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Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have an extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLIs) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), -15, and -18 to generate memory-like (ML) NK cells with enhanced antileukemia responses. We treated 9 pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells in a phase 1 trial. Patients received fludarabine, cytarabine, and filgrastim followed 2 weeks later by infusion of donor lymphocytes and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical and matched-related and -unrelated donors. After infusion, donor-derived ML NK cells expanded and maintained an ML multidimensional mass cytometry phenotype for >3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered interferon-γ production. After DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for >3 months, with 1 patient in remission for >2 years. No significant toxicity was experienced. This study demonstrates that, in a compatible post-HCT immune environment, donor ML NK cells robustly expand and persist with potent antileukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at https://clinicaltrials.gov as #NCT03068819.

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

To the editor: Quantitative response evaluation in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) relies on the morphologic quantification of bone marrow (BM) blasts. This process is subject to the operator-dependent quality of BM collection and the interobserver variability

The underlying molecular effects of decitabine treatment in MDS and AML patients are poorly understood. To systematically uncover the global genomic and transcriptomic alterations induced by decitabine in vivo, we evaluated a series of primary bone marrowf samples from patients treated with decitabine 20 mg/m2 on days 1-10 in 28 day cycles (NCT01687400). Samples were evaluated with: bulk-RNA sequencing (Day 0 & 10, n=29), single-cell RNA sequencing (Day 0 & 10, n=4), whole-genome bisulfite sequencing (WGBS, Day 0, 10, & 28, n=29), and exome sequencing (Day 0 & 28 n=44). Cases were selected for RNA-Seq and WGBS based on adequate sample availability, and founding clone mutations that were present in at least 75% of the cells in each sample on day 0. As expected, interpatient heterogeneity dominated global transcriptional variance across the bulk-RNA sequencing dataset, due to the unique mutational and cellular heterogeneity of each sample. We identified 638 significantly differentially expressed genes (DEGs) between day 0 and day 10. MSigDB-geneset analysis identified significant pathway enrichment of the day 10 upregulated genesets for myeloid maturation, immune activation, and cell cycle regulation, while down-regulated genesets were enriched for erythroid pathways, oxidative phosphorylation, cellular senescence, and metabolic processes (FDR < 0.05). Upregulated genes were strongly associated with increased expression in external datasets of flow-sorted mature myeloid cells (i.e. Bloodspot), whereas downregulated genes correlated with decreased expression in mature populations, further suggesting that decitabine induces myeloid maturation in AML and MDS patients. Application of the Telescope computational pipeline identified increased overall expression of endogenous retroviruses (ERVs) at day 10 (p < 0.01). Interpatient heterogeneity was again evident, and ERV activation was limited to 18 ERV families (FDR In summary, this study provides insights into in vivo mechanisms of decitabine activity in AML and MDS patients, which includes global hypomethylation, myeloid maturation, induction of an inflammatory response, activation of specific ERV families, and strong interpatient heterogeneity. However, no specific transcriptional or epigenetic biomarkers emerged that could serve as effective predictors of decitabine responses. Disclosures Welch: ArcherDx: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Notable labs: Research Funding; Janssen research: Research Funding.

Background. Acute Myeloid Leukemia (AML) is genetically and epigenetically heterogeneous. Most AML samples display clonal heterogeneity at presentation, which evolves with therapeutic interventions. To better understand the epigenetic consequences of clonal heterogeneity, we are using single-cell RNA-sequencing (scRNA-seq) to characterize expression heterogeneity in AML. To date, scRNA-seq has had limited utility in applications where it is essential to link transcriptional heterogeneity to genetic variation, because it has been difficult to identify specific mutations in individual cells using scRNA-seq data alone. To address this limitation, we developed an approach to use scRNA-seq data to identify expressed mutations in individual AML cells, and link these variants to the expression heterogeneity in the same samples. Methods. We generated duplicate cDNA libraries for each of 5 cryopreserved bone marrow samples from adult patients with de novo AML, using the 10x Genomics Chromium Single Cell 5' Gene Expression workflow for Single Cell RNA Sequencing. Single cell libraries were sequenced to yield a median of 20,474 cells per sample, and 192,427 reads per cell. Transcript alignment, counting, and inter-library normalization were performed using the Cell Ranger pipeline (10x Genomics). The Seurat R package was used for further normalization, filtering, principal component analysis, clustering, and t-SNE visualization. A nearest-neighbor algorithm was developed to assign each cell in the data set to the most transcriptionally similar hematopoietic lineage. For each case, we performed whole genome sequencing (WGS) to identify germline and somatic variants, and define clonal architecture. We then developed bioinformatic methods to determine which cells harbor these mutations, assign those cells to mutationally-defined subclones, and link mutations to defined expression clusters. Results. WGS identified 25-56 coding mutations per sample; we were able to identify 22%-46% of these mutations in at least one cell in the scRNA-seq data, including point mutations (e.g. DNMT3A, U2AF1, TP53, IDH1, IDH2, SRSF2, CEBPA, and others) and indels (e.g. FLT3-ITD, NPMc). Although the libraries were 5' biased, expressed mutations could be identified at long distances from the 5' end of transcripts; for example, an expressed DNMT3AR882H mutation (2.646 Kb from the initiating codon) was easily detected (Fig 1c). The frequency of detected mutations in the single-cell data varied widely (range: 1-1564 cells; median: 11 cells), and as expected, depended heavily on the expression level of the gene, and the size of the clone containing the mutation. Regardless, a median of 1378 cells (6.7%) had at least one identifiable mutation in the 5 samples. Using these data, we were able to 1) distinguish AML cells from normal cells in bone marrow samples (Fig 1a/b), 2) identify major subclones within the AML samples (Fig 1c/d), and 3) identify mutation-specific and subclone-specific expression profiles. In 2 samples with mutationally-defined subclones (one with a CEBPAR142fs mutation, and the other with a GATA2R361C mutation), subclone-specific gene expression profiles were clearly detected in the scRNA-seq data, and could be directly associated with cells containing the mutant transcription factors. In the case with the subclonal GATA2R361C mutation, cells with that mutation were restricted to a subset of expression clusters (Fig 1d). In this subset, we identified an expression signature that is supported by pre-existing knowledge of the GATA2/SPI1 transcriptional regulatory circuit. In addition, we observed that expression heterogeneity frequently occurs independent of mutations defined by specific subclones. For instance, the GATA2R361C subclone contained additional heterogeneity (5 independent expression clusters) that could not be accounted for by mutations (Fig 1a/d). Moreover, the other 3 cases exhibited extensive expression heterogeneity within the AML cells that was not explained by genetically defined subclones. In sum, scRNA-seq data, when adapted to detect mutations, has dramatically improved our understanding of the expression heterogeneity of AML, which arises from two main sources: 1) cell-type composition of the sample, and 2) expression variation among the AML cells themselves (caused by both mutation-associated and mutation-independent factors). Disclosures Williams: 10x Genomics: Employment, Equity Ownership. Fiddes:10x Genomics: Employment, Equity Ownership. Church:10x Genomics: Employment, Equity Ownership.

There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy.

Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARα fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARα+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARα, as assessed by serial dilution experiments, was 10−4. All patients were in hematologic remission and tested PCR− at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR− in ≥2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARα during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up. © 1998 by The American Society of Hematology.

BCR-ABL antisense oligodeoxynucleotides (ODN) have provided evidence of antileukemia effect when tested in vitro against Philadelphia-positive (Ph-pos) cells and in vivo when injected into leukemic mice. On the basis of the results obtained in vitro at diagnosis, eight patients with chronic myelogenous leukemia (CML) were selected and submitted to autologous bone marrow transplantation (ABMT) with bone marrow (BM) cells purged in vitro with junction-specific (J-sp) BCR-ABL antisense ODN at the time of transformation in accelerated phase or during second chronic phase. Mononuclear BM cells were treated in vitro for 24 or 72 hours with 150 μg/mL of antisense ODN yielding a median recovery of 47.6% mononuclear cells, 48.8% CD34+ cells, and 20.3% clonogenic cells. After a conditioning regimen including busulphan and etoposide, the reinfused treated cells allowed engraftment and hematologic reconstitution in all patients. Evaluation of the antileukemic effect by standard cytogenetic analysis and fluorescence in situ hybridization showed a complete karyotypic response in two cases and a minimal or no response in the other six. The patient autografted in second chronic phase died in blast crisis 7 months after ABMT; of the seven patients autografted in transformation, three developed blast crisis 21 to 39 months after reinfusion, one died from unrelated BMT complications 30 months after ABMT, and three are in persistent second chronic phase 14 to 26 months after autograft. The low toxicity of the protocol and the hemopoietic reconstitution observed in all patients make this approach feasible; the marked karyotypic response observed in some patients and the duration of the second chronic phase show that ODN-mediated BM purging and autograft is a promising treatment for this high-risk group of CML.

*The 2 first authors contributed equally to the work. Background. The best post-remission treatment for younger acute myeloid leukemia (AML) patients has remained controversial (Cornelissen JJ &Blaise D, Blood 2016, 127, 62-70) and there is paucity of studies comparing intensive chemotherapy to autologous (auto) or allogeneic (allo) bone marrow transplantation (BMT). Aim. The main objective of this study was to provide a long-term follow-up evaluation of patients previously enrolled in the pivotal EORTC/GIMEMA AML-8A (Zittoun et al., New England Journal of Medicine 1995, 332, 217-223). Methods. The EORTC/GIMEMA AML-8A prospectively assessed the impact of 3 post-remission treatments on disease-free survival (DFS) and overall survival (OS) in younger ( Results. In the current report, median follow-up was 11.1 (range, 0-28) years. For the whole population (n=422), the 5-, 10 and 15-year OS rates from inclusion were 35%, 32% and 31%, respectively. After the completion of ICT1, 168 patients were allocated to theallo-BMT arm, while 254 patients were randomized to auto-BMT (n=128) or ICT2 (n=126). DFS from CR was longer afterallo-BMT than auto-BMT and DFS from CR was longer after auto-BMT than ICT2, due to a lower relapse incidence (P Conclusions. This long-term follow-up of the EORTC/GIMEMA AML-8A study confirms a better DFS with allo-BMT or auto-BMT when compared to ICT2 for AML patients in first CR. Further, this long-term follow-up study revealed that the vast majority of patients alive in first CR at 5-year remains disease-free survivors 5 years later. Although indications of allogeneic hematopoietic stem cell transplantation (allo-HCT) are nowadays largely driven by cytogenetic/molecular AML profile, long-term results of AML8A study demonstrate that auto-BMT remained superior to ICT2 in younger AML patients not candidate for an allo-HSCT. Disclosures Efficace: TEVA: Consultancy, Research Funding; Seattle Genetics: Consultancy; Bristol Myers Squibb: Consultancy; Lundbeck: Research Funding. Martinelli:Celgene: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau.

Acute leukemia with the t(11;17) expressing the PLZF-RARαgene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient withPLZF/RARα APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.

Hybrid fusion genes are specific tumor markers of several leukemic subtypes. The use of reverse transcription-polymerase chain reaction (RT-PCR) to amplify chimeric cDNAs allows sensitive detection of the neoplastic clone for diagnostic and monitoring studies in these leukemias. Nonetheless, the clinical relevance of minimal residual disease (MRD) evaluation by PCR remains controversial. In this study, 9 patients (pts) with acute promyelocytic leukemia (APL) in long-term remission for 4 to 12 years were analyzed for the presence of MRD by RT- PCR amplification of the specific PML/RAR-alpha fusion gene. Seven pts had been treated with conventional chemotherapy (CHT) alone, 1 had undergone allogeneic bone marrow transplantation (BMT), and 1 autologous BMT as consolidation therapy after CHT. In 8 cases, the presence of the t(15;17) rearrangement could be documented in diagnostic BM specimens by cytogenetic and/or molecular analysis. A two- rounds “nested” RT-PCR assay with sensitivity levels of 1 in 10(5) was used to analyze BM samples collected at 32 to 141 months from the achievement of complete remission (CR). In no cases were residual PML/RAR-alpha transcripts detectable in these remission controls. All patients are in unmaintained CR at 48 to 154 months from CR and at 6 to 17 months from PCR evaluation. These results suggest that long-term survival of APL is associated with eradication of cells carrying the specific PML/RAR-alpha rearrangement, indicating that PCR negativity should be considered the therapeutic goal in these patients. Our findings further strengthen the clinical relevance of PCR monitoring studies in APL, as opposite to other leukemic subtypes (chronic myeloid leukemia and acute myeloid leukemia-M2) in which the prognostic significance of PCR evaluation is unclear.

Seventy patients with acute promyelocytic leukemia (APL) were characterized at the DNA level using genomic retinoic acid receptor- alpha (RAR-alpha) probes on Southern blot experiments. Sixty-two cases were defined as M3 according to the French-American-British (FAB) criteria, and eight had a diagnosis of microgranular or variant (M3v) APL. The use of two restriction enzymes and three probes exploring the second intron of the RAR-alpha gene allowed us to detect specific abnormal DNA fragments in every case, with clustering of rearrangements within the 20-kb intronic region between RAR-alpha exons II and III. A more detailed mapping of APL breakpoints was performed in 52 cases in which three EcoRI subregions of the RAR-alpha second intron were analyzed with corresponding probes. Comparison of clinical and hematological features in the three subgroups of patients with distinct RAR-alpha breakpoints did not show significant differences regarding age, peripheral blood (PB) counts, presence of coagulopathy, or FAB classification (M3 v M3v). Interestingly, a significant difference was observed in the M/F ratio of the three subgroups, with a higher incidence of rearrangements at the 5′ end of the RAR-alpha second intron in female patients, and more frequent 3′ breakpoints in males. The results of this study indicate that a unique genomic alteration consistently occurs on the 17q- derivative of the APL specific t(15;17) aberration. Moreover, the clinical relevance of RAR-alpha gene analysis both at diagnosis and in follow-up studies is further emphasized.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists—in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.

To determine how AML subclonal architecture changes during decitabine treatment, and whether specific mutations might correlate with sensitivity vs. resistance to decitabine, we performed exome sequencing at multiple time points during single agent decitabine therapy. We enrolled 69 patients with either AML (age ≥ 60, or with relapsed/refractory disease, N = 45) or MDS (N = 24) on a phase I clinical trial. All subjects were treated with decitabine 20 mg/m2 on days 1-10 of 28 day cycles. With a median follow-up of 13.7 months, the intention to treat clinical response (complete remission with or without complete count recovery: CR/CRi) is 40%, with survival correlating with response (median survival - CR/CRi: 583 days; partial response/stable disease (PR/SD): 260 days; progressive disease (PD) or failure to complete cycle 1: 36 days, p < 0.0001). We performed exome sequencing on unfractionated bone marrow cells at diagnosis (day 0), cycle 1 day 10, cycle 1 day 28, cycle 2 day 28, and, when possible, during remission and at clinical relapse/progression. We have completed sequencing analysis for the first 34 cases (outcomes: 5 CR, 15 CRi, 3 PR, 8 SD, and 3 PD). Several important themes have emerged, as follows: 1) We correlated mutation status at diagnosis with clinical response. All six patients with TP53 mutations obtained clinical CR or CRi, and exome analysis demonstrated near complete elimination of the TP53- associated founding clones by the end of cycle 2 (p < 0.03). Long-term outcomes were similar in these patients compared with other patients who achieved CR/CRi: four patients relapsed after 8, 9, 10, or 17 cycles; 1 patient is doing well post-transplant; and one patient died of an infectious complication after cycle 2. No other mutations were significantly associated with clinical response or with consistent mutation clearance. 2) We observed a reduction in blast counts, which preceded mutation elimination in fourteen cases with CR, CRi or PR. This suggests that decitabine may induce morphological blast differentiation in vivo prior to mutation elimination. 3) In eight of nine cases with a clinical response followed by relapse, clinical progression was associated with expansion of a pre-existing subclone. We have not yet observed any recurrent mutations that reliably predict whether a subclone will contribute to relapse. Intriguingly, in two of these cases, the relapse-associated subclone was detectable at diagnosis and was eliminated more slowly than the founding clone mutations, suggesting that this subclone harbored intrinsic decitabine-resistance. 4) Complete remission can occur with concomitant non-malignant, clonal hematopoiesis. In three cases with a CR, a new clonal population was selected for during the remission. In two of these cases, there were no shared mutations between the founding clone and the emergent, non-malignant, clonal hematopoiesis, suggesting that these clones were unrelated. 5) Mutational architecture is generally stable, but differential chemo-sensitivity can be detected even between subclones in the same patient. In ten cases with PR or SD, we observed minimal shifts within the mutational burden over the course of eight weeks, suggesting that "clonal drift" is a relatively slow process. However, in four cases with SD, what appeared clinically to be simple persistent disease was in fact a dynamic elimination of one subclone, and its replacement by a different subclone. Similarly, in three cases with CRi, we observed rapid clearance of a subclone with slower clearance of the founding clone, again suggesting differential chemo-sensitivity among subclones. 6) Finally, we correlated pharmacologic markers with clinical outcomes. We observed no correlation between steady-state plasma decitabine levels and clinical responses. Using Illumina 450k methylation arrays, we observed a correlation between response and the extent of decitabine-induced hypomethylation in total bone marrow cells that persisted on cycle 1 day 28 (p < 0.01), but not on cycle 1 day 10 (p < 0.1). In summary, these data reveal that response to decitabine is associated with morphologic blast clearance before mutations are eliminated, that relapse is associated with subclonal outgrowth that may be identified early in the treatment course, and that TP53 mutations may be predictive of rapid clinical responses, although, like most responses to decitabine, these are not necessarily durable. Disclosures Off Label Use: Decitabine treatment of AML.. Uy:Novartis: Research Funding. Oh:CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Abboud:Novartis: Research Funding; Gerson Lehman Group: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Merck: Research Funding; Teva Pharmaceuticals: Research Funding. Cashen:Celgene: Speakers Bureau. Schroeder:Celgene: Other: Azacitidine provided for this trial by Celgene; Incyte: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

Our group (Welch, Cell 2012) previously showed that hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations with age. This produces a genetically heterogeneous HSPC population with each HSPC possessing its own unique set of mutations. Later work from our group (Xie, Nature Medicine 2014) and others (Genovese, Jaiswal, NEJM 2014) demonstrated that some these mutations may provide HSPCs with a fitness advantage, allowing them to clonally expand over time in healthy individuals. We recently published data (Wong, Nature 2015) suggesting that cytotoxic therapy can select for HSPC clones with TP53 mutations, resulting in their clonal expansion and contributing to the subsequent development of therapy-related AML/MDS. From these data, we hypothesized that the intensive cytoreductive chemotherapy used to treat AML poses a significant selection pressure on a patient's non-malignant HSPC population, favoring HSPCs with specific somatic mutations and potentially resulting in oligoclonal hematopoiesis even after elimination of the founding AML clone. To test this hypothesis, we performed enhanced exome sequencing on cryopreserved bone marrow cells from 25 adult de novo AML patients (who received a "7+3" regimen for induction of remission) at time of their initial diagnosis, at first morphologic remission (~day 30), and at long-term follow up (at first relapse or during a prolonged first remission) (Klco, JAMA, in press). In 15 patients, we observed genetic clearance of the AML founding clone at the time of first morphologic remission (defined as all AML founding clone mutations declining to a variant allele frequency (VAF) < 2.5%). Surprisingly, in 5 of the 15 patients exhibiting clearance of their AML founding clone, we observed a concomitant expansion of a non-malignant clonal population during cytoreductive therapy, resulting in long-lived clonal hematopoiesis. Somatic mutations harbored by these expanding hematopoietic clones were validated with a high-coverage PCR-based sequencing approach. In contrast to the studies highlighting clonal hematopoiesis in individuals unexposed to chemotherapy, patients with evidence of persistent clonal hematopoiesis after cytoreductive therapy (median age = 52.2 years) were similar in age to patients without such evidence (median age = 54.1 years). The majority of these "rising clones" harbored somatic mutations in genes frequently mutated in AML such as DNMT3A, TET2 and TP53. Using next-generation sequencing and droplet digital PCR, we determined that in all of the patients with an expanding non-malignant clone, the clone was, in fact, present in the initial AML diagnosis sample at very low VAFs (0.007-0.75%). These populations rapidly expanded with chemotherapy, comprising 13-57% of the total hematopoietic population upon its completion. In all 4 of cases with sample availability, these clones remained at an expanded level a year or more after initial chemotherapy exposure. These results suggest that certain non-malignant HSPCs, having previously acquired specific aging-related somatic mutations, may gain a competitive fitness advantage after cytoreductive therapy, expand, and persist long after the completion of chemotherapy. Two of the five patients with clonal non-leukemic hematopoiesis post-chemotherapy relapsed. In both patients, the relapsed AML clone evolved from the original AML founding clone and did not involve the non-malignant clonal population, which also persisted at relapse. Both patients re-achieved morphologic remission with salvage therapy. A post-salvage therapy bone marrow sample was available in one of the cases. Interestingly, it showed that the patient's non-malignant clonal population expanded even further with salvage therapy, eventually comprising almost 80% of the total bone marrow cells. These results show that non-malignant oligoclonal hematopoiesis is common in AML patients after cytoreductive chemotherapy, with non-malignant HSPCs carrying certain somatic mutations often gaining a fitness advantage and expanding. The long-term clinical consequences of oligoclonal hematopoiesis after cytoreductive chemotherapy are unknown but are likely to be different from oligoclonal hematopoiesis developing in healthy elderly individuals. Additional studies will be required to define the mechanisms by which certain HSPCs gain a fitness advantage after cytoreductive chemotherapy. Disclosures No relevant conflicts of interest to declare.

An unexpectedly high incidence of blast transformation after splenectomy has been reported in patients with myelofibrosis with myeloid metaplasia. However, whether this was associated with spleen removal after adjustment for risk factors was not determined. We conducted a multicenter historical cohort study of patients with myelofibrosis with myeloid metaplasia diagnosed from January 1970 through January 1994. A total of 549 patients (325 men and 224 women from 22 to 92 years of age; median age, 63 years) were included in the final data set. The Cox's proportional-hazards model was used to identify factors associated with blast transformation and death. To further adjust for factors related to spleen removal assignment, a propensity score for splenectomy was estimated using recursive-partitioning analysis. Blast transformation developed in 78 patients (14.2%). Patients who underwent splenectomy developed more blast transformations than those who were not splenectomized (23 of 87 [26.4%] v 55 of 462 [11.9%]; P.001). The cumulative incidence of blast transformation 12 years after diagnosis was 27.0% in nonsplenectomized patients and 55.0% in splenectomized ones (P = . 01). The risk factors independently predictive of blast transformation included prior splenectomy (relative risk = 2.61), platelet count less than 100 x 10(9)/L at diagnosis (relative risk = 2.45), and the presence of blasts in peripheral blood at diagnosis (relative risk = 2.31). The relative risk of blast transformation in splenectomized patients increased from 2.2 at 48 months from diagnosis to 14.3 at 12 years. Patients with the same propensity score for splenectomy showed a higher risk for blast transformation on the basis of having undergone splenectomy (P = .02). In conclusion, the risk of blast transformation is significantly increased in subjects who underwent splenectomy and appears to be independent of factors related to spleen removal assignment.

Reverse-transcription polymerase chain reaction (RT-PCR) of the PML/RAR alpha fusion gene may predict relapse in acute promyelocytic leukemia (APL) patients in hematologic complete remission (CR). We have prospectively studied by RT-PCR 15 PML/RAR alpha+ APL patients undergoing autologous bone marrow transplantation (ABMT) in second CR. The median time of first CR duration was 12 months (range, 6 to 40). All patients were reinduced with all-trans retinoic acid (ATRA), followed in 12 of 15 cases by mitoxantrone and Ara-C as consolidation. Fourteen patients received the BAVC (BCNU, Ara-C, m-AMSA, and VP-16) schedule as conditioning regimen. Unpurged marrows were collected immediately before conditioning treatment, analyzed by RT-PCR, and reinfused at median of 2 months (range, 2 to 7) from the achievement of second CR. Seven patients were PCR+ and eight PCR for PML/RAR alpha in their pretransplant marrows. All seven patients of the former group remained PCR+ during the follow-up and relapsed at a median time of 5 months (range, 2 to 9) from ABMT and 9 months (range, 4 to 14) from second CR. Of the eight PCR- patients, all remained PCR- during the follow-up controls. One patient relapsed at 10 months from ABMT, one died of a secondary (PML/RAR alpha-) leukemia, and six are in hematologic and molecular remission at a median time of 28 months (range, 15 to 60) after ABMT and 32 months (range, 17 to 62) from second CR. Our results indicate that, in APL patients in second CR, ABMT with PML/RAR alpha- marrow cells is likely to result in prolonged clinical and molecular remissions. Conversely, patients who test PCR+ after reinduction necessitate the use of alternative aggressive approaches, including unrelated allogeneic transplant.

Two hundred fifty-three patients with newly diagnosed acute promyelocytic leukemia (APL) were eligible to enter the multicentric GIMEMA-AIEOP "AIDA" trial during the period July 1993 to February 1996. As a mandatory prerequisite for eligibility, all patients had genetic evidence of the specific t(15;17) lesion in their leukemic cells confirmed by karyotyping or by reverse transcription-polymerase chain reaction (RT-PCR) of the PML/RAR alpha fusion gene (the latter available in 247 cases). Median age was 37.8 years (range, 2.2 to 73.9). Induction treatment consisted of oral all-trans retinoic acid (ATRA), 45 mg/m2/d until complete remission (CR), given with intravenous Idarubicin, 12 mg/m2/d on days 2, 4, 6, and 8. Three polychemotherapy cycles were given as consolidation. Hematologic and molecular response by RT-PCR was assessed after induction and after consolidation. At the time of analysis, 240 of the 253 eligible patients were evaluable for induction. Of these, 11 (5%) died of early complications and 229 (95%) achieved hematologic remission. No cases of resistant leukemia were observed. Of 139 cases studied by RT-PCR after induction, 84 (60.5%) were PCR-negative and 55 (39.5%) PCR-positive. One hundred sixty-two patients were evaluable by RT-PCR at the end of consolidation. Of these, 159 (98%) tested PCR-negative and 3 (2%), PCR-positive. After a median follow up of 12 months (range, 0 to 33), the estimated actuarial event-free survival for the whole series of 253 eligible patients was 83% +/- 2.6% and 79% +/- 3.2% at 1 and 2 years, respectively. This study indicates that the AIDA protocol is a well-tolerated regimen that induces molecular remission in almost all patients with PML/RAR alpha-positive APL. Preliminary survival data suggest that a remarkable cure rate can be obtained with this treatment.

Abstract 1871 Introduction: In a randomized phase 3 study, the use of VTD as induction therapy prior to and consolidation therapy following double ASCT increased the rate of complete or near complete response (CR/nCR) and extended PFS in comparison with TD given as induction and post-ASCT consolidation therapy in 474 newly diagnosed MM patients (Cavo et al, Lancet 2010). However, the specific impact of VTD consolidation on improved clinical outcomes was not defined. Methods: To address this issue, we performed a per-protocol analysis of 321 patients who received the entire treatment program, including the two pre-planned cycles of consolidation therapy with either VTD (160 of 236 patients, 68%) or TD (161 of 238 patients, 68%). By study design, two 35-d cycles of VTD (V 1.3 mg/m2 on days 1, 8, 15, and 22; T 100 mg/d on days 1–35; D 40 mg on the days of and after each V administration) or TD (at the same doses as in VTD) were given as consolidation therapy to patients randomly assigned to each of the two respective induction regimens. Patient and disease characteristics at baseline were comparable in the two groups of patients. Results: The rates of CR and CR/nCR were significantly higher after consolidation therapy with VTD compared with TD (CR: 61% vs 47%; p=0.012; CR/nCR: 73% vs 61%; p=0.020). The impact of VTD consolidation on post-ASCT enhanced rates of CR and CR/nCR was confirmed by the McNemar test (CR: p=0.0009; CR/nCR: p=0.004) which conversely failed to demonstrate a significant increase in the frequencies of CR (p=0.052) and CR/nCR (p=0.110) with TD consolidation therapy. The absolute probability of upgrading from less than CR before consolidation to CR after consolidation was 31% with VTD and 17% with TD (Pearson chi-square test: p=0.03). Ninety six percent of patients who upgraded from less than CR to CR after VTD consolidation were in nCR (44%) or VGPR (52%) before starting consolidation therapy. A landmark analysis (with the landmark set as the start of consolidation therapy) was performed to compare time to progression (TTP), PFS, and overall survival (OS) between treatment groups. With a median follow-up of 30 months, the estimated 3-year probability of relapse or progression was 38% with VTD and 52% with TD (p=0.039 by Kaplan-Meier analysis) (HR: 0.68, 95% CI: 0.47–0.98, p=0.041). PFS was significantly longer for patients receiving VTD consolidation than for those treated with TD (3-year estimates: 62% vs 46%; p=0.025) (HR: 0.66, 95% CI: 0.46–0.95, p=0.027), a gain particularly evident for patients who failed to achieve CR after ASCT (3-year PFS estimates: 66% vs 43%; HR: 0.54, 95% CI: 0.32–0.91, p=0.022). Superior PFS with VTD vs TD consolidation was retained across poor prognosis subgroups, including patients with t(4;14) and/or del(17q) (HR: 0.44, p=0.004), del(13q) (HR: 0.44, p=0.002), β2-microglobulin >3.5 mg/L (HR: 0.57, p=0.025), lactate dehydrogenase >190 U/L (HR: 0.57, p=0.005), or ISS stage 2 and 3 (HR: 0.57, p=0.021). In a multivariate regression analysis, the most important and independent variables positively correlated with PFS were VTD consolidation therapy (p=0.002), double ASCT (p=0.001), low β2-microglobulin (p Conclusions: In comparison with TD, post-ASCT consolidation therapy with the triplet VTD regimen significantly increased the rates of CR and CR/nCR, and extended landmarked TTP and PFS. Superior PFS with VTD consolidation was maintained across poor prognosis subgroups, including those with advanced ISS stage and/or high-risk cytogenetic profiles. In a multivariate regression analysis VTD consolidation was confirmed to be an independent variable favorably affecting PFS. The superior activity seen with VTD versus TD as induction therapy before ASCT was retained despite the same triplet regimen being used as post-ASCT consolidation. Disclosures: Cavo: Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria; Genzyme: Honoraria. Off Label Use: Bortezomib and Thalidomide as post autotransplantation consolidation therapy in myeloma. Patriarca:Schering-Plough: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Roche: Honoraria. Petrucci:Celgene: Honoraria; Janssen: Honoraria. Di Raimondo:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau. Palumbo:Merck: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria. Offidani:Celgene: Honoraria; Janssen: Honoraria. Baccarani:Bristol-Meyers Squibb: Honoraria, 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.

Abstract 5172 The introduction of JAK-2 V617F mutation in the diagnostic process of patients with erythrocytosis has led to a better definition of Polycythemia Vera (PV) patients, who had the mutation in > 90% of cases: however, in the real-life many patients with otherwise unexplained Hb and Ht elevation and lack of JAK-2 V617F mutation are located in the gray zone of the so-called Idiopathic Erythrocytosis (IE). In order to highlight clinical features of patients with IE, we revised 247 patients with primary erythrocytosis (i.e. excluding cases with respiratory and/or severe cardiovascular diseases) diagnosed in 2 Hematological Institutions in Rome from 6/1985 to 12/2010 with a evaluable JAK-2 mutational status at diagnosis or during the follow-up. Were considered as “PV JAK-2 positive” all patients with presence of mutation V617F at diagnosis or during follow-up. Among the patients who resulted JAK-2 V617F negative, were considered as “PV JAK-2 negative” all patients classifiable as PV according to PVSG criteria (if diagnosed before 1/2002) or WHO 2002 criteria (if diagnosed after 1/2002): the remaining patients were classified as “IE”. With these criteria, 181 patients (73.2%) were PV JAK-2 positive, 26 (10.5%) PV JAK-2 negative and 40 (16.3%) IE: the main clinical features of these 3 groups at diagnosis and during the course of disease are summarized in the table.PV JAK-2 positivePV JAK-2 negativeIENo of patients1812640Male87 (48%)22 (84%)38 (95%)Median age yrs (IR)59.3 (49.7–68.5)47.9 (42.6–64.5)57.8 (44.2–65.9)Median WBC x 109/l (IR)10.0 (8.4–13.0)10.6 (6.3–12.5)7.3 (6.4–8.3)Median PLTS x 109/l (IR)481 (349–651)214 (189–344)205 (165–236)No with palpable spleen63 (35.0%)7 (27.0%)2 (5.0%)Previous thrombosis29 (16.0%)2 (7.6%)1 (2.5%)Thrombosis during disease32 (17.7%)1 (3.8%)4 (10.0%)Fibrotic/Blastic evolution12 (6.6%)00 Comparing the 3 groups for the above features, there was a very significative (p Disclosures: No relevant conflicts of interest to declare.