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Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML posttreatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody–oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A- and TET2-mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.Significance:This study offers a proof of principle of patient-personalized customized single-cell proteogenomics in AML including whole-genome sequencing–defined structural variants, currently unmeasurable by commercial “off-the-shelf” panels. This approach allows for the definition of genetic and immunophenotype features for an individual patient that would be best suited for measurable residual disease tracking.

Supplementary Data

Fig. S1, S2, S3 and Table S1.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the MYC oncogene combined with p53 loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non–small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.

RNA-Seq results: DE genes in GBM1B cells with +/- 24h EGF treatment (related to Figure 4).

Figure S1-S8 and legend for Table S1-S8

Hyperactivated EGFR signaling is a driver of various human cancers, including glioblastoma (GBM). Effective EGFR-targeted therapies rely on knowledge of key signaling hubs that transfer and amplify EGFR signaling. Here we focus on the transcription factor TAZ, a potential signaling hub in the EGFR signaling network. TAZ expression was positively associated with EGFR expression in clinical GBM specimens. In patient-derived GBM neurospheres, EGF induced TAZ through EGFR–ERK and EGFR–STAT3 signaling, and the constitutively active EGFRvIII mutation caused EGF-independent hyperactivation of TAZ. Genome-wide analysis showed that the EGFR–TAZ axis activates multiple oncogenic signaling mechanisms, including an EGFR–TAZ–RTK positive feedback loop, as well as upregulating HIF1α and other oncogenic genes. TAZ hyperactivation in GBM stem-like cells induced exogenous mitogen-independent growth and promoted GBM invasion, radioresistance, and tumorigenicity. Screening a panel of brain-penetrating EGFR inhibitors identified osimertinib as the most potent inhibitor of the EGFR–TAZ signaling axis. Systemic osimertinib treatment inhibited the EGFR–TAZ axis and in vivo growth of GBM stem-like cell xenografts. Overall these results show that the therapeutic efficacy of osimertinib relies on effective TAZ inhibition, thus identifying TAZ as a potential biomarker of osimertinib sensitivity.Significance:This study establishes a genome-wide map of EGFR–TAZ signaling in glioblastoma and finds osimertinib effectively inhibits this signaling, justifying its future clinical evaluation to treat glioblastoma and other cancers with EGFR/TAZ hyperactivation.

Response criteria for patients treated for acute myeloid leukemia (AML) based on cytomorphology are inadequate. Many patients achieving a complete remission by such criteria will later relapse. Patients with AML in such remissions who test negative using higher sensitivity measures of residual disease burden (measurable residual disease [MRD]) have on average lower relapse rates and better survival than those testing positive. This association has raised the possibility that these technological advances in measurement of tumor burden could be used to optimize the drug development and regulatory approval processes in AML. The heterogeneous genetic etiology, diverse immunophenotypic profiles, related precursor states and polyclonal architecture however combine to make the development of standardized and validated MRD assessments for AML challenging. Current and future methods to measure residual disease in AML, performance characteristics of testing currently in use, and potential uses for optimized AML MRD tests including as a surrogate endpoint are discussed.

BackgroundPrevention of relapse for adults with Acute Myeloid Leukemia (AML) in first remission (CR1) is the most common indication for allogeneic hematopoietic cell transplantation (alloHCT). Measurable residual disease (MRD) detection has been associated with higher relapse rates, but testing is not standardized. Establishment of validated AML MRD criteria would allow harmonization across centers facilitating clinical trials and generalizable practice guidelines.MethodsPatients aged 18 or older who underwent first alloHCT forFLT3, NPM1, IDH1, IDH2and/orKITmutated AML in CR1 were eligible for this multicenter study. Residual mutations detected in remission using ultra-deep error-corrected next-generation DNA-sequencing (NGS-MRD) associated with increased relapse risk were validated in a second independent cohort. The impact of baseline characteristics on NGS-MRD results was evaluated.ResultsPre-conditioning CR1 blood samples from 1075 patients were tested. Detection of residualNPM1and/orFLT3-ITD mutations before alloHCT was associated with worse outcomes after transplantation in both discovery (patients transplanted 2013-2017) and validation (patients transplanted 2018-2019) cohorts. In multivariate Cox regression analysis detection of residualNPM1m (relapse; HR: 4.9, 3.5-7.0, PFLT3-ITD (relapse: HR: 4.2, 3.1-5.7, PConclusionsDetection of residualNPM1and/orFLT3-ITD mutations in the blood of patients undergoing alloHCT for AML in CR1 is associated with increased relapse and worse survival. In those undergoing reduced intensity conditioning, melphalan may reduce the risk associated with testing NGS-MRD positive prior to transplantation.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML posttreatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody–oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A- and TET2-mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts. Significance: This study offers a proof of principle of patient-personalized customized single-cell proteogenomics in AML including whole-genome sequencing–defined structural variants, currently unmeasurable by commercial “off-the-shelf” panels. This approach allows for the definition of genetic and immunophenotype features for an individual patient that would be best suited for measurable residual disease tracking.

ImportancePreventing relapse for adults with acute myeloid leukemia (AML) in first remission is the most common indication for allogeneic hematopoietic cell transplant. The presence of AML measurable residual disease (MRD) has been associated with higher relapse rates, but testing is not standardized.ObjectiveTo determine whether DNA sequencing to identify residual variants in the blood of adults with AML in first remission before allogeneic hematopoietic cell transplant identifies patients at increased risk of relapse and poorer overall survival compared with those without these DNA variants.Design, Setting, and ParticipantsIn this retrospective observational study, DNA sequencing was performed on pretransplant blood from patients aged 18 years or older who had undergone their first allogeneic hematopoietic cell transplant during first remission for AML associated with variants in FLT3, NPM1, IDH1, IDH2, or KIT at 1 of 111 treatment sites from 2013 through 2019. Clinical data were collected, through May 2022, by the Center for International Blood and Marrow Transplant Research.ExposureCentralized DNA sequencing of banked pretransplant remission blood samples.Main Outcomes and MeasuresThe primary outcomes were overall survival and relapse. Day of transplant was considered day 0. Hazard ratios were reported using Cox proportional hazards regression models.ResultsOf 1075 patients tested, 822 had FLT3 internal tandem duplication (FLT3-ITD) and/or NPM1 mutated AML (median age, 57.1 years, 54% female). Among 371 patients in the discovery cohort, the persistence of NPM1 and/or FLT3-ITD variants in the blood of 64 patients (17.3%) in remission before undergoing transplant was associated with worse outcomes after transplant (2013-2017). Similarly, of the 451 patients in the validation cohort who had undergone transplant in 2018-2019, 78 patients (17.3%) with residual NPM1 and/or FLT3-ITD variants had higher rates of relapse at 3 years (68% vs 21%; difference, 47% [95% CI, 26% to 69%]; HR, 4.32 [95% CI, 2.98 to 6.26]; P P Conclusions and RelevanceAmong patients with acute myeloid leukemia in first remission prior to allogeneic hematopoietic cell transplant, the persistence of FLT3 internal tandem duplication or NPM1 variants in the blood at an allele fraction of 0.01% or higher was associated with increased relapse and worse survival compared with those without these variants. Further study is needed to determine whether routine DNA-sequencing testing for residual variants can improve outcomes for patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by abnormal clonal proliferation of myeloid progenitor cells found predominantly within the bone marrow (BM) and blood. Recent studies suggest that genetic and phenotypic alterations in the BM microenvironment support leukemogenesis and allow leukemic cells to survive and evade chemotherapy-induced death. However, despite substantial evidence indicating the role of tumor–host interactions in AML pathogenesis, little is known about the complex microenvironment of the BM. To address this, we performed novel proteomic profiling of the noncellular compartment of the BM microenvironment in patients with AML (n = 10) and age- and sex-matched healthy control subjects (n = 10) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). We show that proteomic assessment of blood or RNA-sequencing of BM are suboptimal alternate screening strategies to determine the true proteomic composition of the extracellular soluble compartment of AML patient BM. Proteomic analysis revealed that 168 proteins significantly differed in abundance, with 91 upregulated and 77 downregulated in leukemic BM. A highly connected signaling network of cytokines and chemokines, including IL-8, was found to be the most prominent proteomic signature associated with AML in the BM microenvironment. We report the first description of significantly elevated levels of the myelosuppressive chemokine CCL23 (myeloid progenitor inhibitory factor-1) in both AML and myelodysplastic syndrome patients and perform functional experiments supportive of a role in the suppression of normal hematopoiesis. This unique paired RNA-sequencing and proteomics data set provides innovative mechanistic insights into AML and healthy aging and should serve as a useful public resource.

Hyperactivated EGFR signaling is a driver of various human cancers, including glioblastoma (GBM). Effective EGFR-targeted therapies rely on knowledge of key signaling hubs that transfer and amplify EGFR signaling. Here we focus on the transcription factor TAZ, a potential signaling hub in the EGFR signaling network. TAZ expression was positively associated with EGFR expression in clinical GBM specimens. In patient-derived GBM neurospheres, EGF induced TAZ through EGFR–ERK and EGFR–STAT3 signaling, and the constitutively active EGFRvIII mutation caused EGF-independent hyperactivation of TAZ. Genome-wide analysis showed that the EGFR–TAZ axis activates multiple oncogenic signaling mechanisms, including an EGFR–TAZ–RTK positive feedback loop, as well as upregulating HIF1α and other oncogenic genes. TAZ hyperactivation in GBM stem-like cells induced exogenous mitogen-independent growth and promoted GBM invasion, radioresistance, and tumorigenicity. Screening a panel of brain-penetrating EGFR inhibitors identified osimertinib as the most potent inhibitor of the EGFR–TAZ signaling axis. Systemic osimertinib treatment inhibited the EGFR–TAZ axis and in vivo growth of GBM stem-like cell xenografts. Overall these results show that the therapeutic efficacy of osimertinib relies on effective TAZ inhibition, thus identifying TAZ as a potential biomarker of osimertinib sensitivity. Significance: This study establishes a genome-wide map of EGFR–TAZ signaling in glioblastoma and finds osimertinib effectively inhibits this signaling, justifying its future clinical evaluation to treat glioblastoma and other cancers with EGFR/TAZ hyperactivation.

Genetic mutations associated with acute myeloid leukemia can also be detected in age-related clonal hematopoiesis, making confident assignment of detected variants to malignancy challenging particularly in the post-treatment setting. This has implications for measurable residual disease monitoring, where the relationship between sequencing and flow cytometry is also imperfect. We show, using whole-genome-sequencing informed patient-personalized single-cell DNA and antibody-oligonucleotide sequencing, that it is possible to resolve immunophenotypic identity of clonal architecture.

PURPOSE Patients with myelodysplastic syndrome (MDS) are at risk of relapse after allogeneic hematopoietic cell transplantation. The utility of ultra-deep genomic testing to predict and the impact of conditioning intensity to prevent MDS relapse are unknown. METHODS Targeted error-corrected DNA sequencing was performed on preconditioning blood samples from patients with MDS (n = 48) from the Blood and Marrow Transplant Clinical Trials Network 0901 phase III randomized clinical trial, which compared outcomes by allogeneic hematopoietic cell transplantation conditioning intensity in adult patients with < 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment. Clinical end points (53-month median follow-up) included transplant-related mortality (TRM), relapse, relapse-free survival (RFS), and overall survival (OS). Of the 48 patients examined, 14 experienced TRM, 23 are relapse-free, and 11 relapsed, of which 7 died. RESULTS Using a previously described set of 10 gene regions, 42% of patients (n = 20) had mutations detectable before random assignment to reduced intensity conditioning (RIC) or myeloablative conditioning (MAC). Testing positive was associated with increased rates of relapse (3-year relapse, 40% v 11%; P = .022) and decreased OS (3-year OS, 55% v 79%, P = .045). In those testing positive, relapse rates were higher (3-year relapse, 75% v 17%; P = .003) and RFS was lower (3-year RFS, 13% v 49%; P = .003) in RIC versus MAC arms. Testing additional genes, including those associated with MDS, did not improve prognostication. CONCLUSION This study provides evidence that targeted DNA sequencing in patients with MDS before transplant can identify those with highest post-transplant relapse rates. In those testing positive, random assignment to MAC lowered but did not eliminate relapse risk.

The powerful “graft versus leukemia” effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic cell transplantation in acute myeloid leukemia (AML) provides rationale for investigation of immune-based therapies in this high-risk blood cancer. There is considerable pre-clinical evidence for potential synergy between PD-1 immune checkpoint blockade and the hypomethylating agents already commonly used for this disease. We report here the results of 17-H-0026 (PD-AML, NCT02996474), an investigator sponsored, single-institution, single-arm open-label ten-subject pilot study to test the feasibility of the first-in-human combination of pembrolizumab and decitabine in adult patients with refractory or relapsed AML (R-AML). In this cohort of previously treated patients, this novel combination of anti-PD-1 and hypomethylating therapy was feasible and associated with a best response of stable disease or better in 6 of 10 patients. Considerable immunological changes were identified using TCRβ sequencing as well as single-cell immunophenotypic and RNA expression analyses on sorted CD3+ T cells in patients who developed immune-related adverse events (irAEs) during treatment. Clonal T cell expansions occurred at irAE onset; single-cell sequencing demonstrated that these expanded clones were predominately CD8+ effector memory T cells with high cell surface PD-1 expression and transcriptional profiles indicative of activation and cytotoxicity. In contrast, no such distinctive immune changes were detectable in those experiencing a measurable anti-leukemic response during treatment. Addition of pembrolizumab to ten-day decitabine therapy was clinically feasible in patients with R-AML, with immunological changes from PD-1 blockade observed in patients experiencing irAEs.One Sentence SummaryAML patients receiving a novel combination of a PD-1 immune checkpoint inhibitor with a hypomethylating agent demonstrated clear evidence of induced immunological responses in those developing autoimmune toxicity during treatment but not in those demonstrating an anti-leukemic response.

Background: Chromosome 7 (chr7) abnormalities are commonly seen in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome and are associated with poor prognosis. Flow cytometry (FCM) is typically used clinically to quantify residual malignancy during treatment but the relationship of cell surface immunophenotype with genetic features is incompletely defined. Single-cell RNA sequencing (scRNA-seq) with oligonucleotide-conjugated antibodies may be able to integrate cytogenetic genotypes found within leukemic clones with specific transcriptomic and immunophenotypic signatures. Methods: Bone marrow (BM) aspirate was collected from 12 AML patients with known abnormalities on chr7 according to cytogenetics and 3 healthy donors (HDs). We previously established a reference for normal immune cells where we assessed the BM of 20 HDs (PMID:30518681). HDs in present study were selected from this same cohort. Multi-parameter FCM was performed as previously described and scRNA-seq with 31 antibodies (10x Genomics, 3'v2) was performed on BM mononuclear cells. Data from 16 samples (including 1 patient replicate) were mapped onto the atlas with dimension reduction in gene expression (GEX) by principal component analysis and Uniform Manifold Approximation and Projection (UMAP). Cell clusters were constructed and surface proteins were utilized to determine cellular annotation. Patient GEX profiles were compared to those of HDs, and two algorithms were used to identify malignant cells with chromosomal abnormalities. Alterations of large chromosomal segments were identified by Hidden Markov Model. Clinical cytogenetics and expression matrices from the model output were combined to annotate individual cells as malignant or normal for all potential regions. Machine learning classifiers were applied to predict malignant cells using protein expression and important proteins were selected by models with measure for significant features. Results: After quality control, 132,658 cells were included, of which 43,441 and 12,572 cells from AML patients and HD respectively had additional cell surface immunophenotyping data. Samples were well-integrated by UMAP (Figure 1A) and cell annotation overlapped with previously reported annotations by GEX of HDs (Figure 1B), with 15,270 malignant cells identified (Figure 1C). Malignant groups were created if more than 20 cells shared the same chromosomal alterations. Among all 12 patients, 3 of them only had monosomy 7 as the sole genetic aberration, while 9 patients had abnormalities on other chromosomes. The correlation between cell types showed distinct features among patients, and cells in different groups had different protein expression profiles. For example, 22% of the cells (n = 432) from the myeloid population of patient 1 (n = 1957) were identified as normal and 77% had a loss of chr7 (Figure 2A). Clustering in lower resolution separated them into two groups: CD11b+CD33+ (n = 1026) and CD133+CD34+ (n = 931). The CD133+CD34+ group had a higher percentage of malignant cells overall (74% vs. 83%) while a subset of the CD11b+CD33+ group (CD16+CD13+) had the highest percentage of cells with losses on both chr2 and chr7 (60%). Data from patient 2 also suggested that cells with different immunophenotypes were estimated to have different chromosomal changes (Figure 2B). While 69% of the malignant cells had a loss on chr7, most of those without changes on chr7 were from CD45+CD11b+ group where 62% of the cells did not have a detectable change on chr7. The top significant proteins for distinguishing malignant from normal cells among all patients were CD117, CD33, CD34, CD44, and CD47. FCM intensity was compared with the scRNA-seq immunophenotyping data, confirming the similarity in distribution. The scRNA-seq data with immunophenotyping can capture and recapitulate the leukemic immunophenotype, further linking it with copy number changes to reveal potential subclonal structures. Conclusion: By comparing the expression profile of AML patients with abnormalities on chr7 to HDs, this study provides evidence that leukemic immunophenotype is correlated with chromosomal structural changes. The experiments on a single-cell level were able to identify clones in higher resolution and revealed potential cell surface protein markers that could be used clinically to identify specific malignant populations in patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding. Hourigan: Sellas: Research Funding.

BACKGROUND: The pathogenesis of acute myeloid leukemia (AML) is often attributed to the presence of somatic allelic variant(s) in hematopoietic stem/progenitor cells. However, malignant clones may have heterogenous cell-surface immunophenotypes including overlap with non-malignant cells. While leukemia-associated immunophenotypes and difference from normal approaches are used for flow cytometric assessment during and after treatment, such analysis may underrepresent true leukemia disease burden. Assessments of AML measurable residual disease (MRD) using flow cytometry and molecular methods have been reported as discrepant. Single-cell RNA sequencing experiments have recently attempted to distinguish malignant cells based on gene expression and/or immunophenotypic profiles alone. We hypothesized that single-cell genotyping of mutated transcript(s) coupled with broad surface proteome and transcriptome profiling could provide an integrated multimodal method for AML characterization. METHODS: We adapted the previously reported "genotyping of transcriptomes" (PMID: 31270458) to identify cells carrying the NPM1 type A mutation commonly seen, and typically stable throughout the disease course, in AML. Healthy human peripheral blood mononuclear cells (PBMC) were mixed with an AML cell line carrying NPM1 type A mutation (OCI-AML3) at 7:3 ratio and labelled with 163 oligo-tagged antibodies. Single cell 3'v3 gene expression- (GEX), antibody derived tag- (ADT) and genotyping of NPM1 (GNPM) -libraries (10X Genomics) were sequenced on the NovaSeq 6000 (Illumina). Results were processed using Seurat 4.0 toolkit. RESULTS: In total, 72% (n=1680) of barcoded cells could be genotyped for NPM1. Of the genotyped cells, 59% (n = 986) were not NPM1 mutated. Visualization using Uniform Manifold Approximation and Projection (UMAP) showed separation of healthy PBMCs and OCI-AML3 cells using protein data, confirmed by annotation using NPM1 genotyping (Figure 1). We found a significant positive correlation between mRNA and corresponding cell surface protein expressions in non-mutated (Pearson's coefficient, r = 0.502, p = 6.87e-11) and NPM1 mutated (r= 0.392, p= 7.5e-7) cells. Compared to non-mutated, NPM1 mutated cells showed nearly 14-fold higher NPM1 transcript levels. In addition, a total 63 proteins were highly expressed on the surface of NPM1 mutated cells (Figure 2). Among these, CD33 and CD36 showed maximum 8-fold increase in expression. Other highly expressed proteins with at least >2.5-fold change were cell adhesion molecules (including CD328, CD155, and CD56), extracellular matrix binding proteins (CD49a/b) and interleukin receptor (CD123). CONCLUSION: Overall, our results demonstrate proof of principle that high-throughput cell surface proteome, transcriptome and genotyping analysis can be simultaneously performed to comprehensively and confidently characterize individual AML cells. Patient-specific multiomics data with broad cell-surface proteomic screening may allow novel target identification for monitoring and/or therapeutic intervention. Ongoing work will now use this methodology to characterize a cohort of NPM1 mutated AML patient samples. Figure 1 Figure 1. Disclosures Hourigan: Sellas: Research Funding.

PURPOSE Patients with acute myeloid leukemia (AML) in remission remain at risk for relapse even after allogeneic hematopoietic cell transplantation (alloHCT). AML measurable residual disease (MRD) status before alloHCT has been shown to be prognostic. Whether modulation of the intensity of the alloHCT conditioning regimen in patients with AML who test positive for MRD can prevent relapse and improve survival is unknown. METHODS Ultra-deep, error-corrected sequencing for 13 commonly mutated genes in AML was performed on preconditioning blood from patients treated in a phase III clinical trial that randomly assigned adult patients with myeloid malignancy in morphologic complete remission to myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC). RESULTS No mutations were detected in 32% of MAC and 37% of RIC recipients; these groups had similar survival (3-year overall survival [OS], 56% v 63%; P = .96). In patients with a detectable mutation (next-generation sequencing [NGS] positive), relapse (3-year cumulative incidence, 19% v 67%; P < .001) and survival (3-year OS, 61% v 43%; P = .02) was significantly different between the MAC and RIC arms, respectively. In multivariable analysis for NGS-positive patients, adjusting for disease risk and donor group, RIC was significantly associated with increased relapse (hazard ratio [HR], 6.38; 95% CI, 3.37 to 12.10; P < .001), decreased relapse-free survival (HR, 2.94; 95% CI, 1.84 to 4.69; P < .001), and decreased OS (HR, 1.97; 95% CI, 1.17 to 3.30; P = .01) compared with MAC. Models of AML MRD also showed benefit for MAC over RIC for those who tested positive. CONCLUSION This study provides evidence that MAC rather than RIC in patients with AML with genomic evidence of MRD before alloHCT can result in improved survival.

Successful clinical remission to therapy for acute myeloid leukemia (AML) is required for long-term survival to be achieved. Despite trends in improved survival due to better supportive care, up to 40% of patients will have refractory disease, which has a poorly understood biology and carries a dismal prognosis. The development of effective treatment strategies has been hindered by a general lack of knowledge about mechanisms of chemotherapy resistance. Here, through transcriptomic analysis of 154 cases of treatment-naive AML, three chemorefractory patient groups with distinct expression profiles are identified. A classifier, four key refractory gene signatures (RG4), trained based on the expression profile of the highest risk refractory patients, validated in an independent cohort (n = 131), was prognostic for overall survival (OS) and refined an established 17-gene stemness score. Refractory subpopulations have differential expression in pathways involved in cell cycle, transcription, translation, metabolism, and/or stem cell properties. Ex vivo drug sensitivity to 122 small-molecule inhibitors revealed effective group-specific targeting of pathways among these three refractory groups. Gene expression profiling by RNA sequencing had a suboptimal ability to correctly predict those individuals resistant to conventional cytotoxic induction therapy, but could risk-stratify for OS and identify subjects most likely to have superior responses to a specific alternative therapy. Such personalized therapy may be studied prospectively in clinical trials.

Several prospective randomized trials comparing conditioning intensity before allogeneic hematopoietic cell transplantation (HCT) have been performed, with conflicting results. Although reduced-intensity conditioning (RIC) leads to lower treatment-related mortality (TRM), this is offset by higher rates of relapse. Long-term follow-up of randomized comparative trials are limited. Here we present long-term follow-up of a randomized comparison of myeloablative conditioning (MAC) compared with RIC before HCT for acute myelogenous leukemia (AML) or myelodysplasia (MDS). Long-term comparative analyses of overall survival, relapse, and relapse-free survival were performed. Patients age 18 to 65 years with

New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from 20 healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated a strong correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference data set and highlights opportunities for further refinement.

New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from twenty healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated good correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference dataset and highlights opportunities for further refinement.

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

Acute myeloid leukemia (AML) is a genetically heterogenous and often fatal cancer of the hematopoietic system. Even after achieving an initial complete remission, more than half of such AML patients experience clinical relapse due to the persistence of “minimal” residual disease (MRD). Ex-vivo studies have hypothesized that the interactions between residual leukemic cells and the local microenvironment in the bone marrow may play a key role in their survival and chemoresistance. Therefore, we performed for the first time a global examination of the proteomic profile of the bone marrow microenvironment in AML patients using a highly multiplexed method based on the ability of slow off-rate modified aptamers (modified small single-stranded oligonucleotides) to bind target proteins with high specificity and affinity at slow dissociation rates. Ten relapsed/refractory adult AML patients and age-matched healthy subject controls were recruited, under an IRB-approved protocol, for research bone marrow aspirate (BMA) and blood serum collection. The supernatant resulting from centrifugation of BMA and serum samples were processed and analyzed on a SOMAscan™ hybridization microarray platform for the detection and quantification of 1,305 target proteins (Somalogic, CO). Data was corrected using Hybridization Control and Median Signal Normalization methods. Significant differences were found between AML and healthy donor bone marrow, such that 133 analytes were differentially expressed in the AML group (Wilcoxon rank sum test FDR p1.5); 85 over-expressed and 48 under-expressed. In addition to proteins already known to be elevated in AML patients (eg: Erythropoietin, Thrombopoietin, Hepcidin and Ferritin) and dysregulation of pathways previously identified as disease relevant (eg: Arginase), we also discovered multiple statistically significant differences in levels of soluble proteins that are not currently known to be associated with AML pathogenesis or treatment. Comparative analysis between blood serum and bone marrow determined that 82 of the 133 candidates have differential expression that was specifically restricted to the bone marrow. The STRING database was queried for pathway analysis of enriched protein sets in the AML group and clustered analytes with molecular functions including cytokine activity (GO:0005125, n=11, p=1.93e-08), cytokine receptor binding (GO:0005126, n=12, p=3.17e-08) and signal transducer activity (GO:0004871, n=19, p=9.23e-05). Using a high-throughput proteomic technology we have identified an AML bone marrow microenvironment-specific profile comprised of both proteins with known implications in leukemic pathogenesis and also several novel candidates from biologically plausible pathways that, once validated, may provide mechanistic insight and opportunity for therapeutic targeting. Citation Format: Bogdan Popescu, Katherine Lindblad, Giovanna Fantoni, Gege Gui, Janet Valdez, Meghali Goswami, Christin DeStefano, Catherine Lai, Angélique Biancotto, Julián Candia, Foo Cheung, Julie Thompson, Laura W. Dillon, Christopher S. Hourigan. Highly multiplexed proteomic assessment of the human acute myeloid leukemia bone marrow microenvironment [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 3756.

Background: The powerful "graft versus leukemia" effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic stem cell transplantation in reducing relapse of high-risk acute myeloid leukemia (AML) provides the rationale for the investigation of immune-based therapy in those with relapsed or refractory AML. There is considerable pre-clinical evidence for potential synergy between PD-1 checkpoint blockade and hypomethylating agents. Aims: To determine the feasibility of a novel combination of pembrolizumab and decitabine in relapsed/refractory adult AML patients. Methods: 17-H-0026, (PD-AML, NCT02996474) was an investigator sponsored, single-institution, single-arm open-label ten subject study approved by the NHLBI IRB and conducted in accordance with the Declaration of Helsinki (FDA IND: 131826). Pembrolizumab 200 milligrams was administered intravenously on day 1 of every three-week cycle, with decitabine 20 milligrams per meter squared administered on days 8-12 and 15-19 (i.e.: total of 10 days) of alternative cycles starting with cycle 1. Up to eight cycles (24 weeks) of therapy were given. Results: Ten high-risk patients (median age 62, range 30-81) were enrolled, seven with refractory disease (including two with therapy-related myeloid neoplasm) and three with early relapse (less than 6 months from completion of last therapy). This novel combination therapy was well tolerated, with a toxicity profile largely consistent with that expected from decitabine. No grade 5 adverse events occurred. Most grade 4 adverse events were hematological. Non-hematological grade 4 events were seen in only two subjects; febrile neutropenia, hypotension and sepsis in one, and sepsis in the other. Two patients suffered from hypothyroidism as an immune-related adverse event (after 2 and 4 cycles respectively) and a third patient developed central diabetes insipidus thought possibly associated with pembrolizumab. In summary, 4 of the 10 patients had stable disease at the end of 8 cycles (24 weeks), 4 progressed prior to cycle 8, 1 patient was taken off study due to grade 4 toxicity (sepsis) in cycle 5 in a morphological leukemic free state (MLFS) and 1 patient achieved an MRD negative complete response at the end of 8 cycles. Specifically, a 74 year-old male patient, enrolled at second relapse, achieved a CR3 lasting 337 days (compared with prior CR2 of 185 days) which was MRD negative at the end of eight cycles, and was still alive 14 months from start of treatment. A 81 year old male patient, with refractory treatment related myeloid neoplasm achieved a MRD-negative MLFS but was removed from study early due to grade 4 toxicity. The 4 patients with stable disease at the end of planned eight cycles included a 71 year-old male patient in early first relapse who decreased blasts from 4.4% at enrollment to 0.1% at the end of 8 cycles. Median overall survival for patients on this study was 7 months (range 2 to ongoing at 14 months) with a median time of follow-up in survivors of 13 months (range 7-14 months). Updated survival data will be reported at the meeting. Additional planned laboratory correlates include assessment of changes in leukemic clonality, T-cell receptor repertoire diversity and bone marrow-resident immune cell profiles during therapy. Conclusion/summary: This first proof of principle study demonstrates the feasibility of the combination of pembrolizumab and decitabine in relapsed/refractory adult AML patients. . Table. Table. Disclosures No relevant conflicts of interest to declare.