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Allogeneic hematopoietic stem cell transplantation (SCT) after a patient with acute myeloid leukemia (AML) achieves a remission from intensive chemotherapy (IC) is given with curative intent. Recently, venetoclax-based regimens have become the standard of care for patients with newly diagnosed AML who are unfit for IC. If these patients achieve remission, they may also be considered for potentially curative consolidation with SCT. There are limited data comparing outcomes after SCT with these different induction strategies. The purpose of the current study was to evaluate depth of remission before SCT and outcomes after SCT in adults with nonrelapsed/refractory AML receiving pre-SCT therapy with either venetoclax/azacitidine (ven/aza) or IC. This was a retrospective, single-institution analysis of 169 patients receiving SCT in first remission after IC or ven/aza. Patient demographics and AML risk features were collected, as well as pre-SCT measurable residual disease (MRD) assessed by flow cytometry and molecular methods. Relapse, transplantation-related mortality, incidence of acute and chronic graft-versus-host-disease (GVHD), and death from any cause were also recorded. Descriptive and survival statistics were applied to these data to compare IC and ven/aza groups. Cox proportional hazard models were used for univariate and multivariate analyses. We demonstrate that despite differences in baseline factors between these groups, outcomes were similar. Relapse-free and overall survival, as well as cumulative incidences of transplantation-related mortality, relapse, and acute and chronic GVHD were comparable between groups. Exploring survival in younger (65 years) versus older (≥65 years) patients by treatment group did not alter these results. Finally, although pre-SCT MRD by flow cytometry was significantly predictive of post-SCT relapse and survival in the IC + SCT patients, it was not significantly predictive of relapse and survival in the ven/aza + SCT patients. Although these findings require prospective validation in a larger cohort of patients, they suggest that ven/aza followed by SCT is a reasonable management strategy for transplantation candidates at any age.

B-lymphoblastic leukemia/lymphoma (B-ALL) is the most common pediatric malignancy and the most commonly diagnosed adult lymphoblastic leukemia. Recent advances have broadened the spectrum of B-ALL, with DUX4 gene fusions implicated in a subclass occurring in adolescents and young adults and harboring a favorable prognosis. DUX4 fusions have been challenging to identify. We aimed to determine whether expression of the DUX4 oncoprotein, as detected by targeted immunohistochemistry, might serve as a surrogate for molecular detection of DUX4 fusions in B-ALL. A cohort of investigational B-ALLs was generated with enrichment for DUX4 fusions by the inclusion of cases with characteristic demographic features and immunophenotypic properties. B-ALLs with mutually exclusive cytogenetics were collected. Immunohistochemical staining by a monoclonal antibody raised against the N-terminus of the DUX4 protein was performed. N-DUX4 immunohistochemistry demonstrated strong, crisp nuclear staining in blasts of seven investigational cases, six of which had nucleic acid material available for molecular evaluation. Five of these cases demonstrated RNA-seq DUX4-fusion positivity. One N-DUX4 immunohistochemistry positive case lacked a definitive DUX4-fusion by RNA-seq, though demonstrated a gene expression profile characteristic of DUX4-rearranged B-ALLs, a CD2+ immunophenotype, and a lack of staining by C-terminus DUX4 antibody immunohistochemistry. At least 83.3% [5/6] positive predictive value. N-DUX4 immunohistochemistry was negative in blasts of three RNA-seq DUX4-fusion-negative cases (3/3; 100% negative predictive value). B-ALLs with mutually exclusive cytogenetic profiles were all N-DUX4 negative (0/10, specificity 100%). N-DUX4 immunohistochemistry is reliable for the distinction of DUX4-rearranged B-ALLs from other B-ALLs. We recommend its use for subclassification of B-ALLs in adolescents and young adults and in B-ALLs that remain "not otherwise specified."

Background Myelodysplastic syndrome (MDS) arises from and is perpetuated by malignant stem cells. Curative therapy requires eradication of this population, but this is not achieved with currently available medical therapies. MDS stem cells possess unique molecular properties that can be therapeutically exploited, but in general these strategies have not been explored clinically. Our recent in vitro studies showed that combination treatment with the protein synthesis inhibitor omacetaxine (oma) and the hypomethylating agent (HMA) azacitidine (aza) efficiently targeted MDS stem cells with minimal impact on normal stem cells. Based on these findings we conducted a Phase 1 clinical trial for MDS patients with concomitant oma + aza (NCT03564873). Methods Patients were eligible if they had newly diagnosed MDS with ≥5% blasts. Aza was given at 75 mg/m 2 IV days 1-7 of a 28-day cycle; oma was given subcutaneously twice daily on days 1-7 of a 28-day cycle with 4 dose cohorts (0.75, 1.0 and 1.25 mg/m 2, and a de-escalation cohort of 0.5 mg/m 2). Dose escalation was based on a 3+3 design. Bone marrow biopsies occurred at baseline and day 8 and 28 of cycle 1, and at the conclusion of odd-numbered cycles. The primary endpoint was the maximum tolerated dose (MTD) of oma with aza. Responses were assessed according to 2006 IWG criteria with hematologic improvement (HI). Using pre- and post-treatment patient samples, advanced single cell techniques including mass cytometry and antibody-based single-cell RNA sequencing (CITE-Seq) were used to elucidate mechanisms of sensitivity and resistance. High sensitivity DNA sequencing techniques were used to understand clonal evolution and detect measurable residual disease (MRD). Results Nine patients were required to complete phase 1. See Table 1 for baseline characteristics. Two patients in cohort 1 experienced dose limiting toxicity (DLT) (grade 3 hypoxia and grade 3 respiratory failure). De-escalation to cohort 0 resulted in one DLT event (grade 4 GI bleed) among the six patients treated in this cohort. The MTD was established as 0.5 mg/m 2 oma days 1-7. Adverse events are detailed in Table 2. Median number of cycles was 2 (1-3). 8/9 patients achieved a marrow CR; 5 had HI. 6 patients proceeded to transplantation and none have relapsed with median time from transplant of 772 (183-1158) days. Median response duration is 587 days (29-938). 5 remain alive. MRD negativity, as measured with droplet digital PCR (sensitivity up to 0.1%), was achieved in 2 out of 4 patients measured. Both MRD negative patients had complete clearance of mutated splice factor genes; we previously showed no ability to clear these mutations (0/13 patients) using venetoclax+aza in AML (Nature Medicine (2018) 24:1859-1866). Baseline analysis of patient specimens by single cell transcriptomics demonstrates a distinct population of MDS stem cells (Figure 1A). This subset has overlapping transcriptional properties with known AML stem cell profiles as highlighted by increased geneset expression. Within the stem cell compartment, we observed up-regulation of protein synthesis pathways (Figure 1B) (e.g. KEGG ribosome pathway in MDS stem cells), indicating that oma/aza treatment may specifically target the malignant stem cell population. Subsequent studies are investigating the use of CITEseq as a means to evaluate drug response, which will provide very high resolution analyses of transcriptional signatures in MDS patients. By specifically monitoring eradication of MDS stem cells during the course of treatment, it should be possible to predict therapeutic efficacy and response duration while elucidating genes that correspond to oma/aza sensitivity and resistance. Conclusions Phase 2 of this study is ongoing; 23 newly-diagnosed high risk MDS patients will be enrolled with a primary endpoint to determine the overall response rate. In addition a ten-patient HMA failure expansion cohort will be enrolled to determine preliminary toxicity and efficacy assessments. Correlative studies will seek to determine the impact of this regimen on the MDS stem cell population and the mechanism of this impact. Figure 1 Figure 1. Disclosures McMahon: Takeda: Membership on an entity's Board of Directors or advisory committees. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding. Pollyea: Foghorn: Honoraria; Gilead: Consultancy, Honoraria; Celgene: Honoraria; Bristol Myers Squibb: Honoraria; Genentech: Consultancy, Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Honoraria; Aprea: Honoraria; Karyopharm: Consultancy, Honoraria; Kiadis: Honoraria; Novartis: Consultancy, Honoraria; Syndax: Honoraria; Syros: Consultancy, Honoraria; Takeda: Honoraria; Teva: Research Funding. OffLabel Disclosure: Omacetaxine for MDS

Chimeric antigen receptor (CAR) T cell therapy is a rapidly evolving immunotherapeutic treatment modality for adult and pediatric patients with a variety of cancers, which has been most extensively investigated in B-cell malignancies. Given that CAR T cell immunotherapy involves changing the genetic composition of a patient's T cells, this living drug presents unique safety and quality control challenges. Vector copy number (VCN), a measurement of transgene copies within a CAR T cell product, is a product-specific characteristic that must be quantified prior to patient administration as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T cell products has been performed via qPCR. qPCR is reliable along a broad range of concentrations but has inherent limitations in its lower limit of detection and limit of quantification. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared to qPCR include simplicity, reproducibility, lower limit of detection, and definitive quantification. In this present study, we developed an assay for analysis of the novel bicistronic UCD19x22 CAR T cell construct, which was developed in the laboratory of Dr. Terry Fry at the University of Colorado and will be moving in to clinical trials later this year. Custom primer-probe assays were designed using Primer Express v3.0.1 and the ThermoFisher Custom TaqMan Assay Design Tool. As an internal control, forward and reverse primers as well as a VIC-labeled probe specific to human albumin (NCBI gene 213, HGNC:399) were designed. Primers and a FAM-labeled probe assay, specific for the bicistronic CD19x22 CAR T cell product, were designed at the junction site between the two distinct CARs. This study compares two different digital PCR modalities: (1) droplet digital PCR (ddPCR) via the BioRad QX200 system which utilizes water-in-oil droplet partitions and (2) the QIAcuity digital PCR system utilizing a nanoplate-based partitioning platform. While dPCR is a newer methodology compared to ddPCR, the two apply parallel procedures, data generation, and analyses. The primer/probe assay was validated with qPCR, dPCR and ddPCR using patient samples from preclinical CAR T cell manufacturing production runs, as well as Jurkat cell subclones which stably express this bicistronic CAR T product. We successfully developed an assay to specifically detect and quantify our bicistronic CD19xCD22 CAR transgene. ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product without any signal detected in samples containing untransduced T cells or T cells transduced with CD19 only CARs. Additionally, our assay gives accurate, precise, and reproducible CAR T cell VCN measurements across qPCR, dPCR, and ddPCR modalities. We demonstrate that digital PCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, and that specific assays can be developed for detection of unique constructs. Future studies will evaluate the utility of this assay with digital PCR modalities in measuring CAR T cell persistence in clinical trial patient samples after receiving this novel CAR T cell product. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.