The Role of Nucleophosmin 1 (NPM1) Mutation in the Diagnosis and Management of Myeloid Neoplasms

Nucleophosmin (NPM1) is a multifunctional protein with both proliferative and growth-suppressive roles in the cell. In humans, NPM1 is involved in tumorigenesis via chromosomal translocations, deletions, or mutation. Acute myeloid leukemia (AML) with mutated NPM1, a distinct diagnostic entity by the current WHO Classification of myeloid neoplasm, represents the most common diagnostic subtype in AML and is associated with a favorable prognosis. The persistence of NPM1 mutation in AML at relapse makes this mutation an ideal target for minimal measurable disease (MRD) detection. The clinical implication of this is far-reaching because NPM1-mutated AML is currently classified as being of standard risk, with the best treatment strategy (transplantation versus chemotherapy) yet undefined. Myeloid neoplasms with NPM1 mutations and <20% blasts are characterized by an aggressive clinical course and a rapid progression to AML. The pathological classification of these cases remains controversial. Future studies will determine whether NPM1 gene mutation may be sufficient for diagnosing NPM1-mutated AML independent of the blast count. This review aims to summarize the role of NPM1 in normal cells and in human cancer and discusses its current role in clinical management of AML and related myeloid neoplasms.

Azacitidine to Consolidate and Deepen the Therapeutic Response Achieved by Intensive Induction Treatment in a Young Patient Affected by NPM1mut-AML Who Has Become Ineligible for High-Dose Consolidation

Acute myeloid leukemia (AML) is the most common leukemia in adults. In spite of the most recent discoveries about the molecular landscape of this disease, the treatment of elderly and unfit young patients continues to be a great challenge. The hypomethylating agents (HMA) still represent an effective therapeutic option for these categories, especially for the low-risk subgroups. We report the case of a young patient with NPM1^mut-AML who underwent a first cycle of intensive induction treatment, achieving a complete remission, but suffered from a serious life-threatening neurologic toxicity. Due to the ineligibility to further lines of intensive chemotherapy, we decided to consolidate the response with azacitidine, administered according to the regular schedule. The minimal residual disease (MRD), monitored through the NPM1 mutation at diagnosis, progressively decreased and became undetectable after 36 cycles of hypomethylating therapy. After 1 year from discontinuation of azacitidine, MRD remains undetectable. Therefore, HMA might still represent a feasible and effective option for patients with low-risk AML, especially when the standard chemotherapy is not indicated, or as maintenance therapy in nontransplantable patients.

Targeting CD33 for acute myeloid leukemia therapy

Background The aim of this study was to analyze the level of CD33 expression in patients with newly diagnosed AML and determine its correlation with clinical characteristics. Methods Samples were collected for analysis from AML patients at diagnosis. We evaluated the level of CD33 expression by flow cytometry analysis of bone marrow. Chi-square or t- tests were used to assess the association between the high and low CD33 expression groups. Survival curves were generated by the Kaplan-Meier and Cox regression model method. Results In this study we evaluated the level of CD33 expression in de novo patients diagnosed from November 2013 until January 2019. The mean value of 73.4% was used as the cutoff for the two groups. Statistical analysis revealed that 53 of the 86 (61.2%) AML patients were above the mean. Although there was no statistical significance between CD33 expression level and gene mutation, FLT3 mutation (P = 0.002) and NPM1 mutation (P = 0.001) were more likely to be seen in the high CD33 group. The overall survival (OS) was worse in the high CD33 group (39.0 m vs. 16.7 m, x2 = 13.06, P < 0.001). The Cox survival regression display that the CD33 is independent prognostic marker (HR =0.233,p = 0.008). Univariate analysis showed that the high expression of CD33 was an unfavorable prognostic factor. Of the 86 patients, CD33-high was closely related to the patients with normal karyotype (x2 = 4.891,P = 0.027), high white blood cell count (WBC, t = 2.804, P = 0.007), and a high ratio of primitive cells (t = 2.851, P = 0.005). Conclusions These findings provide a strong rationale for targeting CD33 in combination with chemotherapy, which can be considered a promising therapeutic strategy for AML.

Effective Menin inhibitor-based combinations against AML with MLL rearrangement or NPM1 mutation (NPM1c)

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

Umbilical Cord Blood Microtransplantation in Newly Diagnosed, Elderly Patients with Acute Myeloid Leukemia

Objective: We observed the efficacy and safety of umbilical cord blood microtransplantation (UCBMT) in the treatment of newly diagnosed with acute myeloid leukemia (AML) in the elderly. Methods: Prospective one-arm phase I clinical study. The patients should meet the following criteria: 60-80-year-old; newly diagnosed AML; receive the treatment of chemotherapy combined with UCBMT. Result: In total, 11 patients newly diagnosed with AML received chemotherapy in combination with UCBMT, from November, 2019 to January, 2021, including 7 males and 4 females. The average age was 71 (60-80). For the patients, 7 cases with normal chromosome karyotype, and 2 cases with +8 chromosome, 1 case with 7q- chromosome, and 1 case with karyotype of monomer. In the 7 patients with normal chromosome karyotype, 3 cases were FMS-liketyrosine kinease 3 (FLT3) positive (2 of them in combination with nucleophosmin 1 (NPM1) mutation); in addition, in 4 patients of the 7, one showed double mutation of CEBPA, one showed NPM1 mutation, one showed IDH1 mutation, and one showed IDH2 mutation. In 4 patients with chromosomal abnormalities, one patient showed no special gene, one patient showed ASXL1 mutation, one patient was IDH1 mutation, and one patient was TP53 mutation. All of the patients were treated with IA (IDA 8-10 mg/ m 2/day x 3 days, cytarabine 100 mg/ m 2/day x 7 days) for inducing chemotherapy. For the patients with 60-70-year-old, they were treated with IDA (8 mg/ m 2/day); and for the patients with 70-80-year-old, they were treated with darubicin (10 mg/ m 2/day). In the consolidation phase, the patients were treated with cytarabine (1 g/ m 2, q12h) for 3 consecutive days. There were 3 courses of consolidation chemotherapy. Next, patients received single unrelated cord blood transplantation (UCBT) from China's public umbilical cord blood bank, HLA matching was performed for all patients before treatment. A total of 4 units of UCB with HLA 0-3/6（HLA-A,-B,-DR）matching and the ABO blood type matched with the patient were transfused after induction and consolidation chemotherapy for 24-48 hours, then with follow-up. At the same time, the immunological characteristics of these patients were fully analyzed. We demonstrated that, 8 of 11 patients received one course of induction chemotherapy, and achieved a complete response. The complete response rate was 72.7%. What's more, the median time for neutrophils ≥ 0.5 x 10 9/L and platelete ≥ 20 x 10 9/L was 12 days. There were no treatment-related deaths during induction therapy. The median follow-up was 14 (7-31) months. 1 patient showed monomer karyotype with P53 gene mutation, and got complete remission after one course of induction chemotherapy with IA. However, the patient died for AML recurred. For the other 10 patients, they were alive, and the OS of 1 year was 89.8%. Moreover, we found the expression of PD-1 on CD8 +T cells decreased, while the expression of CD38 increased after therapy. Besides, the proportion of NKp30 +NK cells, as well as the IFN-γ +TFN-α +NK cells increased significantly. Conclusion: UCBMT therapy for newly diagnosed elderly AML patients could accelerate the recovery of hematopoietic function and improve the safety of chemotherapy. This method is effective and worthy of further promotion. Disclosures No relevant conflicts of interest to declare.

An Open-Label Phase II Trial of the Combination of Decitabine, SQ Bortezomib and Pegylated Liposomal Doxorubicin for the Treatment of Patients with Relapsed/Refractory Acute Myelogenous Leukemia

Background Relapsed/refractory (R/R) acute myeloid leukemia (AML) remains a challenge to cure. Prior studies of hypomethylating agent (HMA) decitabine (DEC), proteasome inhibitor bortezomib (BTZ), and anthracycline (AC) pegylated liposomal doxorubicin (PLD) monotherapy, as well as DEC + BTZ and BTZ + PLD regimens, have demonstrated safety and modest activity in R/R AML. Inhibition of NF-κB signaling by BTZ and DEC could prevent AC resistance resulting from NF-κB activity. Thus, we hypothesized that the DEC + BTZ + PLD (DBP) regimen would have activity in R/R AML. Methods We performed a phase II trial of DBP, with a safety lead-in cohort, in patients aged 18-90 with R/R AML. The original protocol called for 1-4 28-day (D) cycles of induction with intravenous (IV) DEC 20mg/m 2 on D1-10, subcutaneous (SQ) BTZ 1.3mg/m 2 on D1, 4, 8, and 11, and IV PLD 40mg/m 2 on D4. Dose-limiting toxicity (DLT) of grade 3 peripheral neuropathy (G3 PN) in the first 2 patients led to a revised schedule of BTZ on D5, 8, 12, and 15 and PLD on D12, eliminating simultaneous DBP dosing on any 1 day. Patients achieving a bone marrow blast count &lt;5% after any course of induction proceeded to the continuation regimen: 28-D cycles of DEC on D1-5, BTZ on D1 and 8, and PLD on D12. Treatment continued until progression, intolerance, bone marrow transplant (BMT), study withdrawal, or administration of 12 cycles. Patients reaching lifetime maximum AC exposure could remain on trial with PLD removed from their regimen. Primary endpoint was objective response rate (ORR), defined as complete remission (CR) + CR with incomplete hematological recovery (CRi) + partial remission. Response was based on International Working Group criteria and determined by blood count values between cycles. Secondary endpoints of overall and event-free survival (OS, EFS) were estimated by Kaplan-Meier method. Toxicity was monitored per Common Terminology Criteria for Adverse Events (AEs) v4.03. Results Ten patients were enrolled from May 2016 to February 2018, after which the sponsor closed the protocol. Median age was 57 years [range 27-69]. Patients were 50% female, 60% White, 10% African American/Black, 30% other/mixed race, and 40% Hispanic/Latino, with median baseline ECOG score of 1 [0-1] and median 2 [1-3] lines of prior therapy. Sixty percent had de novo and 40% had secondary disease. By WHO subtype, 30% had AML with MRC, 20% NPM1 mutation, 10% inv(3), 10% therapy-related, and 30% not otherwise specified. European LeukemiaNet 2017 risk was favorable in 20%, intermediate in 40%, and adverse in 40%. Median number of cycles completed was 2 [1-7] with a median time on study of 100.5 days [35-678]. One patient achieved CR and 2 achieved CRi for an ORR of 30%. An additional patient likely had a CR with &lt;5% blasts and count recovery but had a suboptimal aspirate differential. Including this unconfirmed CR, ORR was 40%. An additional 2 (20%) achieved morphological leukemia-free state (MLFS). Of the 6 patients with any response (CR + CRi + MLFS), 2 achieved best response after cycle 1, 2 after cycle 2, 1 after cycle 3, and 1 after cycle 4. Relapse occurred in 2 of 5 (40%) while on study, at 425 days after CRi and 83 days after MLFS. All 3 patients with prior HMA exposure were non-responders. All patients discontinued treatment. Reasons included BMT (40%), AE (30%), progression (20%) and insurance loss (10%). Half planned to bridge to BMT as next-line therapy following study treatment. When taken off study, 50% were alive while 20% had died from AML complications, 20% from graft-versus-host-disease post-BMT, and 10% after relapse post-BMT. Median OS was 6.67 months (95% confidence interval [CI] 6.07 to not reached [NR]). Median EFS was 3.22 months (95% CI 1.50 to NR), with a maximum EFS of 16.93 months. Following G3 PN in the first 2 patients, no DLTs occurred on the modified regimen. Seventy percent of patients experienced at least possibly related G3+ AEs or serious AEs (SAEs). Of the 22 related G3+ AEs, anemia and decreased platelet count were seen in 50% and dizziness in 20%. Of the 22 related SAEs, anorexia, fatigue, PN, febrile neutropenia, and bacteremia were most common, each occurring in 20%. Conclusion The DBP triplet demonstrated preliminary anti-AML activity in a R/R AML patient cohort. Staggered dosing was better tolerated than simultaneous DBP. DBP may serve as an effective bridge to BMT for some patients. This study supports further evaluation of DBP, or related combinations, in R/R AML. Disclosures Rosenberg: Takeda, Janssen: Speakers Bureau. Abedi: Seattle Genetics: Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Speakers Bureau. Tuscano: BMS, Seattle Genetics, Takeda, Achrotech, Genentech, Pharmacyclics, Abbvie: Research Funding. Jonas: AbbVie, BMS, Genentech, GlycoMimetics, Jazz, Pfizer, Takeda, Treadwell: Consultancy; 47, AbbVie, Accelerated Medical Diagnostics, Amgen, AROG, Celgene, Daiichi Sankyo, F. Hoffmann-La Roche, Forma, Genentech/Roche, Gilead, GlycoMimetics, Hanmi, Immune-Onc, Incyte, Jazz, Loxo Oncology, Pfizer, Pharmacyclics, Sigma Tau, Treadwell: Research Funding; AbbVie: Other: Travel reimbursement. OffLabel Disclosure: Bortezomib is FDA-approved for the treatment of multiple myeloma in patients who have already been treated with 2 lines of prior therapy and progressed on the most recent therapy. Decitabine is indicated for treatment of patients with myelodysplastic syndromes. Doxorubicin is approved in AML among other cancers.

The Impact of BMI on Next Generation Sequencing Panels for Patients with Myelodysplastic Syndromes

Introduction: The usage of cytogenetics and next generation sequencing (NGS) has become more prominent in recent years for risk stratification of patients (pts) with myelodysplastic syndromes (MDS). Management of these patients is a multi-faceted endeavor which requires understanding of how MDS genetics interacts with a patient's lifestyle and co-morbidities. Obesity rates in the United States continue to rise; the current rate in Virginia is 30% (Centers for Disease Control and Prevention, CDC). This study analyzed whether body mass index (BMI) at diagnosis was correlated with the number or type of MDS mutations seen on initial NGS and whether obesity influenced outcomes stratified by individual mutations. Methods: Adult pts diagnosed with MDS at the University of Virginia from August 2012 to December 2019 who had cytogenetics and NGS panels done prior to initiation of MDS therapy were included. Any patient without the aforementioned initial diagnostic information was excluded. MDS disease characteristics, treatment type, comorbidities, and patient demographics were assessed including weight, height, and BMI (categories defined per CDC) at time of diagnosis. The primary aim of this analysis was to evaluate the association of BMI with the number and type of mutations on NGS panel at diagnosis. Wilcoxon-Mann-Whitney tests were used to explore the difference in BMI by presence of 13 mutations of interest: ASXL1, DNMT3A, FLT3, IDH1, IDH2, NPM1, NRAS, SETBP1, SF3B1, SRSF2, TET2, TP53, and U2AF1. The Spearman correlation coefficient was used to assess the relationship between BMI and total number of mutations. Kaplan-Meier estimates, log rank tests, and Cox proportional hazard models were used for time-to-event analyses. All analyses were performed using SAS 9.2 (SAS Institute, Cary, NC). Results: 143 MDS pts met inclusion criteria with a median age at diagnosis of 69 (range 39-87) and 60.7% (n=88) were male. The median weight was 81.3 kg and the median BMI at diagnosis was 27.7 kg (range 16.04-52.66). There were 29 pts in the normal BMI class (20%), 4 pts in the underweight class (3%), 38 pts in the obese class (26%), and 72 pts in the overweight class (50%). By IPPS-R risk scoring system, 64 pts were low or very low risk (44%), 39 pts intermediate risk (27%), and 41 pts high or very high risk (29%). There was no difference in the median number of mutations across BMI groups with a spearman correlation coefficient of 0.05 (Figure 1). However, the median BMI was significantly lower in patients with the NPM1 mutation versus those without (p=0.04), diagnosed prior to the WHO re-classification of these patients to acute myeloid leukemia (AML). Presence of the NPM1 mutation in MDS is also associated with worse PFS and an increased hazard ratio of 9.4 (p=0.0003), likely behaving similarly to AML. In comparison, presence of the SF3B1 mutation was found to be protective wherein patients who lacked the mutation had an increased hazard ratio of 6.0 (p=0.0133). BMI alone did not predict for progression free survival (PFS) or overall survival (OS); this was also true regardless of patient residency based on zip code. However, obese patients exhibiting the NPM1 and SF3B1 had better PFS (Figure 2). Moreover, the landmark analysis indicates among patients who are alive and free of progression at 20 months after MDS diagnosis, overweight patients exhibit better PFS than normal/underweight or obese patients (p=0.0192) (Figure 3). Conclusion: While BMI in pts with MDS does not appear to predict the gross number of NGS mutations, NPM1 mutations occurred more often in pts with low BMIs. Overweight pts who did not progress by 20 months have a better PFS. Prospective and larger retrospective studies are needed to expand on the findings that BMI may have an unexplored potential in stratifying pts with newly diagnosed MDS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation. Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 &gt; 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker. The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group. Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. Disclosures Eguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.

Single-Cell Proteomics Identifies Leukemia Landscape Associated with Clinical Outcomes in R/R AML Treated with MDM2i (Milademetan) and FLT3i (Quizartinib): Putative Role of CD68 and Diversity Index

Distinct mutations could differentially regulate cellular programs and alter the proteomic landscape in AML. Sequential acquisition of various mutations not only leads to clonal diversification but also alters the leukemia proteomic landscape through activation of mutation-specific gene programs. Characterization of AML proteomic profiles and diversity could be utilized as a measure of genetic imprint on leukemia proteome to inform clinical decision-making. We reasoned that diverse leukemia-specific proteomic profile could be indicative of the presence of multiple mutations activating numerous pathways, thus leading to a heterogenous clonal composition and more likely to therapy resistance. We aimed to test this hypothesis by assessing the proteomic profiles of FLT3-ITD AML patients treated with MDM2i (Milademetan) plus FLT3i (Quizartinib) (NCT03552029) and interrogate the association between proteomic landscape and therapy response. We assessed single-cell proteomic profiles of 35 sequentially collected samples for six selected patients treated with MDM2i+FLT3i using CyTOF, enabling us to assess expression of 51-parameters across leukemia compartments and identify leukemic clones with distinct proteomic profiles. Three patients achieved CRi while three patients did not respond. This allowed us to start interrogating proteomic signatures for their ability to predict response to therapy. We performed single-cell analysis and interrogated the phenotypic profiles of leukemia compartments to assess leukemia hierarchies, defined by spatial organization of leukemic subpopulations, and whether mutations in AML were associated with unique phenotypes. Notably, we found that NPM1-mutant (Mt) leukemia cells lacked CD34 expression, expressed high levels of CD99 and had patchy c-kit expression. Despite lacking a canonical marker, CD34, high-dimensional analysis positioned NPM1-Mt leukemia cells spatially in close proximity to CD34+ leukemia cells (NPM1 WT), indicating that NPM1 WT and Mt leukemia cells are closely related. CD34+ expressing cells most likely serve as the founding clone and acquisition of NPM1 mutation led to emergence of CD34- leukemia clones. As expected, all three patients who achieved CRi were NPM1-Mt and NPM1-Mt leukemia cells in CRi patients expressed CD68. Importantly, we also observed that CD68+ leukemia cells were eradicated in a NR patient where only a fraction of leukemia cells expressed CD68. This suggests that NPM1 mutations could activate unique cellular programs and induce distinct differentiation states (CD68), which could sensitize leukemia cells to MDM2+FLT3 inhibition. Altogether, NPM1 mutation status and CD68 expression level were associated with therapy response. Next, we mapped the response kinetics and quantified survived leukemia cells across multiple timepoints. Strikingly, MDM2i+FLT3i almost completely eliminated circulating blasts in responders (R) by day 8 while leukemia blasts persisted in NR (median blast %: 0.11 in R vs 19.8 in NR). This indicates that assessment of therapy response as early as day 8 could provide insights into the overall response and identify patients who will fail to achieve CR. Importantly, patients with reduced leukemia blasts at day 8 were also leukemia-free in BM at the end of cycle 1. Lastly, we sought to investigate the association between proteomic landscape diversity and therapy response, and quantified the number of leukemia subpopulations by unsupervised clustering. The median number of subpopulations detected in R vs NR at baseline were 3 and 9, respectively. We also utilized the inverse Simpson index to quantify the proteomic diversity of leukemia compartments and to further investigate the association between proteomic diversity and therapy outcome in an unbiased manner. The median diversity indices in R vs NR were 64 vs 212, revealing that patients with CR had restricted pre-treatment proteomic diversity. These findings suggest that a pre-treatment diverse phenotypic landscape could portend poor therapeutic outcome. Altogether, single-cell proteomic analysis identified correlates associated with overall clinical response in AML patients treated with MDM2i+FLT3i. Further validation is needed in a larger cohort of patients. Such approaches could be utilized in clinical-trial settings to predict therapy response with targeted agents and inform clinical decision-making. Disclosures Lesegretain: Daiichi-Sankyo Inc.: Current Employment. Daver: Amgen: Consultancy, Research Funding; Glycomimetics: Research Funding; Trovagene: Consultancy, Research Funding; Hanmi: Research Funding; Genentech: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Novimmune: Research Funding; ImmunoGen: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Astellas: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Andreeff: AstraZeneca: Research Funding; Glycomimetics: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Oxford Biomedica UK: Research Funding; Medicxi: Consultancy; Syndax: Consultancy; Karyopharm: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Research Funding; Senti-Bio: Consultancy; Amgen: Research Funding.

IDH1/2 Mutations Are Maintained in a Subset of Patients with Acute Myeloid Leukemia in Complete Remission and Do Not Correlate with Residual Disease

Rationale: Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations may be the nearest signal to in vivo leukemogenesis that we are able to see in overt acute myeloid leukemia (AML). Without immediate transforming activity and slow, metabolism-related effect, IDH1/2 mutations may be considered within the early events that made a myeloid stem cell a malignant one. Methods: With the aim to investigate the behavior of IDH1/2 mutation in AML patients, we prospectively and longitudinally collected clinical data and samples of DNA extracted from bone marrow of consecutive patients. The study was approved by local Ethical Authority (012/2009/U/Tess, 01/2011/U/Tess, 10/2011/U/Tess and 253/2013/O/Tess). Droplet digital polymerase chain reaction (DdPCR) was performed with Bio-Rad's Qx200 DdPCR System© according manufacture instructions. DdPCR for all the samples were performed in duplicate. Serial dilutions were used to identify sensitivity limit of the method for each mutation included in the study. Results : We analyzed 106 samples from 23 patients (median of 5 samples per patient). At database cut-off, 19th September 2020, median follow-up was 23.8 months (IQR 19.5 - 38.8). Fourteen patients were female. Median age at AML diagnosis was 59 years (IQR: 47.5 - 66). Most of the patients had normal karyotype (15/23, 65%) and 1 patient (4%) received diagnosis of AML after a previous myelodysplastic syndrome. Five out of 22 patients tested (23%) harbored FLT3 ITD, 8/21 (38%) had a NPM1 mutation. Almost the entire population (20/23, 87%) received intensive chemotherapy as induction regimen and 55% of these patients obtained complete remission (CR) after induction. Ten out of 23 patients (45%) harbored an IDH1 mutation, 2/23 (9%) R132G and 8/23 (35%) R132H; 13/23 patients (57%) harbored an IDH2 mutation, 7/23 R172K, 1/23 (4%) R172S and 5/23 (22%) R140Q. Most of the samples were collected in complete remission (CR) (54.5%). Other samples were collected at the time of AML diagnosis (15.1%) or relapse/stable disease (37.3%). Median IDH1/2 fractional abundance was 45.3% (IQR 28.6 - 46.8) at diagnosis, 39.5% (IQR 29.5 - 48.0) at relapse/refractory, and .10 % (IQR: .05 - 17.24) in CR. Few cases showed a fractional abundance below 20% at relapse and only one case was IDH1 negative (during an extramedullary relapse). The fractional abundance of IDH1/2 mutation in CR present a bi-modal trend, allowing us to define 2 groups by k-means stratification. Patients in group 1 (42 samples) tend to have a mutation specific fractional abundance that varies with disease burden (figure B, blue box). On the contrary, at the different time points patients in group 2 (group center 45.58%, 12 samples) have fractional abundance values over 30%, comparable to the levels at diagnosis, even in the absence of any other evidence of AML (figure B, orange box). For NPM1, IDH1/2 positive patients, in 18 timepoints, NPM1 qPCR minimal residual disease (MRD) was compared with IDH1/2 fractional abundance selecting the best possible threshold (best possible threshold .083, predicted sensitivity 75%, predicted specificity 60%; R2= .763, 95% C.I.: .521 - .1.00; asymptotic significance = .062). Finally, we selected 15 patients in which we were able to determine IDH1/2 mutation fractional abundance after induction therapy. Our results indicate that IDH1/2 ddPCR positivity does not impact on prognosis in our patient set. Conclusion: Our study shows that IDH1/2 mutations are maintained throughout time in a subset of patients with IDH1/2 positive AML in CR and that they do not univocally correlate with levels of residual disease. In these AML patients, the persistence of IDH1/2 mutations may be part of a more complex process involving clonal hematopoiesis. In such setting, further studies on the biological and clinical significance of IDH1/2 mutations persistence are warranted. Figure 1 Figure 1. Disclosures Martinelli: Abbvie: Consultancy; Astellas: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Incyte: Consultancy; Stemline Therapeutics: Consultancy. Papayannidis: Pfizer, Amgen, Novartis: Honoraria. Cavo: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; Novartis: Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Curti: Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.