To improve the tolerability of post-transplant maintenance and outcomes despite poor risk disease genetics, we conducted a phase 1 study of venetoclax/FluBu2 RIC transplantation with tacrolimus/methotrexate GVHD prophylaxis followed by all-oral venetoclax/decitabine-cedazuridine (ven/dec-c) maintenance in poor-risk MDS/AML patients (N=30). 58% had prior venetoclax exposure and 63% were TP53-mutated; 15/19 had TP53 multi-hit state. At a median of +55 days, pre-emptive maintenance therapy with venetoclax (400 mg on days 1-14) and dec-c (decitabine 35 mg/cedazuridine 100 mg on days 1,3,5 or 1,2,3) was initiated for eight 42-day cycles in 26/30 (87%) patients (remaining 3 relapsed early, 1 withdrew). On maintenance, grade 3-4 neutropenia (96%) occurred though infections were rare (N=2). No DLTs occurred. 6-month acute GVHD grade II-IV rate was 13%. 1-year moderate/severe chronic GVHD rate was 31%. At a median follow up of 25.1-months (range,15-33), median OS and PFS were not reached. On maintenance, 2-year OS was 77% (95%CI,55-89), PFS 62% (95%CI,38-79), NRM 0%, and cumulative incidence of relapse 38% (95%CI,18-59). Exploratory studies identified 96% had pre-transplant NGS-MRD+, favorable survival in those with non-TP53 MRD+, and delayed conversion on maintenance in 11/18 (61%) in those with TP53 MRD+. PROs assessed in first 6-months of maintenance were stable except for emotional function, which improved (P=0.008). Trial is registered at clinicaltrials.gov/NCT03613532.

Assessment of deconvolution performance of synthetic chimeric cell populations in in-silico mixing experiments.

Extraction of mitochondrial DNA mutations from bulk RNA-seq data in CLL.

Longitudinal changes of somatic nuclear and mitochondrial DNA mutations in genetically stable CLL.

ABSTRACT:The mechanisms that lead to extramedullary tropism of acute myeloid leukemia (eAML) remain obscure and no specific therapeutic approaches for this entity exist. Because the long-term survival of eAML is poor, a deeper understanding of the immune microenvironment and leukemia phenotypes underlying this entity is warranted. Here, we performed bulk and single-cell transcriptome profiling of 23 eAML biopsies from 10 patients with isolated extramedullary disease in skin and subcutaneous tissue. Unlike normal healthy skin, we found leukemia cutis to be heavily immune infiltrated; in extramedullary relapse after allogeneic stem cell transplantation, >90% of T/natural killer cells were donor derived. eAML-associated T cells expressed a clear signature of T-cell exhaustion, dissimilar to leukemia-associated immune populations in bone marrow relapse (n = 7) but related to acute and chronic skin inflammation. Furthermore, HLA class II was downregulated in 4 of 7 leukemia cutis specimens, consistent with an immune escape phenotype in eAML. Extramedullary and bone marrow-resident leukemia cells differed with regard to the expression of 8 homing receptor molecules (ICAM1 [encoding CD54], PECAM1 [CD31], ITGA4, ITGA6, ITGAL, ITGB4, ITGA5, and ITGAV). Serial samples obtained from 1 leukemia cutis throughout consecutive immune checkpoint blockade with ipilimumab followed by nivolumab showed a consistently high degree of overlap between local and circulating T-cell receptor sequences, suggesting that only a minority of eAML-associated T cells are leukemia specific. Our analysis reveals eAML to associate with complex changes in leukemia and T-cell gene expression profiles that suggest multiple potential avenues for therapeutic targeting.

Relapse remains the main cause of death following allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk myeloid malignancies. Post-transplant maintenance therapy may reduce relapse risk and improve outcomes. We previously showed that post-transplant azacitidine + venetoclax (ven/aza) maintenance after reduced intensity conditioning (RIC) HCT with tacrolimus/methotrexate (Tac/MTX) graft-versus-host disease (GVHD) prophylaxis is feasible, safe, and associated with encouraging outcomes. The ability to deliver prophylactic maintenance after modified RIC conditioning including post-transplant cyclophosphamide/tacrolimus/mycophenolate mofetil (PTCy/Tac/MMF) GVHD prophylaxis has not yet been shown. We sought to determine the extent to which PTCy/Tac/MMF GVHD prophylaxis affected the safety, tolerability, and preliminary efficacy of venetoclax/FluBu2 RIC HCT followed by ven/aza maintenance in patients with high-risk MDS and AML. Venetoclax was administered at 400 mg daily dose on days -8 to -2, overlapping with FluBu2. Eligible patients included those with adverse risk AML (by ELN 2017/2022 or therapy-related AML) or adverse risk MDS (IPSS Intermediate-2/High or MDS with high-risk mutations). We compared clinical outcomes with our previously published venetoclax/FluBu2 RIC HCT Tac/MTX cohort. This phase 1 trial was registered at clinicaltrials.gov/NCT03613532. Twenty-three patients enrolled in the PTCy cohort including 13/23 (57%) whose disease harbored TP53 mutations (12/13 multi-hit). At pretransplant screening (within 28 days of day -8), multi-parameter flow cytometry-measurable residual disease was detected in 11/20 (55%) patients in CR. With this study regimen, neutrophils engrafted at a median of 15 days and platelets engrafted at a median of 22 days from transplant. 100-day nonrelapse mortality (NRM) was 0%, 6-month grade II-IV acute GVHD rate was 4.4% (95% confidence interval [CI] .30 to 18.8) and 1-year chronic GVHD was 9.1% (95% CI: 1.4 to 26.1). 100-day event-free survival (EFS) was 59.1% (95% CI: 41.7 to 83.7). With median 14.9 months follow-up (range: 7.34 to 21.6), 1 year overall survival (OS) was 59.1% (95% CI: 41.6 to 84.0), progression-free survival (PFS) was 47% (95% CI: 30.2 to 73.1), relapse was 48.7% (95% CI: 26.4 to 67.8), and GVHD-free relapse free survival (GRFS) was 37.7% (95% CI: 21.9 to 64.7). We further compared safety and efficacy with another prospective cohort enrolled with identical inclusion criteria who underwent ven/FluBu2 HCT with Tac/MTX GVHD prophylaxis (n = 27). No differences were observed in 100-day cumulative incidence of grade ≥2 infections, though the PTCy cohort had numerically increased bacterial (26% versus 15%) and viral (17% versus 7%) infections. We did not identify differences in OS (P = .33), PFS (P = .55), relapse (P = .76), NRM (P = .28), GRFS (P = .97), EFS (P = .6), acute GVHD (P = .12), or chronic GVHD (P = .41). Furthermore, no significant differences were detected in FACT-BMT quality of life scores when comparing longitudinal changes between pretransplant and post-transplant/premaintenance timepoints by GVHD prophylaxis cohort. We demonstrate acceptable safety and encouraging efficacy of ven/FluBu2 with PTCy in an exceedingly high risk cohort and equivalent outcomes in comparison with Tac/MTX GVHD prophylaxis. Subtle differences in tolerability however highlight the need for additional studies to confirm the optimal backbone for poor-risk MDS/AML cases.

Co-evolution of somatic nuclear and mitochondrial DNA mutations in AML.

Identification of cell types and tracking of mitochondrial DNA mutations in immunosuppression tapering (IST) cohort using ASAP-seq.

In patients with myelodysplastic syndromes (MDS) treated with hypomethylating agents (HMA) and proceeding to allogeneic stem-cell transplantation (alloHSCT), pretransplant risk assessment remains dominated by morphologic remission–based frameworks despite uncertain prognostic value in the transplant setting. We retrospectively analyzed 280 HMA-treated MDS patients undergoing alloHSCT at DFCI and Moffitt between 2013-2021 to evaluate the predictive performance of IWG 2023 response criteria and to develop an alternative framework integrating functional marrow reserve (FMR) with baseline molecular risk. Progression-free survival (PFS) was the primary outcome. Median overall survival (OS) was 52 months, with 5-year PFS and OS of 43% and 48%, respectively. IWG 2023 response categories showed substantial overlap in outcomes, and assessment by composite complete remission status did not discriminate PFS (5-year PFS 46% vs 40%; p=0.16). In contrast, preconditioning hemoglobin and platelet counts were strongly associated with outcomes, whereas marrow blasts and absolute neutrophil count were not. Recursive partitioning identified clinically relevant platelet and hemoglobin thresholds, enabling construction of FMR strata with markedly distinct 5-year PFS (70%, 40%, and 24% for favorable, intermediate and poor risk, respectively; p<0.0001). Baseline IPSS-M independently predicted outcomes (5-year PFS 58%, 45%, and 29%; p=0.003) and further refined prognostication when integrated with FMR. In multivariable analysis, the integrated IPSS-M/FMR model remained independently associated with PFS (HR 3.00–8.31 across risk tiers; p<0.004) and showed superior discrimination compared with IWG 2023 and classical transplant variables (PFS c-index 0.676). These findings support an integrated framework that outperforms remission-based criteria for post-alloHSCT risk prediction in HMA-treated MDS.

Background Effective TCR-directed cell therapies in Multiple Myeloma (MM) require discovery of shared, tumor-restricted antigens with matched TCRs. Cryptic peptides from untranslated regions, aberrant splicing, and retroelements are promising candidates; however, systematic mapping of their cognate TCR:antigen pairs in MM remains limited. Here, we apply high-throughput antigen prioritization with DNA barcoded (dCODE) MHC-I Dextramer technology to validate epitopes and map TCR:antigen pairs, including cryptic epitopes from untranslated regions. Methods We developed a discovery framework integrating whole exome sequencing, RNA-seq, ribosome profiling, and single-cell (sc) immunoprofiling to identify antigens that are transcribed, translated, and predicted to be antigenic. We applied this to bone marrow (BM) from 10 HLA-A*02:01 MM patient samples from the BMT CTN 1401 trial (NCT02728102). Peptides were prioritized using a machine learning model trained on validated antigens. Prioritized antigens were confirmed for plasma cell specificity via sc RNA-seq, and MHC-I presentation potential assessed using public immunopeptidomic datasets. Top antigens were validated in patient BM and matched with cognate TCRs using sc multiomics with dCODE MHC-I Dextramers. Results Of >2,000 cryptic and mutation-derived peptides, 63 cancer-restricted antigens were prioritized, including 6 shared retroelement-derived epitopes from L1, L2, and ERV1 subfamilies. The L2 element antigen was consistently detected across 10 patients, highlighting its potential as a broadly relevant target. Many additional antigens arose from non-canonical ORFs in plasma cell-associated genes, including BCMA and SLAMF7. dCODE MHC-I Dextramer profiling of patient BM samples revealed distinct phenotypic distributions: viral antigen-specific TCRs (influenza, CMV) were enriched in CD8 tissue-resident and terminal effector memory subsets, whereas cryptic tumor antigen-specific TCRs (ERV1/L2, SLAMF7, BCMA) were enriched in early effector-memory and activated states, suggesting a proliferative potential and the ability to generate terminal effectors with strong cytotoxic function. In this pilot, >6,000 viral-/cryptic antigen-TCR pairs were mapped, representing the largest dataset of its kind in MM, with additional patients ongoing. Conclusion This study provides the first single-cell antigen-TCR atlas of cryptic antigens in MM, uncovering shared, tumor-restricted epitopes from retroelements and non-canonical ORFs and their cognate TCRs. By integrating genomics-driven discovery with dCODE MHC-I Dextramer profiling, we demonstrate a scalable, clinically relevant pipeline linking cryptic antigens to functional TCR states. These findings establish cryptic antigens as a class of actionable targets and deliver a blueprint for vaccine- and T cell-based immunotherapies in MM.

Identification of high-confidence mitochondrial DNA mutations for Tapestri analysis.

Mutations targeted using single cell DNA sequencing in AML1012.

ABSTRACT:Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic stem cell transplant (HSCT) portends a dismal prognosis. One approach for reinvigorating a graft-versus-leukemia response is consolidation with donor lymphocyte infusions (DLI) or second HSCT (HSCT2). However, the role of DLI/HSCT2 in patients who achieve complete remission (CR) after salvage therapy is unclear. In this retrospective study, we evaluated the outcomes of 464 patients with post-HSCT AML relapse, focusing on those who achieved CR before consolidation with cellular therapy. In multivariable analysis (MVA), achieving CR after post-HSCT1 relapse was associated with improved overall survival (OS; hazard ratio [HR], 0.42; P< .0001). Of 133 patients (29%) who achieved CR after posttransplant AML relapse and before cellular therapy, 64 received DLI, 28 underwent HSCT2, and 41 received neither. Four-year outcomes from CR for the entire cohort (n = 133) were: OS 29%, relapse-free survival (RFS) 22%, cumulative incidence of relapse 58%, and nonrelapse mortality (NRM) 20%. In MVA, there was no association between receipt of DLI (HR, 0.87; P = .59) or HSCT2 (HR, 1.08; P = .83) and OS. Furthermore, we did not identify a benefit with DLI or HSCT2 with respect to RFS, relapse, or NRM. Patients with donor chimerism <90% at the time of CR had reduced 4-year OS (20% vs 32%; P = .03), as did measurable residual disease-positive patients (17% vs 62%; P = .024). Our results question the benefit of consolidation with DLI or HSCT2 in patients with AML who achieve CR, and we identify high-risk subgroups that should be the focus of future studies with larger cohorts.

Relapse of acute myeloid leukemia (AML) following allogeneic hematopoietic cell transplantation (allo-HCT) remains a life-threatening complication and is influenced by the underlying biology of the AML and possibly by genetic alterations. In this retrospective multicenter study, we evaluated mutational dynamics of AML cells in 57 patients with relapse after allo-HCT. We observed that 68% of patients exhibited genetic instability, characterized by acquisition or loss of mutations, most frequently involving FLT3-ITD, NRAS, and KRAS, while founding lesions such as DNMT3A were usually retained. Clonal evolution patterns varied, with constant profiles (35.0%), linear (29.8%), branching (22.8%), and parallel (12.3%) evolution. However, these evolutionary categories were not associated with differences in progression-free or overall survival. In contrast, relapse timing was highly prognostic: early relapse ( ≤ 6 months) conferred a significant higher mortality risk compared to late relapse, independent of evolution model. Our findings indicate that while relapse after allo-HCT in AML is genetically diverse, timing of recurrence remains the most critical determinant of outcome. Given that certain genetic changes may inform therapeutic options, these findings highlight the relevance of longitudinal molecular monitoring especially during the early post-transplant period.

Deconvolution and identification of AML phenotypes in bone marrow using ASAP-seq.

BMT CTN 1506 ("MORPHO") was a phase 3 study of post-hematopoietic cell transplantation (HCT) maintenance with gilteritinib versus placebo for patients with FLT3-ITD-mutated acute myeloid leukemia (AML) in first remission. Subgroup analysis indicated a significant benefit of post-HCT gilteritinib for participants in North America, but no benefit for those in Europe or Asia. We conducted a post-hoc analysis of the data focusing on days from AML diagnosis to HCT, pre-HCT FLT3 inhibitor use, and FLT3-ITD measurable residual disease (MRD). Participants transplanted < 120 days from AML diagnosis and/or those treated with FLT3 inhibition pre-HCT were more likely to have improved survival from post-HCT gilteritinib. Pre-HCT MRD levels were higher (P = 0.001) in participants transplanted within 120 days from diagnosis and in those treated with a FLT3 inhibitor pre-HCT and transplanted within 120 days (P = 0.008). Pre-HCT MRD was dependent on both FLT3 inhibitor use and time to HCT, as participants treated with successive courses of chemotherapy + FLT3 inhibition had successively lower MRD by the time of HCT. Time from AML diagnosis to HCT and pre-HCT FLT3 inhibitor use both appeared to impact MRD levels immediately prior to HCT, and geographic differences in these two practice patterns likely accounted for the observed regional differences in benefit from post-HCT gilteritinib. Increasing the number of courses of treatment pre-HCT may lower MRD sufficiently to eliminate the need for post-HCT inhibition, but with a presumed risk of some patients experiencing early progression. This trial was registered at www.clinicaltrials.gov as #NCT02997202.

Identification of AML subclones using mitochondrial DNA (mtDNA) mutations.

Blood and bone marrow counts of immunosuppression tapering (IST) cohort from routine clinical measurements.

Abstract Background: Multi-specific immune engagers show therapeutic promise but are limited by systemic toxicities (e.g., cytokine release syndrome, CRS), short half-lives, and poor infiltration into the immunosuppressive tumor microenvironment (TME). Tumor-homing bacteria offer a targeted delivery strategy capable of overcoming restricted infiltration and TME-mediated suppression. Building on our E. coli K-12 surface display platform for localized cytokine delivery (Yang et al., Nat Biotechnol, 2024), we developed Live Immune Modulating Engagers (LIME), engineered E. coli co-displaying scFvs against tumor-associated antigens and effector-cell receptors (T or NK). We hypothesize that dual-display LIME enhances anti-tumor immunity by enabling localized effector-cell activation and precise tumor-immune cell bridging. Methods: E. coli was engineered to display scFvs targeting TAAs (CD19, Mesothelin, DLL3, etc.) and immune receptors (CD3, NKG2D, etc.) using optimized outer-membrane scaffolds, confirmed by flow cytometry. In vitro co-cultures with primary human T or NK cells assessed binding, activation (CD25/CD69), and cytotoxicity (7-AAD/Annexin V). In syngeneic A20 and KPC tumor models, mice received intravenous LIME ± anti-PD-1 (i.p.) or RMC-7977 (oral). In DLL3+ H69 xenografts, NSG mice were infused with human CD3+ T cells followed by LIME, benchmarked against the FDA-approved engager Tarlatamab. Tumor growth was monitored and analyzed by flow cytometry, multiplex ELISA, and CFU quantification. Results: In vitro, LIME activated T or NK cells and enhanced killing across a broad panel of tumor cell lines, including Small cell lung cancer, Glioblastoma, Pancreatic ductal adenocarcinoma, etc. In vivo, CD19 LIME cured ∼34% of A20-bearing mice and achieved ∼80% cures when combined with anti-PD-1, generating durable immune memory. Mesothelin LIME significantly inhibited KPC tumor progression, with extended survival upon RMC-7977 co-treatment. DLL3 LIME provided superior tumor control compared with Tarlatamab in H69 xenografts. Mechanistically, LIME reprogrammed the TME by activating T and NK cells, driving macrophage repolarization toward an M1 state, and enhancing DC activation in draining lymph nodes. Bacteria remained enriched in tumors, and no evidence of CRS was observed in plasma. Conclusions: LIME represents a versatile platform that enables localized, immune engagement, overcomes major barriers in solid tumors, and outperforms existing immune engagers while maintaining a favorable safety profile. Citation Format: Shaobo Yang, Anna Clara Bader, Stephanie Sendker, Ashley Hu, Alice Chen, Daniel Chen, Hetal Nath, Eden Bobilev, Michal Sheffer, Veronica W. Hui, Tereza Kochs, Andreia Maia, Fuguo Liu, Xingyu Deng, Maily Nguyen, Mila Stanojevic, Mubin Tarannum, Alaa Ali, Roman Shapiro, Young Rock Chung, Erin M. Parry, Julissa G. Tello, Stephanie K. Dougan, Marco Campisi, David A. Barbie, John Koreth, Robert J. Soiffer, Catherine J. Wu, Jerome Ritz, Rizwan Romee. Reprogramming of the non-pathogenic E. coli surface architecture to create next-generation immune engagers for potent cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4904.

Differential phenotypes of physiologic (donor-) and malignant hematopoietic (recipient-derived) cells.