogenase at time of sampling was 300 U/L (range 116-2,142). Median bone marrow blasts at time of sampling were 3% (range 0-82%). Molecular sequencing and karyotype +/- 30 days of sample collection were available for 18 (43.9%) and 27 (65.9%) of specimens, respectively. The most common mutations identified were JAK2 (50%), ASXL1 (27.8%), SRSF2 (22.2%), CALR (16.7%), and TP53 (16.7%). Normal karyotype was reported in 37% of the samples (one was consistent with donor chimerism post-allogeneic SCT), 33.3% had complex karyotype, 11.1% had del 5q or 7q/-7, and 18.5% had del 13q or 20q. CD123 expression on the CD45(dim) gated blasts ranged from 0.3-95.6% with a median value of 11.02%. CD123 expression is presented in figure 1 box plot. Figure 2a shows correlation between CD123 expression and AP/BP compared to chronic phase disease (p < 0.01). Figure 2b shows CD123 expression correlation with ASXL1 compared to unmutated ASXL1 (p =0.023). There were no statistically significant associations between CD123 expression and any other tested variables. Conclusions: These single-institution data suggest that high CD123 expression on blasts is associated with leukemic transformation and high-risk disease. This is demonstrated by correlation of higher expression in AP and BP MPN as well as correlation with ASXL1 mutation. CD123 expression in MPNs has been previously reported and explored as a therapeutic target in a single phase I trial, though these data suggest that specifically targeting high risk disease, defined by blasts >10% or the presence of ASXL1 mutation, may be more effective with greater target availability. This is an especially important subpopulation of patients with MPNs in need of more effective and less toxic therapy than current standards of care that typically include cytotoxic chemotherapy.

P103 - Topic: AS06-Prognosis/AS06b-Predictive factors of response to treatment: AUGMENTED ACTIVITY OF THE ALVOCIDIB AND 5-AZACYTIDINE COMBINATION IN MYELODYSPLASTIC NEOPLASMS IS ASSOCIATED WITH THE PRESENCE OF ASXL1 MUTATIONS

P103 - Topic: AS06-Prognosis/AS06b-Predictive factors of response to treatment: AUGMENTED ACTIVITY OF THE ALVOCIDIB AND 5-AZACYTIDINE COMBINATION IN MYELODYSPLASTIC NEOPLASMS IS ASSOCIATED WITH THE PRESENCE OF ASXL1 MUTATIONS

Molecular Predictors of Response and Survival in Patients with Relapsed/Refractory Acute Myeloid Leukemia Following Venetoclax Plus Hypomethylating Agent Therapy

Introduction Venetoclax (Ven) in combination with hypomethylating agents (HMA) is frequently used for treatment of relapsed/refractory AML patients in routine practice. However, limited data exists on molecular predictors of response and survival following Ven+HMA therapy in the relapsed/refractory setting. A prior retrospective study demonstrated superior response with TET2and ASXL1 mutations (AJH, 2019). Accordingly, our primary objective was to determine the impact of mutations on response and survival in relapsed/refractory AML patients receiving Ven + HMA. Methods Patients with relapsed/refractory AML, excluding post-transplant relapse, receiving Ven+HMA outside clinical trials at the Mayo Clinic were retrospectively recruited after institutional review board approval. Cytogenetic and molecular studies were performed at the time of AML diagnosis by conventional karyotype, and next-generation sequencing (42-gene panel), respectively. All patients received either azacitidine 75 mg/m2 days 1-7 or decitabine 20 mg/m2 days 1-5 with Ven dose adjusted based on azole antifungal prophylaxis. Response was assessed according to the 2017 European Leukemia Net (ELN) criteria. Patient characteristics 87 relapsed/refractory AML patients (median age 64 years, 62% male, 51% de novo) received Ven+HMA. ELN cytogenetic risk (n=77) included favorable (1%, n=1), intermediate (57%, n=44) or adverse (42%, n=32). Mutations involved TP53 in 20 patients (23%), ASXL1 in 19 (22%), IDH1/IDH2 in 12 (14%), TET2 in 11 (13%), K/NRAS in 11 (13%), NPM1 in 9 (10%), and FLT3-ITD in 7 (8%). Frequently administered prior therapies included 7+3 (n=51), CPX-351 (n=17), enasidenib /ivosidenib (n=6). Prior HMA was documented in 18 (21%) patients. 71 (82%) patients received decitabine and the remainder azacitidine with median Ven dose of 100 mg for a median of 2 cycles. Subsequent targeted therapies following Ven+HMA included enasidenib (n=3) and gilteritinib (n=2). Predictors of response 18 (21%) patients achieved complete remission (CR), 19 (22%) CR with incomplete hematological recovery (CRi), resulting in CR/CRi in 37 (43%). In univariate analysis, age > 65 years (CR/CRi, 61% vs 29%, p=0.003) presence of IDH1/2 (75% vs 37%, p=0.01) and ASXL1 mutations (68% vs 35%, p=0.01) were associated with favorable response; adverse karyotype (25% vs 58%, p=0.004), and presence of TP53 mutations (25% vs 48%, p=0.06) predicted inferior response. Presence of FLT3-ITD (71% vs 40%; p=0.11) NPM1 (67% vs 40%, p=0.12) and TET2 mutations (64% vs 39%, p=0.13) were borderline significant. In multivariable analysis, presence of ASXL1 mutations (OR 3.4) and absence of adverse karyotype (OR 4.5) remained independent predictors of favorable response. Predictors of survival At a median follow up of 5.6 months, 69 (79%) patients have died and 20 (23%) underwent allogeneic transplant. Post-Ven+HMA median overall survival was 5.9 months (CI, 2.5-14.5 months) and longer in transplanted patients (38.7 vs 5.1 months, p<0.0001). Univariate analysis identified CR/CRi (p<0.0001), IDH1/2 mutations (p=0.002) as favorable, and TP53 (p=0.005), and adverse karyotype (p=0.04) as unfavorable risk factors for survival. Of note, two IDH2 mutated patients on enasidenib following Ven+HMA remain alive at 27 and 13 months, respectively. Despite higher CR/CRi, presence of ASXL1 mutation did not impact survival. Multivariable analysis confirmed the negative survival impact of not achieving CR/CRi (HR 3.1, 95% CI 1.9-5.4, p<0.001), absence of IDH1/2 (HR 3.1, 95% CI 1.4-6.9, p=0.01), and presence of TP53 mutations (HR 2.1, 95% CI 1.2-3.6, p=0.01). Accordingly, a three-tiered model was generated by allocating 1 adverse point each for absence of CR/CRi, absence of IDH1/2 and presence of TP53 mutations, resulting in low (0-1 point, n=34, 13.7 months), intermediate (2 points, n=39, 5.1 months) and high risk (3 points, n=14, 1.9 months) categories (p<0.0001) (Figure) which remained applicable in non-transplanted patients (9.2 vs 4.0 vs 1.9 months, p<0.0001). Conclusions The current study identifies presence of ASXL1 mutation and absence of adverse karyotype as predictors of superior response. Survival was positively influenced by presence of IDH1/2, absence of TP53 mutations and achievement of CR/CRi. Our findings require validation in prospective series, which should also take into consideration the survival impact of subsequent targeted therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cooperative Inhibitory Effect of IDH1 Mutation and ASXL1 Mutation on Normal Myeloid Differentiation

Somatic mutations in the additional sex comb-like 1, ASXL1, have been described in various types of myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). In addition, ASXL1 mutations are frequently found in healthy population, which predispose them to not only myeloid malignancies, but also cardiovascular diseases. This condition is known as clonal hematopoiesis with intermediate potential (CHIP), although the full etiology of this preleukemic condition is not elucidated. As for AML, patients with ASXL1 mutations are known to have very poor prognosis compared to non-mutated patients. Although previous studies demonstrated that mutant ASXL1 promoted myeloid leukemogenesis via both loss-of-function and gain-of-function manners, mechanisms how ASXL1 mutations induce myeloid transformation is not fully elucidated. We analyzed publicly available datasets of AML patients, and found that isocitrate dehydrogenase 1 (IDH1) is co-mutated in 13% of AML with ASXL1. As the mechanism by which ASXL1 and IDH1 mutations cooperate to promote AML is not understood, we generated a model of AML with ASXL1 and IDH1 mutations using 32D-cl3 cells and discovered that mutant IDH1 could block myeloid differentiation only when mutant ASXL1 was present. At first, in our experiments, we retrovirally overexpressed mutant (G646WfsX12) or normal form of ASXL1 in 32D-cl3, which is a commonly used IL-3 dependent cell-line of murine myeloid precursor cell. We found that Gr.1 expression levels were decreased in these cells compared to 32D-cl3 with mutant ASXL1 (11.1% vs 26.5%, respectively; p=0.014), and that the proportion of cells in which Gr1 expression was induced by treatment with G-CSF was significantly lower (29.1% vs 54.8%, respectively; p=0.003). Next, we generated a tetracycline-inducible mutant (R132H) form of IDH1 lentiviral vector, and transfected them to these 32D-cl3 cells. We found that tetracycline-inducible mutant IDH1 inhibited G-CSF induced Gr.1 expression in the presence of mutant ASXL1 (7.1% with doxycycline vs 29.9% without doxycycline, respectively; p<0.01), while in the absence of mutant ASXL1, forced expression of mutant IDH1 could not block G-CSF induced Gr.1 expression (59.6% with doxycycline vs 59.2% without doxycycline, respectively; p=0.035). In accordance to these analyses of surface markers, we found that reduction of expression levels of CEBPβ induced by tetracycline-inducible ectopic expression of mutant IDH1 was only observed in 32D-cl3 cells with mutant ASXL1. Furthermore, we found that growth rate of 32D-cl3 cells with mutant ASXL1 and mutant IDH1 was significantly higher than those with mutant ASXL1 alone. These findings suggested that mutant IDH1 can exert its inhibitory effect of normal myeloid differentiation only when mutant ASXL1 is present. Herein, we hypothesized that mutant ASXL1 and mutant IDH1 had unknown cooperative mechanisms to suppress normal differentiation and promote leukemogenesis, and performed transcriptome analysis of the 32D-cl3 cells treated with G-CSF. Tendency toward decreased expression of genes associated with myeloid differentiation in the presence of IDH1 was only observed in cells with mutant ASXL1. Further, it was revealed that specifically dysregulated genes such as HDAC6 or KMT2A in leukemic stem cells were enriched in 32D-cl3 cells with mutant ASXL1, which suggested that mutant IDH1 could induce leukemogenesis only in the presence of ASXL1. In our study, we found that the presence of IDH1 R132H mutation in 32D-cl3 cells had an inhibitory effect on normal myeloid differentiation only in the presence of ASXL1 mutation. Transcriptome analysis was performed to explore genes dysregulated by cooperation of mutant ASXL1 and mutant IDH1. We demonstrated that there might be cooperative mechanisms between ASXL1 and IDH1 to induce differentiation and promote leukemogenesis. Our study would not only provide a new role for IDH1 mutation in ASXL1 mutated AML, but also identify a new therapeutic target for ASXL1-mutatated AML.

ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis. Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017). Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

P544: MOLECULAR PREDICTORS OF RESPONSE AND SURVIVAL IN TREATMENT-NAÏVE PATIENTS WITH ACUTE MYELOID LEUKEMIA FOLLOWING VENETOCLAX AND HYPOMETHYLATING AGENTS

Background: Venetoclax (Ven) in combination with hypomethylating agents (HMA) is FDA-approved for elderly/unfit AML patients. Limited data exists on molecular predictors of response and survival. In a prior study, response was superior (CR/CRi >80%) with NPM1, IDH1/2, DNMT3A mutations, inferior with TP53, RUNX1, FLT3/ITD, RAS and prolonged survival with NPM1 and IDH2 mutations (2 yr OS 71.8%/79.5%) (DiNardo, Blood, 2020). Aims: Our objective was to determine the impact of mutations on response and survival in treatment-naïve AML patients receiving Ven+HMA. Methods: Treatment-naïve AML patients receiving Ven+HMA outside a clinical trial at the Mayo Clinic were included. Molecular studies were performed by next-generation sequencing. Response was assessed according to the 2017 European LeukemiaNet (ELN) criteria. Standard statistical analyses were performed using JMP Pro (Version 16.0.0). Results: i) Patient characteristics 103 AML patients (median age 74 years, 67% male, 62% de novo) received upfront Ven+HMA. ELN cytogenetic risk included favorable (6%, n=6), intermediate (50%, n=52) or adverse (44%, n=45). Mutations involved TP53 in 25 patients (25%), TET2 in 24 (23%), IDH1/IDH2 in 20 (19%), RUNX1 in 19 (19%), ASXL1 in 18 (18%),), SRSF2 in 18 (18%), K/NRAS in 15 (15%), NPM1 in 13 (13%), DNMT3A in 13 (13%), FLT3-ITD in 10 (10%) patients. ii) Predictors of response 40 (39%) patients achieved complete remission (CR), 20 (19%) CR with incomplete hematological recovery (CRi), resulting in CR/CRi in 60 (58%) patients. In univariate analysis, presence of ASXL1 mutation was associated with favorable response (CR/CRi 83% vs 53%, p=0.01), secondary AML (CR/CRi 49% vs 65%, p=0.09), adverse karyotype (49% vs 67%, p=0.11), presence of TP53 (32% vs 67%, p=0.002) and FLT3-ITD mutations (30% vs 61%; p=0.06) predicted inferior response. In multivariable analysis, including the aforementioned variables, presence of ASXL1 mutation (83% vs 53%; OR 4.5) and absence of TP53 (67% vs 32%; OR 3.3) and FLT3-ITD mutations predicted favorable response (61% vs 30%; OR 6.4). Moreover, in ASXL1 mutated patients, CR/CRi was not impacted by presence of TP53 mutation (100% vs 81%) whereas in ASXL1 unmutated patients, presence of TP53 mutation predicted inferior response (26% vs 63%; p=0.001). Presence of NPM1 (CR/CRi; 69% vs 57%, p=0.41), IDH1/2 (70% vs 55%; p=0.23), DNMT3A (54% vs 59%; p=0.73), RUNX1 (58% vs 58%; p=0.1), RAS (60% vs 58%, p=0.88) did not impact response. iii) Predictors of survival After a median follow up of 6.6 months, 68 patients died and 9 underwent allogeneic stem cell transplant. Median overall survival (mOS) was 8.5 months and longer in transplanted patients (not reached vs 8.4 months, p=0.08). Age-adjusted analysis in 94 patients not transplanted, identified CR/CRi (p<0.0001), NPM1 (p=0.009), IDH1/2 mutations (p=0.02) as favorable, and TP53 (p=0.01), ASXL1 mutations (p=0.17) adverse karyotype (p=0.05) unfavorable risk factors for survival. Despite higher CR/CRi, ASXL1 mutated patients had shortened mOS due to higher relapse. Multivariable analysis confirmed the negative survival impact of not achieving CR/CRi, ASXL1 mutation and adverse karyotype. Accordingly, a three-tiered model was generated by using the three variables (Figure 1). Image:Summary/Conclusion: Our observations differ from those of DiNardo, et al. (Blood, 2020). The current study identifies presence of ASXL1 mutation and absence of TP53 and FLT3-ITD mutations as predictors of favorable response. Additionally, we propose a novel three-tiered survival model based on response, ASXL1 mutation and karyotype.

Real‐world experience with venetoclax and hypomethylating agents in myelodysplastic syndromes with excess blasts

Real‐world experience with venetoclax and hypomethylating agents in myelodysplastic syndromes with excess blasts

Mutation Landscape and Prognostic Correlates of ASXL1 Variants in Primary and Secondary Myelofibrosis

▪Introduction: Myelofibrosis (MF), whether primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocytemia, is characterized by a complex and partially undeciphered molecular architecture. Besides mutations in driver genes (JAK2, CALR, MPL), somatic mutations in selected myeloid-associated genes have been shown to impact prognosis of MF patients (pts). Among these, ASXL1 mutations (ASXL1MTs) are associated with poor outcomes in myeloid malignancies including PMF, where they are included in the category of “high molecular risk” (HMR) mutations along with EZH2MTs, IDH1/2MTs, and SRSF2MTs (Vannucchi AM, Leukemia 2013). However, a recent study (Luque Paz D, Blood Adv 2021) questioned the value of ASXL1MTs in MF. The current study aimed at further characterizing the prognostic role of ASXL1MTs in MF.Methods: After IRB approval, pts with WHO-defined MF were included in the study. Mutational analysis by targeted NGS was performed as previously described (Guglielmelli P, JCO 2017). All deposited variants were manually curated to assess pathogenicity. In this study, we also used the molecular model proposed by Luque Paz et al. that identifies 4 genetic groups: TP53MT; High-risk (≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1/2); ASXL1MT-only; and “Others”.Results: A total of 525 pts were included in the study, including 331 (63%) PMF and 194 (37%) SMF. Median age at diagnosis was 89 (18-90) years, 314 (60%) were male. The median follow-up time was 80 (98% CI, 68-90) months. Overall, 324 (62%) pts were JAK2MT, 126 (24%) CALRMT, 24 (5%) MPLMT, 40 (8%) triple negative (TN), and 11 (2%) double mutated. Among non-driver genes, ASXL1MTs were found in 158 (30%) pts, EZH2MTs in 45 (9%), SRSF2MTs in 37 (7%), NRASMTs in 30 (6%) U2AF1MTs in 27 (5%), TP53MTs and CBLMTs in 25 (5%) each, IDH1/2MTs in 18 (3%), and KRASMTs in 15 (3%). Pts in the HMR category were 125 (38%) in PMF and 63 (32%) in SMF.According to the above model, distribution of pts was as follows: TP53MT n=25 (5%), High-risk n=137 (26%), ASXL1MT-only n=64 (12%), and Others n=299 (57%). Pts in the TP53MT and ASXL1MT-only groups were more likely to be diagnosed with SMF compared to pts in the High-risk and Others groups (44% and 48% vs 28% and 38%, respectively). In addition, the High-risk group was enriched in TN pts (16%), while CALRMTs were more common in the ASXL1MT-only and Others compared to the TP53MT and High-risk groups (25% and 27% vs 12% and 18%, respectively).In univariate analysis, the TP53MT and High-risk groups were associated with the worst overall survival (OS), with median values of 38 (14-110) and 55 (45-85) months (P=.0039), respectively (Fig 1A). Albeit remarkably better, the OS of pts in the ASXL1MT-only group was inferior compared to pts in the Others group (median 124 [91-156] vs 193 [142-NR] months; P=.0118) (Fig 1A).We then analyzed separately PMF and SMF cohorts. In the former, the TP53MT and High-risk groups remained associated with the worst OS (median 58 [20-126] vs 55 [36-85] months), although with no significant difference, likely due to the low frequency (4%) of TP53MTs mutations in PMF (Fig 1B). Concurrently, the negative prognostic impact of the ASXL1MT-only group was confirmed in comparison to the Others group (median 103 [78-NR] vs 320 [178-NR] months; P=.0170). In pts with SMF, while the TP53MT group (6%) had by far the worst OS (median 13 [6-NR] months), the OS of the ASXL1MT-only group (median 141 [56-171] months) was comparable to that of the Others group (median 131 [106-NR] months; P=.5188) and not different from the High-risk group (median 58 [45-174] months; P=.3606) (Fig 1C). In a further analysis including only pts in the High-risk group, ASXL1MTs were found in 62% and 63% of patients with PMF and SMF, respectively. In survival analysis, the presence of ASXL1MTs was associated with an increased risk of death only in PMF (median OS 47 [31-73] vs 102 [34-317] months; P=.0240), unlike in SMF (median OS 90 [47-174] vs 25 [16-338] months; P=.3296) (Fig 1D-E).Conclusion: In the current study, we critically re-addressed the prognostic impact of ASXL1MTs by applying a genetic model recently developed by Luque Paz et al. to our cohort of molecularly annotated, WHO-defined MF pts. Overall, our results confirm that ASXL1MTs -even in the absence of other co-occurring high-risk mutations- harbor a negative prognostic impact mainly in PMF. These findings also reinforce the idea that PMF and SMF represent two different biological entities. [Display omitted] DisclosuresVannucchi: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Prognostic Impact of ASXL1 Mutations and Extent of Bone Marrow Dysplasia in Patients with Who - defined Myelodysplastic Syndrome with Ring Sideroblasts

Introduction:The 2016 World Health Organization (WHO) classification of myeloid neoplasms defines myelodysplastic syndrome, ring sideroblasts (MDS-RS) by the presence of dysplasia in single or multiple cell lineages with at least 15% ring sideroblasts (RS) or as few as 5% RS in the presence of SF3B1 mutations (Arber et al. Blood 2016). MDS-RS is further categorized into: MDS-RS with single lineage dysplasia (SLD, previously refractory anemia with RS) & MDS-RS with multi-lineage dysplasia (MLD, previously refractory cytopenias with MLD & RS). We carried out this study to define clinical & molecular differences between MDS-RS-SLD versus MDS-RS-MLD & assess survival outcomes.Methods:Successive cases of MDS-RS, meeting the 2016 WHO criteria were identified from our institutional database. In compliance with the WHO definition, only de novo cases were included. All patients had bone marrow (BM) biopsies & cytogenetics at diagnosis. Bone marrow slides including Prussian blue stains for RS were re-reviewed. Targeted exome sequencing for the following genes; TET2, ASXL1, DNMT3A, IDH1, IDH2, TP53, SRSF2, SF3B1, SH2B3, NPM1, FLT3, U2AF1, ZRSR2, JAK2, CSF3R, MPL, MFSD11, CEBPA, SETBP1, ZRSR2, RUNX1, IKZF1, CALR, KRAS, NRAS, CBL, PTPN11, STAG2, BCOR & GATA2, were carried out on BM specimens obtained at diagnosis . Statistics were performed via JMP Pro software (version 10).Results:Eighty seven patients with MDS-RS were included in the study, median age 73 years (range: 44-88), 30 (34%) males. Of these, 63 (72%) were categorized as MDS-RS-SLD & 24 (28%) as MDS-RS-MLD. Twenty-two (25%) had an abnormal karyotype, of which 8 (33%) had MDS-RS-MLD (p=0.30). Overall R-IPSS stratification included 2 (2%) very low, 51 (59%) low, 22 (25%) intermediate, 9 (10%) high & 3 (4%) very high risk categories, respectively. Targeted exome sequencing in 82 patients demonstrated the following mutational frequencies; SF3B1 61%, ASXL1 18%, DNMT3A 10%, TET2 5%, TP53 4%, IDH-1 2%, SRSF2 2%, and 1% each for PTPN11, ZRSR2, CSF3R &U2AF1 (Table 1).With the exception for a higher frequency of SF3B1 (63% vs 42%, p=0.02) and DNMT3A (13% vs 0%, p=0.02); and a lower frequency of ASXL1 (10% vs 38%, p=0.005) mutations in the MDS-RS-SLD group, there were no other differences between the two groups.At a median follow-up of 34 months, 77 (89%) deaths & 4 (4.6%) leukemic transformations were documented. Median overall survival (OS) for the entire MDS-RS cohort was 49 months (Range: 0-190); 60 (range: 0-190) and 47 (range 0-123) months for MDS-RS-SLD and MDS-RS-MLD groups respectively (p=0.2). On a univariate survival analysis that included age, sex, hemoglobin (Hb), white blood cell (WBC) count with individual differential counts, platelet counts, peripheral blood blasts, BM blasts, karyotype and the aforementioned gene mutations; only abnormal karyotype (p=0.03), lack of SF3B1 mutations (p=0.03) & presence of ASXL1 mutations (p=0.01, figure 1A) adversely impacted OS. Interestingly, the presence or absence of multilineage dysplasia did not impact outcomes (p=0.18) (Figure 1B). In a multivariate model that included the presence of ASXL1, absence of SF3B1 mutations and abnormal karyotype, only the presence of ASXL1 mutations (p=0.007, HR 2.3, 95% CI 1.2-4.18) retained prognostic significance. Given the small number of events, leukemia free survival analysis was not carried out.Conclusions:In the revised 2016 WHO category of MDS-RS, overall survival was independently and adversely impacted by the presence of ASXL1 mutations, with the extent of bone marrow dysplasia not affecting outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of Somatic Mutations in Clonal Evolution of Chronic Myeloid Leukemia from Chronic Phase to Blast Phase

BackgroundRole of somatic mutations in Chronic Myeloid Leukemia (CML) is fairly underexplored. We hypothesize that clonal evolution of chronic phase CML (CP-CML) to blast phase CML (BP-CML) is molecularly driven thus augmenting on previous findings of ASXL1 being a commonly recurrent mutation in CML. Therefore, early detection of specific somatic mutations can predict progression of disease.MethodsWe performed a retrospective chart review of clinically and molecularly annotated 60 cases of CML from Moffitt Cancer Center. Genotyping was performed using next generation targeted DNA sequencing detecting for common recurrent mutations in hematological malignancies. Fisher's Exact and Student T-test were used to determine statistical significance (p-value of <0.05). All calculated p values were two sided.ResultsTotal 48 patients with CP-CML and 12 patients with BP-CML were included. Median age was 56 and 53.5 years in CP-CML and BP-CML cohort, respectively. Among CP-CML cohort, 37.5% (n=18) had at least 1 mutation with ASXL1 being the most frequently mutated gene (27.7%, n=5) where 80% (n=4) were isolated and only 20% (n=1) had comutation in RUNX1 (Figure 1A). In BP-CML patients, at least 1 mutation was detected in 100% of the cases (n=12) with ASXL1 being the most frequently mutated gene (67%, n=8) (Figure 1B). In contrast to the CP-CML cohort, ASXL1 mutation co-occurred with other mutations in 87.5% of the cases (n=7). The most frequent co-occurring mutations were TET2 (37.5%, n=3) and CUX1 (37.5%, n=3) followed by RUNX1 (25%, n=2), SETBP1 (25%, n=2), TP53 (25%, n=2) and PHF6 (12.5%, n=1). Majority of the BP-CML comprised of myeloid BP (83%) where ASXL1 mutations were exclusively found. Lymphoid BP (16%, n=2) cases revealed lymphoid specific mutations in CDKN2A and IKZF1 genes.In BP-CML compared to CP-CML, presence of ASXL1 mutation was more frequent (OR 17.2, 95% CI 3.77-78.32, p=0.002) with increased frequency of co-occurrence with at least one other mutation (OR 28.0, 95% CI 1.35-580.0, p=0.032). In BP-CML cohort, 83% (n=10) of the patients had additional cytogenetic abnormalities (ACAs) and only 16.5% (n=2) had isolated standard Philadelphia chromosome. In contrast to BP-CML, there was no correlation between presence of ACAs and presence of mutations in CP-CML cohort.ConclusionsAlthough this study is comprised of a small sample size, the findings suggest that the presence of ASXL1 in CP-CML may represent an early event or “first hit”, which then acquires an additional mutation or “second hit”, contributing to progression to advanced phase CML. These findings need to be validated in a large prospective cohort study. [Display omitted] DisclosuresChavez:Abbvie: Speakers Bureau; Janssen: Speakers Bureau; Kite: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Pinilla-Ibarz:Pfizer: Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria; BMS: Honoraria, Speakers Bureau.

Prognostic Factors and Survival Outcomes in Blast Transformed Chronic Myelomonocytic Leukemia: A Mayo Clinic-MD Anderson Cancer Center Study of 171 Cases

Background:Blast transformed (BT) chronic myelomonocytic leukemia (CMML); defined by ≥20% blood or bone marrow (BM) blasts, occurs in 15-30% of patients and is a major cause of mortality. Reported risk factors include high risk karyotype, circulating blast count, circulating immature myeloid cells (IMC), CMML morphological subtype, and the presence of ASXL1 mutations. In the current study, we reviewed consecutive cases of BT CMML from the Mayo Clinic, MN, and the MD Anderson Cancer Center, Texas, USA, with the intent to examine i) prognostic factors, ii) survival trends, and iii) treatment outcomes.Methods:Diagnoses of CMML and BT-CMML were according to the 2016 WHO criteria. Treatment response in BT-CMML patients was assessed according to standard AML response criteria. Statistical analyses considered clinical and laboratory data collected at the time of BT. Survival was calculated from the date of BT to the date of death or last contact.Results:171 cases of BT CMML were identified from the Mayo Clinic (n=84) and MDACC (n=87). The median age at BT was 69 years and 64% were male. Eight one (49%) had received prior hypomethylating agent (HMA) therapy (Decitabine 50, 5-azacitidine 31), with 30% having achieved a complete remission (CR) prior to BT. CMML patients that received prior HMA therapy were more likely to have thrombocytopenia (p=0.03), higher LDH levels (p=0.007), lower frequency of TET2 mutations (p=0.04) and higher risk stratification by the Mayo prognostic model (p=0.009). The two institutional cohorts were comparable with regards to variables, both at CMML diagnosis and BT.Patient characteristics at time of BT: The median time from CMML diagnosis to BT was 12 months (range, 1-36). At BT, 16 (10%) presented with extramedullary disease, 81 (63%) with an abnormal karyotype, 41 (34%) with cytogenetic clonal evolution, with the following mutational distribution; TET2 50%, ASXL1 39%, NRAS 39%, FLT3 ITD 20%, NPM1 6%, RUNX1 6%, and 5% each for IDH1/2, EZH2 and JAK2 . Molecular clonal evolution was seen in 4 (22%) of 18 evaluable cases.Survival trends: After a median follow-up of 4.4 months (range 0-122), 141 (82%) deaths were recorded. Median overall survival (OS) was 6 months with 1- and 3-year survival rates of 25% and 9%, respectively; survival trends were similar (p=0.4) for patients diagnosed prior to and after the year 2006 (figure 1A).Treatment and response rates: Specific treatment for BT-CMML was documented in 157 cases and included induction chemotherapy (n=61), induction chemotherapy followed by stem cell transplant (HCT, n=18), HCT alone (n=3), HMA (n=18), novel therapeutic agents (n=18), and best supportive care (BSC, n=39). Treatment response was assessable in 113, including 76 treated with induction chemotherapy, 18 with HMA and 16 with novel therapeutics; with respective CR rates of 24%, 0% and 41%; with an additional 24%, 16% and 16% of patients achieving CR with incomplete blood count recovery (CRi), with a survival similar to those with CR (figure 1B). HCT was reported in 25 patients, 17 myeloablative and 8 reduced intensity, with a median OS of 7 months.Predictors of survival in BT-CMML: In univariate analysis of variables recorded at time of BT, risk factors adversely impacting survival included older age, lower hemoglobin, PB blast %, adverse cytogenetics at BT, prior exposure to HMA, not receiving induction chemotherapy, not achieving CR or CRi and not receiving HCT; all of these risk factors remained significant when analysis was adjusted for age. In multivariable analysis, lack of prior HMA exposure (p=0.001, HR 5.1; 95% CI 1.9-14.1) and the achievement of CR/CRi (p=0.02, HR 1.4; 95% CI 1.2-3.4) remained significant while HCT lost its significance (p=0.09). Gene mutations, including FLT3 ITD, cytogenetic and molecular clonal evolution at the time of BT did not impact survival. The 3-year survival rates for BT patients receiving induction chemotherapy, induction chemotherapy followed by HCT, HMA, novel agents and BSC were 8%, 20%, 12%, 3% and 0% respectively (figure 1C).Conclusions: Survival in patients with BT- CMML is dismal (≈6 months) and is adversely impacted by prior exposure to HMA and by the failure to achieve CR/CRi. After BT, the use of HMA therapy is largely ineffective (0% CR), with intensive therapies such as induction chemotherapy with or without HCT, failing to achieve durable remissions. [Display omitted] DisclosuresJabbour:Amgen: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Takeda Oncology: Research Funding. Kantarjian:Amgen: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding.

A prognostic score including mutation profile and clinical features for patients with CMML undergoing stem cell transplantation

The inclusion of mutation status improved risk stratification for newly diagnosed patients with chronic myelomonocytic leukemia (CMML). Stem cell transplantation is a potentially curative treatment option, and patient selection is critical because of relevant transplant-related morbidity and mortality. We aimed to evaluate the impact of mutation status together with clinical presentations on posttransplant outcome. Our study included 240 patients with a median follow-up of 5.5 years. A significant association with worse survival was identified for the presence of mutations in ASXL1 and/or NRAS. In multivariable analysis, ASXL1- and/or NRAS-mutated genotype (hazard ratio [HR], 1.63), marrow blasts >2% (HR, 1.70), and increasing comorbidity index (continuous HR, 1.16) were independently associated with worse survival. A prognostic score (CMML transplant score) was developed, and the following points were assigned: 4 points for an ASXL1- and/or NRAS-mutated genotype or blasts >2% and 1 point each for an increase of 1 in the comorbidity index. The CMML transplant score (range, 0-20) was predictive of survival and nonrelapse mortality (P < .001 for both). Up to 5 risk groups were identified, showing 5-year survival of 81% for a score of 0 to 1, 49% for a score of 2 to 4, 43% for a score of 5 to 7, 31% for a score of 8 to 10, and 19% for a score >10. The score retained performance after validation (concordance index, 0.68) and good accuracy after calibration. Predictions were superior compared with existing scores designed for the nontransplant setting, which resulted in significant risk reclassification. This CMML transplant score, which incorporated mutation and clinical information, was prognostic in patients specifically undergoing transplantation and may facilitate personalized counseling.

Allogeneic hematopoietic stem cell transplantation could improve the survival of acute myeloid leukemia patients with ASXL1 mutations

ABSTRACT Objective: To discover the function of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in ASXL1-mutated acute myeloid leukemia (AML) patients. Methods: We analyzed the prognostic value of ASXL1 mutations and explored the role of allo-HSCT in 581 AML patients. Results: According to the definition of intermediate- and adverse-risk AML groups in the European Leukemia Net (ELN), ASXL1-mutated patients had shorter OS and DFS than ASXL1-wild-type patients in the intermediate- and adverse-risk AML groups (3-year OS: 47.5% vs. 60.8%, P<0.001; 3-year DFS: 28.5% vs. 48.9%, P<0.001). Among the cytogenetically normal acute myeloid leukemia (CN-AML), differences were found in both OS (47.4% vs.65.2%, P<0.001) and DFS (21.0% vs. 52.1%, P<0.001) between ASXL1-mutated patients and ASXL1 wild-type patients. In the ASXL1-mutated AML cohort, the patients received allo-HSCT had longer 3-year OS (P=0.0005) and 3-year DFS (P<0.0001) than those who did not receive allo-HSCT. Multivariate analysis revealed that ASXL1 mutation was an independent prognostic factor for OS (HR 2.248, 95% CI 1.155-4.375, P=0.017), and allo-HSCT had a positive impact on OS (HR 7.568, 95% CI 3.597-15.92, P<0.001) and DFS (HR 2.611, 95% CI 1.688-4.039, P<0.001) in ASXL1-mutated patients. Conclusion: The results indicate that the presence of ASXL1 mutations is a factor predictive of poor prognosis in AML patients and allo-HSCT could improve the survival of AML patients with ASXL1 mutations.

ASXL1 mutations, previous vascular complications and age at diagnosis predict survival in 85 WHO-defined polycythaemia vera patients.

Polycythaemia vera (PV) patients have an overall comparatively favourable prognosis, but disease progression is very heterogeneous and life-threatening thrombosis and bleedings are frequent complications in untreated disease. Moreover, transformation to more severe secondary myelofibrosis and acute myeloid leukaemia can occur. The aim of this study was to identify gene mutations that could be used together with clinical data as prognostic markers to guide treatment decisions in PV patients. A well-characterized WHO-defined cohort of PV patients was used. Clinical data and blood values were evaluated and a myeloid sequencing panel was used to screen for additional mutations other than the diagnostic JAK2 V617F and JAK2 exon 12 mutations. In 78% of the PV patients, at least one mutation additional to JAK2 V617F was detected. Additional mutations in genes coding for epigenetic modifiers, like TET2, DNMT3A and ASXL1, were most frequent. When correlated to overall survival, mutations in ASXL1 were significantly associated with inferior survival. In an attempt to obtain prognostic guidance in a larger number of patients, the presence of ASXL1 mutations was combined with age and vascular complications prior to diagnosis. Based on these data we were able to define three risk groups that predicted survival.

Impact of the variant allele frequency of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 on the outcomes of patients with newly diagnosed acute myeloid leukemia.

The impact of the allelic burden of ASXL1, DNMT3A, JAK2, TET2, and TP53 mutations on survival remains unclear in patients with newly diagnosed acute myeloid leukemia (AML). The authors assessed bone marrow aspirates from 421 patients with newly diagnosed AML using next-generation sequencing for ASXL1, DNMT3A, JAK2, TET2, and TP53 mutations, defined as the presence of mutations in ASXL1, DNMT3A, JAK2, TET2, or TP53 with a minimum variant allele frequency (VAF) of 5%. A total of 71 patients (17%) had ASXL1 mutations, 104 patients (25%) had DNMT3A mutations, 16 patients (4%) had JAK2 mutations, 82 patients (20%) had TET2 mutations, and 86 patients (20%) had TP53 mutations. Among patients with each mutation, the median VAF of ASXL1 was 34.31% (range, 1.17%-58.62%), the median VAF of DNMT3A was 41.76% (range, 1.02%-91.66%), the median VAF of JAK2 was 46.70% (range, 10.4%-71.7%), the median VAF of TET2 was 42.78% (range, 2.26%-95.32%), and the median VAF of TP53 was 45.47% (range, 1.15%-93.74%). The composite complete response rate was 60%, and was 77% in patients with AML with and without ASXL1, DNMT3A, JAK2, TET2, or TP53 mutations, respectively (P=.006); the median overall survival was 11months and 27months, respectively (P<.001). Multivariate analysis identified age; an antecedent history of dysplasia; white blood cell count; adverse cytogenetic risk; previous treatment with an FLT3 inhibitor; and the VAF of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 mutations by next-generation sequencing as prognostic factors for overall survival. The VAF of ASXL1, DNMT3A, JAK2, TET2, TP53, and NPM1 mutations is associated with worse prognosis in patients with newly diagnosed AML.

PF678 MOLECULAR PROFILE AND RISK OF THROMBOSIS IN JAK2V617F POLYCYTHEMIA VERA PATIENTS

Background:The pathogenesis of PV‐associated thrombosis is multifactorial. Prior thrombosis, advanced age, leukocytosis, JAK2V617F mutation, and cardiovascular risk factors are the main factors influencing thrombotic risk. However, there is scarce information regarding the role of mutations in non‐driver genes in the development of thrombotic events during clinical follow‐up.Aims:To investigate the influence of the molecular profile characterized at PV diagnosis in the probability of developing thrombosis during clinical evolution.Methods:A total of 127 JAK2V617F PV patients were included in the study. JAK2V617F allele burden was assessed by quantitative allele specific PCR in the peripheral blood granulocytes at the moment of diagnosis. Targeted NGS was performed on DNA extracted from granulocytes also at diagnosis. Amplicon libraries were constructed using QIAseq Targeted DNA custom panel (Qiagen, Hilden, Germany), covering the full exonic regions of 25 commonly mutated genes in myeloid malignancies and were sequenced using either MiSeq or NextSeq (Illumina, San Diego, CA, USA). Time to event curves were drawn by the method of Kaplan and Meier with the log rank test for comparisons.Results:With a median follow‐up of 50 months (range 1–200), fourteen patients developed 20 thrombotic events after diagnosis (10.9%), 17 corresponding to arterial thrombosis (85% of the total thrombotic episodes) and 3 to venous thrombosis. Five patients presented more than one thrombotic event. Cerebrovascular disease was the most frequent condition in the group of arterial thrombosis (41%). Median time between diagnosis and the appearance of the first thrombosis was 28 months (range 5.8–80). Median JAK2V617F allele burden was 54% (range 5–99). The distribution by quartiles of the JAK2V617F mutational load was as follows: 0–24% n = 15 (11.8%), 25–49% n = 43 (33.9%), 50–74% n = 33 (26%), 75–100% n = 36 (28.3%). There was no statistically significant difference in thrombosis free survival according to the baseline mutational load of JAK2V617F. A total of 104 somatic mutations other than JAK2V617F were detected at diagnosis in 61 patients (48%). The mean number of pathogenic additional mutations was 0.8 mutation/patient. The most frequently mutated genes were TET2 (36%), ASXL1 (13.2%) and DNMT3A (12.5%). Overall, no difference was observed in the probability of developing thrombosis during the follow‐up according to the presence or absence of pathogenic non‐driver mutations at diagnosis (p = 0.89)(Figure 1a). Nevertheless, we showed a significant correlation between the presence of ASXL1 mutations and the probability of thrombosis (35% thrombotic events in ASXL1 mutated patients vs 8% in wild‐type ASXL1 patients, p = 0.002) (Figure 1b). No significant difference was observed in thrombosis‐free survival according to mutations in TET2 or DNTM3A genes.Summary/Conclusion:Overall, no significant association was observed between the genetic profile (including JAK2V617F allele burden and pathogenic non‐driver mutations) of PV patients at diagnosis and the probability of thrombosis during clinical evolution. However, the subset of ASXL1 mutated patients showed a higher risk of thrombosis. Additional studies are needed to confirm this finding.imageThis study was supported in part by grants from ISCIII and Spanish Ministry of Health, PI16/0153, 2017SGR205, PT17/0015/0011. Beca Gilead 2016 and Xarxa de Banc de Tumors de Catalunya

Predictors of Spleen and Anemia Response to Specific Drugs in Primary Myelofibrosis

Background:Current drug therapy in primary myelofibrosis (PMF) offers palliative value only, including alleviation of splenomegaly, constitutional symptoms and anemia; hydroxyurea (HU), JAK2 inhibitors and interferon (IFN)-α are used for splenomegaly; JAK2 inhibitors for constitutional symptoms; and immunomodulatory drugs (IMiDs), erythropoiesis-stimulating agents (ESAs) and androgens for anemia. The current study constitutes a retrospective evaluation of specific drug response in anemia or splenomegaly in PMF.Methods:Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). For the purposes of the current study, conventional response criteria were modified to reflect clinical benefit assessment without strict adherence to criteria designed for clinical trials; accordingly, spleen response was evaluated only in patients with palpable splenomegaly and was defined as a minimum 50% reduction in palpable spleen size, regardless of response duration; anemia response was evaluated only in patients with hemoglobin level <10 g/dl and was defined as achieving transfusion-indpendence lasting for at least one month or an increase in hemoglobin of 2 g/dl, regardless of response duration. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all statistical calculations.Results:432 cytogenetically- and molecularly-annotated patients with PMF were accessed in order to identify 333 patients who received first-line treatment with HU (n=97), JAK2 inhibitors (n=41), IFN-α (n=22), IMiDs (n=58), androgens (n=19), ESAs (n=54) and various other drugs (n=42), and were evaluable for response; driver mutational status was JAK2 59%, CALR type 1/like 20%, CALR type 2/like 4%, MPL 7% and triple-negative 10%; karyotype included very high risk (VHR) 6%, unfavorable 18% and favorable 76%; 60% of the patients harbored high molecular risk (HMR) mutations including ASXL1 (45%), SRSF2 (18%), U2AF1Q157 (10%), EZH2 (4%), IDH2 (4%) and IDH1 (2%). MIPSS70+ version 2.0 risk distribution was very high 17%, high 46%, intermediate 21%, low 14% and very low 2%.Overall, 249 patients were evaluable for spleen response, including 218 that were treated with the specific drugs analyzed in the current study. Anemia response was evaluated in 222 patients, including 194 (105 transfusion-dependent) that were treated with the specific drugs analyzed in the current study.Predictors of response to HU:Spleen response to HU was more likely in the presence of HMR mutations (37% vs 11%; p=0.02); however, responses were mixed and limited to presence of ASXL1 (40% vs 17%; p=0.06) or SRSF2 (47% vs 22%; p=0.1) mutations while none of 8 patients with either U2AF1Q157 or IDH2 mutations responded. None of 5 patients with VHR karyotype and only 1 (9%) of 11 patients with platelet count <100 x 109/l responded. Anemia responses to HU were infrequent. Overall response rate to HU was predicted by the absence of U2AF1Q157 mutations (64% vs 0%; p=0.007).Predictors of response to JAK2 inhibitors:Spleen response to JAK2 inhibitors was more likely in female patients (87% vs 50%; p=0.01) and in the presence of CALR type 1/like mutations (90% vs 57%; p=0.06). Anemia responses to JAK2 inhibitors were largely unpredictable. In order to further verify the aforementioned observed associations, we accessed data from a previous formal clinical trial of momelotinib (JAK2 inhibitor); among 91 evaluable patients, spleen response was higher in the presence of CALR mutations (73% vs 37%; p=0.009) and female sex (49% vs 39%, p=NS).Predictors of response to other agents:Spleen response to IFN-α was unlikely in the presence of ≥2% circulating blasts (0/6 responded) or presence of ASXL1 mutations (0/7 responded). IFN-α was ineffective for the treatment of anemia. Spleen responses to treatment with IMiDs, androgens or ESAs were unusual while anemia response to all three agents was not predicted by either genetic or clinical markers.Conclusions:Our observations, which require additional examination in a prospective setting, suggest limited value of genetic and clinical markers in predicting response to currently available drugs for PMF; this is consistent with the non-specific mechanism of action for these drugs. CALR (JAK inhibitors) and U2AF1Q157 (HU) mutations respectively predict favorable or unfavorable spleen response. DisclosuresNo relevant conflicts of interest to declare.

Correlations between Pre-Transplant Treatment and Bone Marrow Response with Post-Transplant Outcomes in Patients with Myelodysplastic Syndrome Undergoing Allogeneic Stem Cell Transplantation

Correlations between Pre-Transplant Treatment and Bone Marrow Response with Post-Transplant Outcomes in Patients with Myelodysplastic Syndrome Undergoing Allogeneic Stem Cell Transplantation

Przeszczepienie allogenicznych krwiotwórczych komórek macierzystych w leczeniu chorych na mielofibrozę

Within Ph-negative myeloproliferative types of cancer, myelofibrosis is the most frequent indication for performing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because this is a high-risk procedure, only those patients with a predicted overall survival below 5 years become eligible, as well as those of intermediate-2 or high disease risk; as defined by the IPSS, DIPSS and DIPSS Plus prognostic indices. It has however recently been recognised that patients with a poor prognosis, who belong to the intermediate-1 disease risk group, need to be distinguished and also considered for allo-HSCT. According to ELN/EBMT recommendations from 2015, this group should include patients requiring red blood cell concentrate transfusions who have more than 2% blasts in the circulation and/or an adverse karyotype. When qualifying patients for transplantation, consideration should also be given to adverse molecular risk factors such as the so-called triple negativity (absence of JAK, CALR and MPL mutations) and the presence of ASXL1 mutation. Over recent years, the number of allo-HSCT performed on patients with MF has significantly risen because the number of transplantations using reduced-intensity conditioning regimens has increased, together with those from unrelated donors (cord blood, haploidentical donors). There is a lack of randomised studies comparing the effectiveness of this transplantation procedure according to the types of conditioning and donor selection.