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Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Supplementary Figure from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

Supplementary Table from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.Significance:Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307

Supplementary Figure 1. SURVEYOR assay assessing indel mutations of sgRNAs that target the N-HINGE-LOOP and the MBM-LBM locus. Supplementary Figure 2. Summary of negative selection experiments with sgRNAs targeting exons encoding specific MLL1 and MLL2 protein domains in a second independent OCI-AML3-Cas9 clone (OCI-AML3-Cas9-C8). Supplementary Figure 3. Dose responsive curves from cell viability assays of murine Npm1^CA/+ FIt3^ITD/+ after 7 days of MI-2-2 or MI-503 treatment and compared to MII-Af9 leukemia cells treated for 7 days with MI-503. Supplementary Figure 4. Apoptosis in OCI-AML3 cells treated for 7 days with MI-503 or DMSO as assessed by Annexin V / SYTOX blue staining. Supplementary Figure 5. Cytospins from cultured murine Npm1^CA/+ FIt3^ITD/+ comparing cells treated for 7 days with MI-503 versus DMSO. Supplementary Figure 6. Kaplan-Meier survival estimates from mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=5 mice/group) and treated in vivo for ten consecutive days with either MI-503 or vehicle control. Supplementary Figure 7. Kaplan-Meier estimates of mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=10 mice/group) that had been pretreated with either 10 days of EPS4777 [10 microM] or vehicle prior to transplantation. Supplementary Figure 8. Schematic of RNA sequencing detecting an MLL-AF6 fusion transcript in the DNMT3A mutated AML cell line OCI-AML2. Supplementary Figure 9. RNA sequencing of the OCI-AML2 cells reveals a HOX gene expression pattern as it is typically found in MLL-rearranged leukemias with predominant HOXA and MEIS1 expression but lack of HOXB cluster expression. Supplementary Figure 10. Isobolograms showing drug synergism for (A) MI-2-2 and EPZ4777 in the OCI-AML3 cells and (B) MI-503 and EPZ4777 in the murine Npm1^CA/+ FIt3^ITD/+ cells. Supplementary Figure 11. ChIP-PCR assessing RNA-Polymerase II enrichment after vehicle, single drug (MI-503 or EPZ4777) or combination treatment at the MEIS1 locus. Supplementary Figure 12. Cytologic analysis of human OCI-AML3 cells treated with DMSO, MI-2-2 (12 microM), EPZ4777 (10 microM), or combination therapy (MI-2-2 + EPZ4777). Supplementary Figure 13. Number of morphologically differentiated cells of two primary AML samples (ID1 and ID3) that had been treated in a human co-culture assay for 10 days with DMSO, EPZ4777, MI-503, or EPZ4777 and MI-503 (combo). Supplementary Figure 14. Engraftment of Npm1^CA/+ Rosa^SB/+ leukemia cells in the peripheral blood of tertiary recipient animals 22 days after transplantations. Supplementary Table 1. MLL1 single guide RNAs (sgRNA). Supplementary Table 2. MLL2 single guide RNAs. Supplementary Table 3. Synergy analysis of OCI-AML3 cells (day 7). Supplementary Table 4. Synergy analysis of Npm1^CA/+ FIt3^ITD/+ leukemias (day 11). Supplementary Table 5. Primary AML samples.

Supplementary Data from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Supplementary Figure from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Purpose:Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis.Patients and Methods:Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis.Results:In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients.Conclusions:Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868

Figure S1: Alisertib treatment did not result in a consistent cytokine response. Figure S2: Alisertib did not have a consistent effect on TGF-b levels. Figure S3: Alisertib induced GATA1 expression in the SET2 megakaryocytic cell line. Figure S4: AURKA inhibition induces megakaryocytic gene expression. Figure S5: Alisertib increases GATA1 expression in the bone marrow of patients on therapy. Table S1: Correlation of Genotype and prior JAK inhibitor therapy with response and adverse events Table S2: Summary of Treatment Related Adverse Events Table S3: Association between GATA1 staining, degree of fibrosis and clinical response

Accurate classification and risk stratification is critical for clinical decision making in AML patients. In the newly proposed World Health Organization (WHO) and International Consensus classifications (ICC) of hematolymphoid neoplasms, the presence of myelodysplasia-related (MR) gene mutations is included as one of the diagnostic criteria of AML, myelodysplasia-related (AML-MR), largely based on the assumption that these mutations are specific for AML with an antecedent myelodysplastic syndrome. ICC also prioritizes MR gene mutations over ontogeny (as defined by clinical history). Furthermore, European LeukemiaNet (ELN) 2022 stratifies these MR gene mutations to the adverse-risk group. By thoroughly annotating a cohort of 344 newly diagnosed AML patients treated at Memorial Sloan Kettering Cancer Center (MSKCC), we show that ontogeny assignment based on database registry lacks accuracy. MR gene mutations are frequently seen inde novoAML. Among MR gene mutations, onlyEZH2andSF3B1were associated with an inferior outcome in a univariate analysis. In a multivariate analysis, AML ontogeny had independent prognostic values even after adjusting for age, treatment, allo-transplant and genomic classes or ELN risks. Ontogeny also stratified the outcome of AML with MR gene mutations. Finally,de novoAML with MR gene mutations did not show an adverse outcome. In summary, our study emphasizes the importance of accurate ontogeny designation in clinical studies, demonstrates the independent prognostic value of AML ontogeny and questions the current classification and risk stratification of AML with MR gene mutations.Key pointsBoth ontogeny and genomics show independent prognostic values in AML.The newly proposed myelodysplasia-related gene mutations are neither specific to AML-MRCWHO2016nor predictive for adverse outcomes.Ontogeny stratifies the outcome of AML with myelodysplasia-related gene mutations.

Clonal haematopoiesis (CH) in patients with acute myeloid leukaemia (AML) may persist beyond attaining complete remission. From a consecutive cohort of 67 patients with nucleophosmin 1-mutated (NPM1mut ) AML, we identified 50 who achieved NPM1mut clearance and had parallel multicolour flow cytometry (MFC) and next generation sequencing (NGS). In total, 13 (26%) cleared all mutations, 37 (74%) had persistent CH frequently involving DNA methyltransferase 3α (DNMT3A,70%), tet methylcytosine dioxygenase 2 (TET2, 27%), isocitrate dehydrogenase 2 (IDH2, 19%) and IDH1 (11%). A small number (

Refereed/Peer-reviewed

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

Background: Measurable residual disease (MRD) is a powerful prognostic factor in AML, including in prediction of outcomes post allogeneic stem cell transplant (alloSCT). However, genomic predictors of successful MRD eradication with chemotherapy prior to alloSCT are unclear. Objectives: Here we provide an integrated analysis of 233 patients (pts) who underwent induction chemotherapy with baseline next-generation sequencing (NGS) followed by serial immunophenotypic monitoring for MRD while patients received additional therapy and alloSCT. Methods: All pts who received anthracycline + cytarabine, +/- investigational agents at Memorial Sloan Kettering Cancer Center starting in April 2014 were retrospectively studied (A). 142 out of 233 pts subsequently underwent alloSCT after induction or additional therapy (A). Immunophenotypic MRD was identified in bone marrow aspirates (BMA) by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Molecular analysis was obtained from pre-induction BMA by NGS using 28 or 49 or 400 gene panels. Results: Patient and treatment characteristics for all pts are detailed in panel (B). Induction chemotherapy resulted in an MRD-CR/CRi and MRD+CR/CRi in 29% and 23% of all pts, respectively (C). Additional therapy included consolidation (n=51), intensive re-induction/salvage (n=47) and non-intensive therapy (n=9). Of 83 AML pts with persistent AML and 58 pts with MRD+CR/CRi after induction (R1), 38/141 (27%) were able to be converted to MRD-CR/CRi. While 33/38 of pts went on to alloSCT after conversion to MRD-CR/CRi, 22 and 36 pts went to alloSCT with persistent AML and MRD+CR/CRi AML, respectively. We focused on pre-induction molecular predictors for achieving an MRD-CR/CRi response prior to transplant for the 142 pts who underwent alloSCT (D). Pts with a NPM1 (79%, Odds ratio [OR] 3.7, p=0.01), IDH1 (92%, OR 3.9, p=0.01) and KRAS (100%, OR 5.0, p=0.03) mutations achieved high rates of MRD-CR/CRi prior to alloSCT. In contrast, RUNX1 (28%, OR 0.2, p=0.01), TP53 (12%, OR 0.1, p=0.02) and SF3B1 (14%, OR 0.1, p=0.04) mutations predicted decreased odds of achieving MRD-CR/CRi prior to alloSCT despite induction and post-induction therapy. AlloSCT resulted in high rates of conversion from MRD+ and persistent disease to MRD negativity. Most pts who entered transplant with CR/CRi MRD+ (28/36, 76%) or persistent AML (14/22, 64%) cleared MRD by the first post-transplant BMA at a median of 32 days (E). Post-alloSCT follow-up indicated value in converting MRD+ to MRD- prior to alloSCT. There was no significant difference in post-transplant cumulative incidence of relapse (F) and OS (G) between early MRD-CR/CRi immediately following induction versus later conversion to MRD-CR/CRi with additional therapy prior to alloSCT. Despite initial post-transplant MRD clearance, pts who entered alloSCT with persistent AML or MRD+ had higher incidence of relapse (p=0.00037, F) and poorer post-transplant OS (p=0.013, G) compared to pts who entered alloSCT with MRD-. Pts with persistent disease prior to alloSCT had shorter duration of MRD- induced by alloSCT compared to pts with MRD-CR/CRi after induction or converted MRD-CR/CRi prior to alloSCT (p=0.0042, H). Importantly, duration of MRD negativity after alloSCT for patients who achieved MRD- prior to alloSCT was not affected by whether patients received induction +/- consolidation (I: treatment type 1-3 from B) vs. induction and salvage treatment for refractory AML (I: treatment type 4-6 from B). Conclusion: We show that transplanted AML pts with specific molecular mutations (RUNX1, SF3B1, and TP53) are unlikely to achieve MRD-CR/CRi after induction, consolidation or salvage therapy, while other mutations (NPM1, IDH1, KRAS) predict high rates of MRD- prior to alloSCT. Additional post-induction therapy may be advantageous for some MRD+ pts to achieve MRD- prior to alloSCT. Post-transplant OS is improved in pts who are MRD- at time of transplant, regardless of whether they required additional therapy beyond induction to achieve this state. AlloSCT is highly effective at eradicating MRD, but post-transplant MRD- is more durable in pts who are MRD- pre-alloSCT. Our results suggest that development of MRD-eradicating therapies has the potential to improve post-transplant outcomes and argues for innovative trials for pts with adverse molecular features currently unlikely to achieve MRD- pre alloSCT. Figure Disclosures Cai: Imago Biosciences, Inc.: Consultancy, Current equity holder in private company; DAVA Oncology: Honoraria. Geyer:Amgen: Research Funding. Glass:Gerson Lehman Group: Consultancy. Stein:Syros: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotheryx: Consultancy; Bayer: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Seattle Genetics: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Gilead: Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Morphosys: Consultancy; Roche: Consultancy, Honoraria, Research Funding. Gyurkocza:Actinium: Research Funding. Perales:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; NexImmune: Membership on an entity's Board of Directors or advisory committees; Cidara Therapeutics: Other; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company; Merck: Consultancy. Papaemmanuil:Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; MSKCC: Patents & Royalties; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria. Giralt:KITE: Consultancy; NOVARTIS: Consultancy, Honoraria, Research Funding; OMEROS: Consultancy, Honoraria; AMGEN: Consultancy, Research Funding; TAKEDA: Research Funding; ACTINUUM: Consultancy, Research Funding; MILTENYI: Consultancy, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria. Tallman:Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Rigel: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Orsenix: Research Funding; Cellerant: Research Funding; Abbvie: Research Funding. Goldberg:AROG: Research Funding; Aprea: Research Funding; ADC Therapeutics: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Pfizer: Research Funding; Celularity: Research Funding.

Purpose: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. Experimental Design: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. Results: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3–32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. Conclusions: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

The goal of therapy for essential thrombocythemia (ET) and polycythemia vera (PV) patients is to reduce thrombotic events by normalizing blood counts. Hydroxyurea (HU) and interferon-α (IFN-α) are the most frequently used cytoreductive options for ET and PV patients at high-risk for vascular complications. Myeloproliferative Disorders Research Consortium 112 was an investigator-initiated, phase 3 trial comparing HU to pegylated IFN-α (PEG) in treatment naïve, high-risk ET/PV patients. The primary endpoint was complete response (CR) rate at 12 months. A total of 168 patients were treated for a median of 81.0 weeks. CR for HU was 37% and 35% for PEG (p=0.80) at 12 months. At 24/36 months, CR was 20%/17% for HU and 29%/33% for PEG. PEG led to a greater reduction in JAK2V617F at 24 months, but histopathologic responses were more frequent with HU. Thrombotic events and disease progression were infrequent in both arms, while grade 3/4 adverse events were more frequent with PEG (46% vs. 28%). At 12 months of treatment there was no significant difference in CR rates between HU and PEG. This study indicates that PEG and HU are both effective treatments for PV and ET. With longer treatment PEG was more effective in normalizing blood counts and reducing driver mutation burden, while HU produced more histopathologic responses. Despite these differences, both agents did not differ in limiting thrombotic events and disease progression in high-risk ET/PV patients. (Funded by the National Cancer Institute, 5P01CA108671-09; clinicaltrials.gov number (NCT01259856). [Abstract copyright: Copyright © 2022 American Society of Hematology.]

Chemotherapy and radiation therapy are the foundations of adjuvant therapy for early-stage breast cancer. As a complication of cytotoxic regimens, breast cancer patients are at risk for therapy-related myeloid neoplasms (t-MNs). These t-MNs are commonly refractory to antileukemic therapies and result in poor patient outcomes. We previously demonstrated that somatic mutations in leukemia-related genes are present in the tumor-infiltrating leukocytes (TILeuks) of a subset of early breast cancers. Here, we performed genomic analysis of microdissected breast cancer tumor cells and TILeuks from seven breast cancer patients who subsequently developed leukemia. In four patients, mutations present in the leukemia were detected in breast cancer TILeuks. This finding suggests that TILeuks in the primary breast cancer may harbor the ancestor of the future leukemogenic clone. Additional research is warranted to ascertain whether infiltrating mutant TILeuks could constitute a biomarker for the development of t-MN and to determine the functional consequences of mutant TILeuks.

Purpose: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. Patients and Methods: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. Results: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. Conclusions: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis. See related commentary by Piszczatowski and Steidl, p. 4868

A limited number of drugs are available to treat patients with polycythemia vera (PV) and essential thrombocythemia (ET). We attempted to identify alternative agents that may target abnormalities within malignant hematopoietic stem (HSCs) and progenitor cells (HPCs). Previously, MDM2 protein levels were shown to be upregulated in PV/ET CD34+ cells, and exposure to a nutlin, an MDM2 antagonist, induced activation of the TP53 pathway and selective depletion of PV HPCs/HSCs. This anticlonal activity was mediated by upregulation of p53 and potentiated by the addition of interferon-α2a (IFN-α2a). Therefore, we performed an investigator-initiated phase 1 trial of the oral MDM2 antagonist idasanutlin (RG7388; Roche) in patients with high-risk PV/ET for whom at least 1 prior therapy had failed. Patients not attaining at least a partial response by European LeukemiaNet criteria after 6 cycles were then allowed to receive combination therapy with low-dose pegylated IFN-α2a. Thirteen patients with JAK2 V617F+ PV/ET were enrolled, and 12 (PV, n = 11; ET, n = 1) were treated with idasanutlin at 100 and 150 mg daily, respectively, for 5 consecutive days of a 28-day cycle. Idasanutlin was well tolerated; no dose-limiting toxicity was observed, but low-grade gastrointestinal toxicity was common. Overall response rate after 6 cycles was 58% (7 of 12) with idasanutlin monotherapy and 50% (2 of 4) with combination therapy. Median duration of response was 16.8 months (range, 3.5-26.7). Hematologic, symptomatic, pathologic, and molecular responses were observed. These data indicate that idasanutlin is a promising novel agent for PV; it is currently being evaluated in a global phase 2 trial. This trial was registered at www.clinicaltrials.gov as #NCT02407080.

Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) characterized by an increased risk of developing venous and arterial thromboses and of evolution to myelofibrosis or acute leukemia (1). MPNs are recognized as a major cause of splanchnic vein thromboses (SVT), which include hepatic, splenic, portal and mesenteric vein thromboses (2). Patients with SVT are at an increased risk of developing bleeding events and arterial thrombotic events (3), as well as recurrent SVT and the use of anticoagulation and cytoreduction do not clearly modify this risk (3–5). Management of SVT in MPN patients remains a challenge and most therapeutic recommendations are based on retrospective analyses (6). Recently, De Stefano reported that hydroxyurea failed to prevent recurrent thromboses in the splanchnic vasculature in patients with a prior SVT (7). We conducted a prospective, open-label, multi-center phase II clinical trial of Pegylated Interferon Alfa-2a (PEG) in 20 subjects with SVT and an MPN, who represented a cohort of patients who participated in the Myeloproliferative Disorders - Research Consortium (MPD-RC) 111 trial (). MPD-RC 111 was a prospective study of patients with high-risk ET/PV who were resistant or intolerant to hydroxyurea (HU), but prior HU therapy was not a requirement for patients to enter the SVT cohort. Here we report the outcomes of the 20 SVT patients who were treated with PEG.

Myeloproliferative neoplasms (MPN) that have evolved into accelerated or blast phase disease (MPN-AP/BP) have poor outcomes with limited treatment options and therefore represent an urgent unmet need. We have previously demonstrated in a multicenter, phase 1 trial conducted through the Myeloproliferative Neoplasms Research Consortium that the combination of ruxolitinib and decitabine is safe and tolerable and is associated with a favorable overall survival (OS). In this phase 2 trial, 25 patients with MPN-AP/BP were treated at the recommended phase 2 dose of ruxolitinib 25 mg twice daily for the induction cycle followed by 10 mg twice daily for subsequent cycles in combination with decitabine 20 mg/m2 for 5 consecutive days in a 28-day cycle. Nineteen patients died during the study follow-up. The median OS for all patients on study was 9.5 months (95% confidence interval, 4.3-12.0). Overall response rate (complete remission + incomplete platelet recovery + partial remission) was 11/25 (44%) and response was not associated with improved survival. We conclude that the combination of decitabine and ruxolitinib was well tolerated, demonstrated favorable OS, and represents a therapeutic option for this high-risk patient population. This trial was registered at www.clinicaltrials.gov as #NCT02076191.

The myeloproliferative neoplasms frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis and an accumulation immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial ROS and stabilization of HIF1a, and downregulation of LKB1 and increased levels of HIF1a were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies. Environmental exposures shape patterns of selection for mutations in clonal hematopoiesis. Cancer therapies promote the growth of clones with mutations that are strongly enriched in treatment-related myeloid neoplasms.

Clonal hematopoiesis (CH) is frequent in cancer patients and associated with increased risk of therapy related myeloid neoplasms (tMN). To define the relationship between CH, oncologic therapy, and tMN progression, we studied 24,439 cancer patients. We show that previously treated patients have increased rates of CH, with enrichment of mutations in DNA Damage Response (DDR) genes (TP53, PPM1D, CHEK2). Exposure to radiation, platinum and topoisomerase II inhibitors have the strongest association with CH with evidence of dose-dependence and gene-treatment interactions. We validate these associations in serial sampling from 525 patients and show that exposure to cytotoxic and radiation therapy imparts a selective advantage specifically in hematopoietic cells with DDR mutations. In patients who progressed to tMN, the clone at CH demarcated the dominant clone at tMN diagnosis. CH mutational features predict risk of therapy-related myeloid neoplasm in solid tumor patients with clinical implications for early detection and treatment decisions.

Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)1–8. Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants9,10. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML11. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies. A new mechanism of acquired clinical resistance in two patients with acute myeloid leukaemia driven by mutant IDH2 is described, in which a second-site mutation on the wild-type allele induces therapeutic resistance to IDH2 inhibitors.

TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone marrow fibrosis (BMF) but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs). Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to MKs decreased pSMAD2 without affecting total SMAD2/3 and led to increased numbers of MKs. Treatment of MF MNCs with AVID200 also led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. A phase 1b trial of AVID200 (NCT03895112) was performed and completed in INT-2/high risk MF patients resistant/intolerant to ruxolitinib (rux); baseline platelet count of ≥ 25 x 10 9/L, and grade 2/3 BMF. Subjects received AVID200 intravenously on Day 1 of a 21 day cycle. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. We previously presented the results of the dose escalation study (Mascarenhas ASH 2020) demonstrating that AVID200 was well tolerated without dose limiting toxicities at 3 tested dose levels (Lots A and B) in dose cohorts of 180 mg (A), 550 mg (A)/70 mg (B), and 180 mg (B). Here we report updated safety and efficacy results of the phase 1b dose expansion stage at the two highest doses tested (70 mg (B) and 180 mg (B). Twenty-two subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 in the dose escalation phase and 12 in the dose expansion phase [Table1]. Median time after rux discontinuation was 7.4 months (0.5-59.9). The most common mutations observed at baseline in this cohort included JAK2V617F (71%), TET2 (29%) ASXL1 (24%) and CALR (19%). (Fig 1) No DLTs were observed and Grade 3/4 AEs were observed in 16 (76.2%) subjects. Grade 3/4 non-hematologic AEs were observed in 8 (38.1%) subjects and included one subject in each case (epistaxis, mucositis, extraocular muscle paresis, fatigue, rash, duodenal hemorrhage, gastric hemorrhage, urinary tract infection, and syncope). Grade 3/4 hematologic AEs were anemia (6; 28.3%) and thrombocytopenia (2; 14.3%) [Table 2]. No fatal events were observed. The median number of cycles received was 5 (range 2 - 13) and 7 (33%) patients received more than 6 cycles. For dose levels 2-3 at cycle 7, a CI was attained in one subject at dose level 2 [anemia, spleen and TSS], 5 subjects had SD, 3 subjects had PD and two subjects with 10% and 15% blasts at screening developed MPN-BP while on study based on central review. Reasons for discontinuation by local PI included PD (n=8), lack of response (n=5), study completed (n=2), other (n=2), patient decision (n=1). Median % change in palpable spleen length was +10% (range -70% to +150%) and TSS change was -50% (-100% to +185.7%) The median platelet count at baseline was 114 x 10 9/L (range: 28-695) and 215 x 10 9/L (range: 66-263) after cycle 6 in 7 evaluable subjects (Fig 2A). Notably, 17 subjects had an increase in platelets from baseline during treatment and two subjects normalized their platelet counts. Maximum changes in platelets from baseline across all cycles was +63.8% [range -15.7%, +505.5%] (Fig 2B). Paired bone marrow biopsy pathology samples for 12 subjects were available for central review and showed no significant changes in BMF score or MK histo topography at end of treatment compared to baseline. All patients had elevated plasma levels of TGF β1, but not TGFβ2/β3 levels as detected by ELISA, which were dramatically reduced 21 days after the last dose of AVID200. AVID200 a TGFβ1/3 protein trap is well tolerated and clinical responses at cycle 7 of therapy in this advanced MF patient population were limited as judged by IWG/MRT response criteria. However, AVID200 therapy resulted in significant reduction in serum TGFβ levels and improvements in platelet counts indicating that TGF β1 plays a pivotal role in MF leading to thrombocytopenia which can be reversed with AVID200 therapy. We conclude that AVID200 may best be employed in combination therapy approaches in thrombocytopenic MF patients. Figure 1 Figure 1. Disclosures Mascarenhas: Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy, Research Funding; Forbius: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galecto: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding. Palmer: PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding; Incyte: Research Funding; CTI BioPharma: Consultancy, Research Funding; Protagonist: Consultancy, Research Funding. Kuykendall: Celgene/BMS: Honoraria; Pharmaessentia: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria. Mesa: Genentech: Research Funding; Promedior: Research Funding; Samus: Research Funding; Gilead: Research Funding; CTI: Research Funding; Abbvie: Research Funding; Sierra Oncology: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Pharma: Consultancy; CTI: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; AOP: Consultancy; La Jolla Pharma: Consultancy; Incyte Corporation: Consultancy, Research Funding. Rampal: Stemline: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment; BMS/Celgene: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Novartis: Consultancy; Disc Medicine: Consultancy; Blueprint: Consultancy; Pharmaessentia: Consultancy; Incyte: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Constellation: Research Funding; Kartos: Consultancy; Sierra Oncology: Consultancy. Gerds: PharmaEssentia Corporation: Consultancy; Sierra Oncology: Consultancy; CTI BioPharma: Research Funding; Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Novartis: Consultancy. Yacoub: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; ACCELERON PHARMA: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dynavex: Current equity holder in publicly-traded company; Cara: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Seattle Genetics: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Hylapharm: Current equity holder in publicly-traded company. Talpaz: Imago: Consultancy; Constellation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Other: Grant/research support ; Celgene: Consultancy. Komrokji: Acceleron: Consultancy; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Jazz: Consultancy, Speakers Bureau. Kremyanskaya: Astellas: Research Funding; Astex: Research Funding; Chimerix: Research Funding; Bristol Myers Squibb: Research Funding; Constellation: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Incyte: Research Funding. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Hoffman: Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding. OffLabel Disclosure: AVID200 is a TGFb trap and is in clinical testing for fibrotic diseases. It does not have an approved indication at this time.

INTRODUCTION: NPM1 mutations (NPM1mut) occur in ∼30% of acute myeloid leukemia (AML) and frequently co-occur with mutations in other genes including those attributed to clonal hematopoiesis (CH) including DNMT3A and TET2, among others. CH mutations may persist beyond attaining NPM1mut-negative remission. Persistent CH may be associated with immunophenotypic alterations in myeloid progenitors detected by flow cytometry (FC) which poses an interpretive challenge in assessment of measurable residual disease (MRD) by FC. The aim of this study was to characterize the immunophenotypic alterations associated with persistent CH in the setting of NPM1mut clearance and to determine their possible clinical or biologic significance. METHODS: The cohort included 67 consecutive patients (pts) with NPM1mut AML treated at our institution between 01/2017 and 11/2019. FC assessment for MRD was performed an eight-color panel using FACSCanto II instruments (BD Biosciences, San Diego, CA) with a sensitivity of 10-3 to 10-4. Whole bone marrow (BM) DNA was interrogated for mutations with an 81-gene myeloid next-generation sequencing (NGS) panel using an Illumina MiSeq sequencer (Illumina, Inc., San Diego, CA, USA) with a sensitivity: 1% variant allelic frequency (VAF). RESULTS: Pts included 26 men and 41 women with a median age of 64 (range, 19-84) years with newly diagnosed NPM1mut AML. AML blasts had the following immunophenotype at baseline: promyelocytic-like phenotype (CD34-, CD117+, HLA-DR-) in 18 (27%), aberrant myeloid CD34-/CD117+/HLA-DR+ in 15 (22%), aberrant myeloid CD34+ in 13 (19%), myelomonocytic in 11 (16%), and monocytic in 10 (15%) cases. All pts had additional co-mutations at baseline (Fig 1). The most frequently co-mutated genes were DNMT3A (58%) FLT3 (51%), TET2 (27%), IDH2 (24%), PTPN11 (19%), IDH1 (18%), NRAS (16%), and SRSF2 (12%). Pts were treated with intensive (35;52%) and non-intensive induction regimens (32; 48%) (Fig 1); 22 (33%) received an allogeneic hematopoietic stem cell transplant as post-remission consolidation. We compared FC and NGS results in follow-up samples in pts achieving NPM1mut negative remission with adequate data available for comparison (n=50). 13 (26%) pts cleared all mutations whereas 37 (74%) had persistent CH. The most common mutations in the setting of residual CH involved DNMT3A (70%), TET2 (27%), IDH2 (19%) and IDH1 (11%). Among 37 pts with residual CH, 19 (51%) had no phenotypic alterations detected by FC while 17 (49%) had myeloid progenitors with alterations in intensity of antigen expression (increased CD13, CD123, CD117 and/or decreased CD38) or deviation from normal maturation but not diagnostic for AML MRD (herein referred to as pre-leukemic (PL) phenotype); 1 sample was MRD+ by FC. Mutation VAF of ≥5% was significantly more common (p=0.008) in cases with FC PL+ (100%) vs cases with normal FC phenotype (63%). IDH2 and SRSF2 mutation were exclusively observed in PL+CH+ cases with the former being statistically significant when compared with the FC-normal group (p=0.016). PL phenotype by FC did not correlate with intensity of induction therapy (41% treated with intensive regimens vs 59% non-intensive). The CH+/PL+ cohort had more pts ≥60 yrs old (67%) but the difference was not significant. There was no significant association between PL+ and residual mutation count. Presence of PL+ phenotype was not associated with a shorter relapse-free survival (RFS) (median not reached for both groups). CONCLUSIONS: Post-remission clonal hematopoiesis in the setting of NPM1mut clearance is common, and may result in immunophenotypic changes in myeloid progenitors, posing interpretive challenges for MRD assessment by FC. These alterations may be attributable to specific CH characteristics, such as IDH2 and SRSF2 mutations and VAF, but are not associated with a shorter RFS and thus should not be interpreted as residual AML or considered a high-risk attribute. Additional studies in other AML subtypes are warranted to further delineate these changes and their clinical significance. Figure 1 Disclosures DiNardo: Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Takeda: Honoraria; Jazz: Honoraria; ImmuneOnc: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; MedImmune: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Short:AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Research Funding. Kadia:JAZZ: Honoraria, Research Funding; Ascentage: Research Funding; Astra Zeneca: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Novartis: Honoraria; Cyclacel: Research Funding; Genentech: Honoraria, Research Funding; Amgen: Research Funding; Abbvie: Honoraria, Research Funding; Cellenkos: Research Funding; Pulmotec: Research Funding; Astellas: Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Konopleva:Sanofi: Research Funding; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ascentage: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; Agios: Research Funding; Ablynx: Research Funding. Kantarjian:Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Ascentage: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Immunogen: Research Funding; Jazz: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Ravandi:Macrogenics: Research Funding; Celgene: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Xencor: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding.

Summary Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo . Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo .

We evaluated the feasibility of ruxolitinib therapy followed by a reduced-intensity conditioning (RIC) regimen for patients with myelofibrosis (MF) undergoing transplantation in a 2-stage Simon phase II trial. The aims were to decrease the incidence of graft failure (GF) and nonrelapse mortality (NRM) compared with data from the previous Myeloproliferative Disorders Research Consortium 101 Study. The plan was to enroll 11 patients each in related donor (RD) and unrelated donor (URD) arms, with trial termination if ≥3 failures (GF or death by day +100 post-transplant) occurred in the RD arm or ≥6 failures occurred in the URD. A total of 21 patients were enrolled, including 7 in the RD arm and 14 in the URD arm. The RD arm did not meet the predetermined criteria for proceeding to stage II. Although the URD arm met the criteria for stage II, the study was terminated owing to poor accrual and a significant number of failures. In all 19 transplant recipients, ruxolitinib was tapered successfully without significant side effects, and 9 patients (47%) had a significant decrease in symptom burden. The cumulative incidences of GF, NRM, acute graft-versus-host disease (GVHD), and chronic GVHD at 24 months were 16%, 28%, 64%, and 76%, respectively. On an intention-to-treat basis, the 2-year overall survival was 61% for the RD arm and 70% for the URD arm. Ruxolitinib can be integrated as pretransplantation treatment for patients with MF, and a tapering strategy before transplantation is safe, allowing patients to commence conditioning therapy with a reduced symptom burden. However, GF and NRM remain significant.

MPN-BP originates from a leukemic stem cell (LSC) that is capable of recreating and serial passaging the leukemia in NSG mice (Wang Blood 2018). The genomic architecture of these LSCs has not been well characterized. We therefore performed mutational profiling using capture-based next generation sequencing of primary MPN-BP patient samples and xenografts following their transplantation into NSG mice. All 7 patients with MPN-BP studied had 2-6 known oncogenic gene mutations. T cell-depleted mononuclear cells (MNC, 1-10×106) containing 50-3850 leukemia initiating cells based upon limiting dilution analysis were transplanted into 1-3 NSG mice per patient sample. 1-7 months after transplantation, the mice were sacrificed. 23.3±5.5% leukemic cell chimerism was detected in each mouse. Leukemic cells from xenografts were then selected and sequenced. As shown in Figure 1, primary MNCs from 6 of 7 patients contained 1-3 mutations involving TET2, JAK2V617F, TP53, MPL, KRAS with a variant allele frequency (VAF) ≥45% marking founder clones. Leukemic cells in primary xenografts possessed each of their founder mutations, suggesting clonality and that MPN-BP originates within the LSC compartment. Additionally 1-2 gene mutations with 0.1-9.6% VAF (subclonal mutations) were present in primary samples from 3 of these 6 patients (Pts 1, 6, 7) but their VAF (15.5-71.1%) in xenografts surpassed that present in primary cells. Moreover, a distinct oncogenic mutation in TP53(p.R175G) not present in Pt 7's primary cells was detected in leukemic cells from one of the 3 individual xenografts (VAF: 51.8%). There was also one patient (Pt 4) with primary cells that did not harbor any founder mutations but contained 4 subclonal mutations (VAF: MYB, 6.9%; KRAS, 9.6%, PTPN11, 29.5%; ASXL1(p.T848fs*19), 35.6%). In the xenografts, the VAFs of MYB (0%) and PTPN11 (12.2%) were reduced, while the VAFs of the other 2 subclonal mutations were increased to 77.4% (KRAS) and 48.8% (ASXL1). These findings suggest that multiple genetically distinct LSC clone/subclones exist in patients with MPN-BP that are capable of engrafting NSG mice and have the potential in the future to participate in leukemia progression and/or relapse. All 7 patients originally had a JAK2V617F+ MPN, but 3 of the MPN-BP cells were JAK2V617F¯. Among the remaining 4 patients whose primary cells retained JAK2V617F, the VAF was decreased in leukemic cells within the xenografts. These findings indicate that in JAK2V617F+ MPNs which evolve to MPN-BP, that the MPN-BP may either arise from stem cells distinct from the original JAK2V617F+ MPN stem cells with the acquisition of additional genetic events or progress from the clonal proliferation of distinct JAK2V617F¯ LSCs. Thus, JAK2V617F might play a limited role in either MPN-BP initiation or its progression. Four patients had distinct oncogenic TP53 mutations in primary cells which represented a clonal (VAF: Pt 2, 63.5%; Pt 5, 59.6%) or a subclonal mutation (Pts 1, 7) with a very low VAF (2.5%, 0.4%). The VAF for TP53 mutations were each increased in paired xenograft from 3 patients (Pt 2: 99.8%; Pt 1: 15.5%; Pt 7: 3.8%). These observations support a consistent proliferative advantage of LSCs carrying TP53 mutations in MPN-BP irrespective of how small the original LSC subclone is in the primary cells. Pt 3 and Pt 4 both had a KRAS mutation in their primary cells (VAF: Pt 3, 45.7%; Pt 4, 9.6%). For Pt 3, although the VAF of the KRAS mutation remained the same in the leukemic cells of the primary NSG recipient (44.4%), it increased to 90.2% in a secondary NSG recipient which was characterized by a higher leukemic cell burden (1.8-fold) and shorter survival (12 days less) than the primary NSG recipient. The VAF of KRAS mutation in Pt 4 also reached 77.4% in leukemic cells in the primary xenograft. These data suggest that acquisition of a KRAS mutation in LSCs may not only promote LSC clonal expansion, but also confer enhanced LSC self-renewal capacity. Finally, 4 distinct TET2 mutations were present as a founder mutation in primary cells of 3 patients but their VAF was unchanged or only slightly increased in leukemic cells in paired xenografts, suggesting that TET mutations are not involved in either LSC clonal expansion or disease progression. We conclude that TP53 and/or KRAS mutations appear to play an important role in the development of MPN-BP and may serve as potential targets for the development of effective treatment. Disclosures Rampal: Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Hoffman:Merus: Research Funding.

Background The Myeloproliferative Neoplasms, including Essential Thrombocythemia (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are stem cell disorders which carry the risk of progression to accelerated phase or blast-phase (MPN-AP/BP). Among the risk factors for transformation to MPN-AP/BP are TP53 mutations. TP53 mutations are a risk factor for progression to MPN-AP/BP in chronic-phase myeloproliferative neoplasms (MPN) and indeed 30% of patients with MPN-LT bear TP53 mutations. A recent study indicated that TP53 mutations may persist at low levels for years without an immediate risk of progression in some chronic-phase MPN patients [Kubesova et al. Leukemia 2017]. These observations raise the question as to whether the types of TP53 mutations, their frequency, or co-occurring variants differ between MPN subtypes. TP53 mutations are characterized by frameshift, missense, nonsense or silent mutations. Mutations in TP53 have traditionally been considered functionally equivalent in many prognostic studies, but an increased understanding of the effects of distinct mutations on TP53 activity has led to the recognition of the distinct functional significance of these different mutations. The understanding of the landscape of TP53 mutations in MPN and the association between different TP53 mutations and mutational burdens among MPN subtypes may allow more accurate prognostic evaluation and personalized treatment for patients. We therefore sought to assess the landscape of TP53 mutations in MPN subtypes. Methods We performed Next-Generation Sequencing (NGS) on a cohort of 651 samples derived from 439 MPN patients with ET, PV, MF and MPN-AP/BP. The data were from 3 targeted panels with 576, 585 and 156 genes (with 91, 241 and 319 samples; median coverage 500x). Samples were obtained from Memorial Sloan Kettering Cancer Center and the Myeloproliferative Neoplasms Research Consortium. Putative oncogenic mutations were called using a combination of 4 variant callers and by comparison to established cancer databases. The final variants were manually reviewed to guarantee high quality of downstream analysis. Results We identified somatic mutations in 428/439 patients (157 MPN-AP/BP, 140 MF, 67 ET and 64 PV). Of these, 55 patients (~13%) had at least one TP53 mutation with variant allele frequency (VAF) >= 2% (median TP53 VAF= 28%). In total, 68 TP53 somatic mutations were identified. Majority of these patients had a single TP53 mutation (46/55) and some had multiple mutations (7 had two and 2 had four mutations). Mutations were enriched in MPN-AP/BP with 45/68 (66%) occurring in this group followed by 10/68 (15%) in ET, 9/68 (13%) in MF and 4/68 (6%) in PV (panel A). Missense mutations were the most common type of TP53 variants and constituted ~80-90% of all mutations in ET, MF and MPN-AP/BP. Most stop-gains and frameshifts were observed in MF or MPN-AP/BP group. Furthermore, 92% of TP53 mutations are localized on DNA-binding domain (exons 5 to 8; panel B). The latter observation is consistent with results from other human cancers and highlights the role of these mutations in reducing TP53 DNA binding affinity. About 8% of mutations occur in the tetramerization domain of TP53, which is also critical for DNA binding, as well as protein-protein interactions and post-translational modification. We did not identify a significant association between a specific TP53 mutation type and any particular MPN subtype. However, we identified a significant association between TP53 VAF and MPN subtype (panel C); TP53 VAF was significantly higher in MPN-AP/BP compared to ET (p =0.0001) and MF (p =0.016). Conclusion TP53 mutations have important prognostic significance in patients with MPN. However, subgroups of patients with TP53 mutant chronic-phase disease have been observed to have relatively stable clinical course. Our data demonstrate that the spectrum of TP53 mutations, in terms of type and location within the gene, does not appear to differ between ET, PV, MF, or MPN-AP/BP. However, we observe significant difference with regard to the VAF of TP53 mutations, with an association of higher VAF and MPN-AP/BP state. This data argues that the VAF may be an important consideration in assessing the prognostic impact of a TP53 mutation identified in an MPN patient. Data regarding copy number variations in this cohort, co-occurring mutations, and the impact of TP53 mutations on treatment outcomes will be presented. Figure Disclosures Papaemmanuil: Celgene: Research Funding. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Levine:Prelude Therapeutics: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees. Mascarenhas:Novartis: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding.

Treatment of polycythemia vera (PV) with the Murine Double Minute 2 (MDM2) antagonist, idasanutlin, in a phase 1 trial was reported by our group to be well tolerated with a high overall response rate (Mascarenhas et al, Blood. 2019 Jun 5). A global, phase 2 trial is currently underway evaluating idasanutlin in hydroxyurea (HU) resistant/intolerant PV patients (NCT03287245). MDM2, a negative regulator of TP53 is upregulated in PV CD34+ cells and inhibition of MDM2 targets PV hematopoietic stem/progenitor cells (HSPC) (Lu et al, Blood. 2014;124(5):771-90). Additional trials of MDM2 antagonists have shown promise, however, there is concern that these agents have the potential to induce TP53 mutations or promote expansion of TP53 mutated clones. Resistance to MDM2 inhibitors has been evaluated in solid tumor cell lines and attributed to either the emergence of de novoTP53 mutations or the selection of TP53 mutated clones. (Michaelis et al, Cell Death Dis. 2011;2:e243; Skalniak et al, Cancers. 2018;10(11)). The effect of MDM2 inhibition on TP53 mutant clones is of particular interest in myeloproliferative neoplasms (MPNs). TP53 mutations have been reported with a low allele burden in ~15% of chronic MPN patients (Kubesova et al, Leukemia. 2018;32(2):450-61), however, TP53 loss of heterozygosity and rapid expansion of TP53 mutant clones is associated with transformation to blast phase (Lundberg et al, Blood 2014,123:2220-8). As reported, in the idasanutlin PV trial, 1/13 patients were identified to have a baseline pathogenic TP53 mutation in hematopoietic cells (VAF 5.5%), using a deep sequencing assay with a limit of detection (LOD) of VAF 0.5%. This patient was a non-responder to idasanutlin and upon treatment had an increasing JAK2V617F and TP53 mutant VAF. End of study hematopoietic cell specimens of all patients were deep sequenced (LOD 0.1%) and revealed that 4 additional patients harbored detectable TP53 mutations after idasanutlin treatment with VAF ranging from 1-12%. In each sample, 1-5 unique TP53 mutations were identified, all within the hotspot domain of the TP53 gene. Deeper sequencing of baseline and follow-up samples revealed these mutations were present at a subclonal level (VAF 0.1-5.5%) and increased over time, indicating that treatment with the MDM2 antagonist promoted expansion of already existing TP53 mutant clones (Table 1, Figure 1). None of the patients who lacked a TP53 mutation at baseline developed a TP53 mutant clone with idasanutlin treatment. There was no clear association of presence of TP53 mutations with prior HU, anagrelide or interferon exposure. There has been careful monitoring of patients to determine whether the expanding TP53 clone has clinical ramifications. Patients were on study for a median of 54 weeks (23-131). The only patient who exemplified resistance to idasanutlin was the single patient with a high burden TP53 mutation (37%). All other patients were taken off study due to patient choice/toxicity. Furthermore, all TP53 mutant and non-mutant patients have had stable disease with no evidence of progression to MF or AML. Sequencing of 2 patients post-discontinuation of idasanutlin revealed that the VAF of the TP53 mutant clones decreased since the agent was discontinued. Updated patient molecular data post-treatment discontinuation will be reported at the meeting. To investigate whether idasanutlin induces de novo TP53 mutations in PV myeloid cells we performed long term HSPC cultures. Mononuclear cells from 6 distinct PV patients were treated continuously with idasanutlin (500 nM) over ~6 weeks and DNA from both treated and untreated colonies were analyzed using next generation sequencing with a LOD of 2% VAF and no TP53 mutations were detected. The combined in vitro and clinical data reveals that treatment with an MDM2 antagonist is not associated with the emergence of de novoTP53 mutations but rather the expansion of prior existing TP53 clones. This does not appear to have clinical repercussions, however, close monitoring of these patients is essential. We recommend that patients be screened for TP53 mutations prior to treatment with an MDM2 antagonist and that if present the TP53 mutant VAF be followed during their treatment course. Resistance to MDM2 inhibition is likely dependent on the TP53 mutant VAF and further studies will need to clarify the ideal dosing schedule of MDM2 antagonists and/or combinatorial therapy to prohibit TP53 mutant clonal expansion. Disclosures Houldsworth: Cancer Genertics: Other: stock in; Sema4: Employment. Rossi:Sema4: Employment. Kiladjian:AOP Orphan: Honoraria, Research Funding; Celgene: Consultancy; Novartis: Honoraria, Research Funding. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding.

The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (>1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

Genome profiling represents a critical pillar for clinical, translational, and basic research studies. Hospitals, core facilities, and research enterprises invest significant resources to generate genomic data sets. Yet, data management and analysis is frequently manual, which demands significant operator time and often results in siloed resources rendering them as single-use assets. Centralization of the genomic capital in a framework that enables automated processing, metadata integration and continuous interrogation maximizes return for investment and serves as the critical catalyst for research innovation, clinical translation and reproducible research. We developed Isabl, a plug-and-play infrastructure for scalable bioinformatics operations. Isabl provides solutions for databasing, assets management, tracking, automated and reproducible data processing. Dynamic reporting and meta-analysis across data assets is enabled. Isabl is built on four main components. First, an individual-centric and extensible relational database with tracking support for samples (temporal, spatial, aliquot), experimental data (assays, platforms, sequencing runs), cohorts (clinical trials, research projects) and versioned bioinformatics applications (assembly aware, tools, results). Second, the database is exposed through a fully featured RESTful API that enables horizontal integration with information systems such as sequencing cores LIMS, variant visualization platforms like cBioPortal, and where applicable, clinical and biospecimen institutional databases. Third, a Software Development Kit (SDK) built for Next Generation Sequencing assets management. The SDK enables automated execution of data import and language-agnostic bioinformatics applications (alignment, variant calling, post-processing) with support for cohort and individual level reporting features. Furthermore, the SDK facilitates dynamic retrieval of results using vertical and horizontal queries (individual and cohort level, respectively). Lastly, Isabl comes with a Single Page Web Application that fosters user interaction with multidisciplinary teams (i.e. researchers, project coordinators, engineers, clinicians) facilitating tracking of analyses, results visualization, and dynamic query processing. Isabl is currently supporting the Memorial Sloan Kettering Genome Pediatrics Precision Medicine Initiative, a prototype platform that delivers integrated, real-time automated reporting of clinical targeted gene re-sequencing, research whole genome and transcriptome profiling data; as well as linked data from pre-clinical models (i.e. PDX) and single cells studies. As an open-source tool, Isabl democratizes access to a purpose built, automated, scalable and fully integrable bioinformatics architecture. Isabl will be available at https://github.com/isabl-io. Citation Format: Juan S. Medina-Martínez, Juan E. Arango-Ossa, Gunes Gundem, Max F. Levine, Minal Patel, Noushin R. Farnoud, Venkata D. Yellapantula, Gao Teng, Joseph G. Mccarter, Elsa Bernard, Franck Rapaport, Dominik Glodzik, Ross L. Levine, Andrew Kung, Elli Papaemmanuil. A plug-and-play infrastructure for scalable bioinformatics operations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5105.

Background: MRD is a powerful prognostic factor in AML. Emerging data indicate that allogeneic stem cell transplant (alloSCT) with MRD results in outcomes equivalently poor to alloSCT with morphologic AML (Araki et al., JCO 2016). Genomic predictors of MRD are unclear, and relative efficacy of therapies for MRD remains elusive. Objectives: Here we provide an integrated analysis of responses for 163 patients (pts) who underwent induction chemotherapy with baseline next-generation sequencing (NGS) followed by serial immunophenotypic monitoring for MRD. Methods:163 patients starting in April 2014 who underwent induction chemotherapy at Memorial Sloan Kettering Cancer Center were retrospectively studied. All received anthracycline + cytarabine, with or without investigational agents. Immunophenotypic MRD was identified in bone marrow aspirates (BMA) by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Molecular analysis was obtained from pre-induction BMA by NGS using 28 or 49 gene panels. Cytogenetics/FISH were performed using standard techniques. Results: Patient characteristics are in Table 1. 7/163 (4.9%) died within 30 days of induction.153 pts had BM biopsy after induction prior to further therapy. 124/153 underwent flow after induction. 65/124 (52.4%) achieved CR/CRi after induction alone, 31/124 (25%) MRD+CR/CRi, and 34/124 (27.4%) MRD-CR/CRi. Pre-induction molecular analysis from 126 suggests that certain cytogenetic and molecular abnormalities correlate with achievement of MRD-CR. (Figure 1) Only 2/25 (8%) with RUNX1, 0/13 with SF3B1, and 0/11 with TP53 mutations achieved MRD-CR/CRi as best response after 1 cycle of induction. Only 3 additional RUNX1, 2 SF3B1, and 0/11 TP53 achieved MRD-CR/CRi as best response after a second cycle of therapy. In contrast, 7/8 with CBF AML (inv16 and no KIT mutation, n=4) or (t(8;21), n=3) achieved MRD-CR/CRi (n=5) or CR without flow (n=2) after 1 cycle of induction. 91/163 (55.8%) underwent alloSCT following induction or additional therapy. Post-alloSCT follow-up indicates potential value in converting MRD+ to MRD-. 84/91 were evaluable for MRD with flow cytometry prior to alloSCT. 41/84 (48.8%) were MRD-, 30/84 (35.7%) MRD+, and 13/84 (15.4%) persistent AML. 13/41 (31.7%) MRD-pre-alloSCT were MRD- post-induction. 28/41 (68.2%) MRD+ or persistent AML converted to MRD- prior to alloSCT following additional therapy. 23/29 MRD+CR/CRi pts after induction were intermediate/unfavorable and therefore transplant candidates. 19/23 MRD+CR/CRi intermediate/unfavorable underwent transplant (9 without post-induction therapy, 10 after consolidation), while 4 did not proceed to transplant due to relapse after induction (n=1), relapse after consolidation (n=2), and patient preference. There was no significant difference in post-transplant OS between early MRD-CR immediately following induction and later conversion to MRD-CR prior to alloSCT (Figure 1B). Post-transplant analysis reveals that most pts who enter transplant with persistent AML (n=13) or MRD+ (n=30) clear MRD (30/43, 69.7%) by the first post-transplant BM (median 32 days, Figure 1C). Despite initial post-transplant MRD clearance, pts who entered alloSCT with persistent AML or MRD+ had poorer post-transplant OS compared to pts who entered alloSCT with MRD- (p=0.02, Figure 1D). Conclusion: Our data show that AML pts with specific molecular mutations (RUNX1, SF3B1, and TP53) are unlikely to achieve MRD-CR/CRi after induction chemotherapy. We further show that additional therapy such as consolidation may be advantageous for some MRD+ pts to achieve MRD-CR prior to alloSCT, although others remain resistant to MRD clearance. Post-transplant OS is improved in pts who are MRD- at time of transplant, regardless of whether they required additional therapy beyond induction to achieve this state. Our results suggest that development of MRD-eradicating therapies after AML induction has the potential to improve post-transplant outcomes. Disclosures Goldberg: AROG: Research Funding; Pfizer: Research Funding; Celgene: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Perales:Takeda: Other: Personal fees; Merck: Other: Personal fees; Abbvie: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Novartis: Other: Personal fees. Tallman:ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding.

Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Myeloproliferative neoplasm-blast phase (MPN-BP) and de novo acute myeloid leukemia (AML) each have distinct mutational patterns and clinical courses. MPN-BP patients have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies. So far, the cellular hierarchy that characterizes MPN-BP and the evolution of various leukemia-initiating clones (LIC) in MPN-BP have not been well delineated. We therefore established an in vivo MPN-BP xenograft model to address these questions. Among the 22 patients with MPN-BP studied 11 were cytogenetically normal while the remainder had multiple chromosomal abnormalities including del(5), del(20q), del(14), +1q, del(17p). 86% of the patients had at least 2 myeloid malignancy gene mutations including JAK2, ASXL1,TET2, MPL, SF3B1, RUNX1, U2AF1, PTPN11, IDH1/2, SRSF2 and TP53. These findings indicate that MPN-BP is characterized by multiple mutational events and cytogenetic abnormalities. T cell-depleted mononuclear cells from 8 of 14 patients engrafted in NSG mice {>0.5% hCD45+ cells in bone marrow (BM)}. Among them, samples from 6 patients resulted in a high degree of hCD45+ cell chimerism (34.6±6.4% in BM) and recapitulated numerous aspects of MPN-BP within 4 months, including the presence of at least 20% hCD45dimCD33+ cells or hCD34+ cells, or at least 20% blasts as detected by morphological examination of the marrow and leukemia cell dissemination to the spleen and PB. These mice had a 2.8±0.6- fold increase in splenic weight as compared to mice receiving PBS alone. The leukemic mice were characterized by reduced blood counts, suggesting that MPN-BP cells suppressed normal murine hematopoiesis, or led to cytopenias due to hyper-splenism. Moreover, the greater degrees of blast cell chimerism and the higher frequency of leukemia initiating cells as determined by limiting dilution analyses correlated with a shorter time to leukemia initiation and an inferior clinical outcome of the transplanted NSG mice. Grafts from each of these 6 MPN-BP patients produced a large number of donor-derived myeloid cells and a smaller number of lymphoid cells (mostly CD3+ and few CD19+). Cells belonging to each of these lineages and leukemic cells in primary recipients produced from Pts 4, 5, 6 and 11 had an identical proportion of chromosomally abnormal and mutated cells as primary cells [Pt 4: JAK2V617F, TET2 and PHF6; Pt 5 and 11: Del (20q), +8; Pt 6: +1q, del(17p)], except that a small proportion of T cells from Pts 5 and 11 lacked chromosomal abnormalities. Furthermore, the degree of MPN-BP engraftment and leukemic cell burden increased with the subsequent 3 serial transplantations even when the recipients received progressively smaller numbers of MPN-BP cells from the prior recipient. Primary Pt 6 originally had a JAK2V617F+ PV but lost JAK2V617F at the time the MPN-BP occurred at which time there were two clonal cell populations, one with +1q (12%) and the other del(17p) (80%), the site of the TP53 gene, as well as normal cells (8%). In the primary recipient NSG the donor derived cells were JAK2V617F- but contained +1q (1%) and del(17p) (98.5%) and cytogenetically normal (0.5%). +1q and JAK2V617F were not observed, while cells containing the TP53 deletion alone were detected in donor derived leukemic cells, mature myeloid and T cells in the secondary and subsequent serial recipients. Furthermore, del(17p) was found in phenotypically isolated HSCs, MPPs, MLPs, CMPs, GMPs, MEPs, and mature T cells within the CD33- cell fraction as well as CD45dimCD33+ AML blasts selected from primary MPN-BP cells from Pt6. However, +1q was found exclusively in purified MLPs and MEP. These observations establish that cytogenetic and mutational events that lead to MPN-BP occur at different stages along the developmental HSC hierarchy and that a small population of normal HSCs persist. Furthermore, in JAK2V617F+ MPNs that develop MPN-BP and lose JAK2V617F, additional cytogenetic events occur at different stages along the JAK2V617F- MPN-BP-stem cell hierarchy. Our ability to serially transplant the LIC from these patients has allowed us to create the first MPN-BP PDX model that will not only extend our understanding of MPN-BP stem cell biology but might also prove useful for screening drugs to treat MPN-BP. Disclosures Rampal: Jazz: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; Stemline: Research Funding; Constellation: Research Funding; Celgene: Honoraria. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Research Funding; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Janssen: Research Funding. Hoffman:Formation Biologics: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Incyte: Research Funding; Janssen: Research Funding.

Background Therapy-related myelodysplastic syndrome (MDS) and acute leukemias represent a major cause of non-relapse morbidity and mortality in childhood cancer survivors, and have been associated with exposure to cytotoxic therapies (e.g. radiation, alkylators, topoisomerase inhibitors). Neuroblastoma (NB) patients receive multimodality therapy with intensive chemotherapy, radiation, and immunotherapy, and have had high rates of treatment-related leukemias (Kushner, Cheung et al. 1998). Whilst specific therapeutic modalities have been associated with distinct cytogenetic and molecular abnormalities, our understanding of the relationships between timing of mutation acquisition, dynamics of clonal selection in relation to specific therapeutic modalities, and how these in unison result in overt leukemia, remains limited. Motivated to study these relationships and inform future screening guidelines, we characterized serial bone marrow (BM) samples obtained during surveillance for NB recurrence and therapy-related leukemias. Methods We studied a total of 219 serial samples from 55 NB patients treated at MSKCC over a 21-year period. These included 19 patients with MDS or leukemic transformation (median time following NB diagnosis 4.4 years), 15 with transient cytogenetic abnormalities (median time to abnormality 3.1 years), and 21 matched controls (median disease-free follow-up 8.1 years). On average, we analyzed 4 samples per transformation patient, representative of pre-treatment timepoints at NB diagnosis, during NB treatment, and throughout follow-up, with a lead time of 18 years - 1 month prior to transformation, and at time of leukemic transformation. Comprehensive genomic profiling with targeted gene sequencing (MSK-IMPACT Heme), RNA-seq, and Archer FusionPlex was performed to capture acquired gene mutations, chromosome-level copy number alterations (CNA), and fusion genes at the time of diagnosis. Backtracking studies were performed in longitudinal samples with complete molecular and clonal characterization. Results We detected at least one disease-defining alteration in all cases with MDS or leukemic transformation at time of diagnosis, with a total of 61 putative oncogenic events across all patients (median 3 alterations per patient, range 1-12). As expected, the most frequent events were MLL fusions (n=6 patients), and mutations in TP53 (n=5 patients). The remaining cases harbored chromosomal aneuploidies or acquired gene mutations in NPM1, IDH1, PTPN11, NRAS, BCOR, CUX1, STAG2, WT1, amongst other genes, at a median variant allele frequency 24% (range 4-68%). Backtracking studies identified at least one of these mutations in 81% of patients at a sampling time point prior to diagnosis. In contrast, only two patients (2/15) from the cohort with transient cytogenetic abnormalities had acquired somatic mutations detected at a median VAF of 3%, with resolution of the molecular alterations in subsequent samples. One of the control cases also had an identified mutation, though this patient died of NB with limited hematologic follow-up. The median time of detection of a putative driver alteration was 6 months prior to leukemic transformation, with the earliest identified at 2.8 years prior to disease transformation. At least 3 cases of MLL fusions were detected 2.2, 14.5, and 20.9 months prior to diagnosis. Mutations in TP53 co-occurred with CNAs in all patients in our cohort, and has been shown to be predictive of chemoresistant disease. Mutations in TP53 were also identified in at least 2 pre-leukemic samples per patient in 4 of 5 cases, at a median VAF 5% (range 5-20%). In all of these cases, TP53 mutations preceded clinically detectable CNA. Genetic evolution led clonal dominance, which, intriguingly, often preceded disease presentation in the context of normal hematopoeisis. We also found evidence of clonal drifts, possibly as a consequence of treatment effects. Conclusions Our preliminary data demonstrate that NB patients at risk of developing secondary leukemia can be identified by molecular profiling of BM aspirates obtained during routine disease surveillance for NB. These findings present an opportunity for the development of early detection studies for patients with pediatric malignancies undergoing intensive therapy and importantly inform studies into mechanisms of leukemic transformation and specific gene-treatment effects. Disclosures Cheung: Ymabs: Patents & Royalties.

Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the Nucleophosmin (NPM1) gene, and FLT3 is concomitantly mutated in approximately 60% of these cases. Little is known about how mutant NPM1 (NPM1mut) cells maintain aberrant gene expression. Here, we demonstrate that the histone modifiers MLL1 and DOT1L control HOX and FLT3 expression and differentiation in NPM1mut AML. Using a CRISPR/Cas9 genome editing domain screen, we show NPM1mut AML to be exceptionally dependent on the menin binding site in MLL1. Pharmacologic small-molecule inhibition of the menin–MLL1 protein interaction had profound antileukemic activity in human and murine models of NPM1mut AML. Combined pharmacologic inhibition of menin–MLL1 and DOT1L resulted in dramatic suppression of HOX and FLT3 expression, induction of differentiation, and superior activity against NPM1mut leukemia. Significance: MLL1 and DOT1L are chromatin regulators that control HOX, MEIS1, and FLT3 expression and are therapeutic targets in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on-target activity and constitutes a novel therapeutic concept for this common AML subtype. Cancer Discov; 6(10); 1166–81. ©2016 AACR. See related commentary by Hourigan and Aplan, p. 1087. This article is highlighted in the In This Issue feature, p. 1069

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease. We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days. Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging >500x for DNA and >6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2. The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival. Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance. These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. Disclosures Stein: Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation.