Demo Faculty

BackgroundDistinguishing donor- vs. recipient-derived myelodysplastic neoplasm (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is challenging and has direct therapeutical implications.MethodsHere, we took a translational approach that we used in addition to conventional diagnostic techniques to resolve the origin of MDS in a 38-year-old patient with acquired aplastic anemia and evolving MDS after first allo-HSCT. Specifically, we used single-cell transcriptional profiling to differentiate between donor- and recipient-derived bone marrow cells and established a strategy that additionally allows identification of cells carrying the MDS-associated U2AF1S34Y variant.ResultsThe patient exhibited mixed donor chimerism combined with severely reduced erythropoiesis and dysplastic morphology within the granulocytic and megakaryocytic lineage along with the MDS-associated U2AF1S34Y …

Relapsed and/or refractory acute myeloid leukemia (AML) post-allogeneic hematopoietic cell transplantation (HCT) is usually fatal. We previously reported that post-HCT immunotherapy with Epstein-Barr virus (EBV)-specific donor CD8+ T cells engineered to express a Wilms Tumor Antigen 1-specific T-cell receptor (T TCR-C4) appeared to prevent relapse in high-risk patients. In this phase I/II clinical trial (NCT01640301), we evaluated safety (primary endpoint), persistence and efficacy (secondary endpoints) of EBV-or Cytomegalovirus (CMV)-specific T TCR-C4 in fifteen patients with active AML post-HCT. Infusions were well tolerated, with no dose-limiting toxicities or serious adverse events related to the product. However, T TCR-C4 cells did not clearly improve outcomes despite EBV-specific T TCR-C4 cells showing enhanced potential for prolonged persistence compared to CMV-specific T TCR-C4. Investigating …

Oral squamous cell carcinoma (OSCC) is highly heterogeneous and metastatic, and the mechanisms driving OSCC development, progression, and metastasis remain elusive. Here, we performed single-cell RNA sequencing on 231,442 cells obtained from the tumor core (TC), tumor periphery (TP), adjacent surrounding tissue (ST), and metastatic lymph node (mLN) samples of 10 patients with human papillomavirus (HPV)-negative OSCC. TP and TC showed no major immune cell phenotype differences. Interestingly, partial EMT (p-EMT) cells showed significant activation of glycolysis and hypoxia signatures, serving as potential biomarkers for clinical outcomes. Moreover, p-EMT scores of epithelial cells positively correlated with M2 scores of tumor-associated macrophages, while the proportion of p-EMT at TP was negatively associated with that of GZMB+ exhausted CD8+ T cells with cytotoxic potential and …

Acute myeloid leukaemia (AML) urgently requires novel therapeutic strategies. However, immunotherapy has so far shown limited results, and the cellular interactions driving immune evasion remain poorly characterized. To address this gap, we applied Xenium imaging-based spatial transcriptomics to 62 formalin-fixed paraffin-embedded bone marrow (BM) biopsies from AML patients (26 children, 18 adults) and non-leukemic controls (9 children, 9 adults), yielding 474,580 high-quality single-cell spatial transcriptomes. This enabled accurate characterization of the BM composition in situ, identifying a substantially higher frequency of stromal cells (median 5%, range 2-19%) and macrophages (3%, 0.3-21%) compared to typical BM aspirate-based analyses. Importantly, fusion probes allowed identification of cells carrying the RUNX1:: RUNX1T1 fusion, and spatial analysis revealed a distinct organization of cellular …

This article presents a novel computational tool, “BoneMarrowMap,” that enables the mapping of leukemic single-cell RNA sequencing datasets to hematopoietic differentiation states. By utilizing BoneMarrowMap for a large-scale single-cell RNA sequencing reanalysis, the authors discover 12 recurrent acute myeloid leukemia differentiation patterns linked to prognosis and treatment and dissect leukemic clonal architectures within individual patients. See related article by Zeng and colleagues, p. XX

Background Distinguishing donor- vs. recipient-derived myelodysplastic neoplasm (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is challenging and has direct therapeutical implications. Methods Here, we took a translational approach that we used in addition to conventional diagnostic techniques to resolve the origin of MDS in a 38-year-old patient with acquired aplastic anemia and evolving MDS after first allo-HSCT. Specifically, we used single-cell transcriptional profiling to differentiate between donor- and recipient-derived bone marrow cells and established a strategy that additionally allows identification of cells carrying the MDS-associated U2AF1S34Y variant. Results The patient exhibited mixed donor chimerism combined with severely reduced erythropoiesis and dysplastic morphology within the granulocytic and megakaryocytic lineage along with the MDS-associated U2AF1S34Y …

Background Cytokine induced memory-like natural killer (CIML NK) cells combined with an IL-15 super-agonist (N-803) are a novel modality to treat relapsed/refractory head and neck cancer. Methods We report data from a phase I trial of haploidentical CIML NK cells combined with N-803 with or without ipilimumab (IPI) in relapsed/refractory head and neck cancer patients after a median of 6 prior lines of therapy. The trial adhered to a 3 + 3 dose de-escalation design, with primary endpoint being safety. High-resolution immunophenotypic and transcriptional profiling characterized the NK cells and their interacting partners in vivo. Results The primary safety endpoint was established, with dose-limiting toxicity in 1/10 patients. A transient disease control rate correlated with donor NK cell expansion, the latter occurring irrespective of IPI. The combination of CIML NK cells with N-803 and IPI was associated with …

Understanding how intratumoral immune populations coordinate antitumor responses after therapy can guide treatment prioritization. We systematically analyzed an established immunotherapy, donor lymphocyte infusion (DLI), by assessing 348,905 single-cell transcriptomes from 74 longitudinal bone marrow samples of 25 patients with relapsed leukemia; a subset was evaluated by both protein- and transcriptome-based spatial analysis. In acute myeloid leukemia (AML) DLI responders, we identified clonally expanded ZNF683+ CD8+ cytotoxic T lymphocytes with in vitro specificity for patient-matched AML. These cells originated primarily from the DLI product and appeared to coordinate antitumor immune responses through interaction with diverse immune cell types within the marrow microenvironment. Nonresponders lacked this cross-talk and had cytotoxic T lymphocytes with elevated TIGIT expression. Our …

Cytokine induced memory-like natural killer (CIML NK) cells combined with an IL-15 super-agonist (N-803) are a novel modality to treat relapsed/refractory head and neck cancer. We report data from a phase I trial of haploidentical CIML NK cells combined with N-803 with or without ipilimumab (IPI) in relapsed/refractory head and neck cancer patients after a median of 6 prior lines of therapy. The primary endpoint was safety, which was established, with dose-limiting toxicity in 1/10 patients. A promising though transient disease control rate of 70% correlated with donor NK cell expansion, the latter occurring irrespective of IPI. High-resolution immunophenotypic and transcriptional profiling characterized the NK cells and their interacting partners in vivo. IPI was associated with contraction of Treg:Tcon, rapid recovery of recipient CD8+ T cells, and accelerated rejection of donor NK cells. These results inform evaluation of CIML NK therapy for advanced malignancies, with considerations for combination with IPI.

High-risk blood malignancies such as acute myeloid leukemia or advanced chronic lymphocytic leukemia remain difficult-to-treat clinical challenges. Immunotherapies including allogeneic stem cell transplantation or targeted immunomodulation using immune checkpoint blockade can provide long-term disease control, but their success rate is still not satisfactory. Single cell genomics is an approach that can effectively monitor immune and leukemia cell dynamics by providing detailed insight into changes in cellular compositions, cell states and interactions. This work provides examples how integrated immune and leukemia monitoring at single cell resolution reveals the determinants of response or non- response to immunotherapy and tracks the clonal evolution of malignant cell populations as therapeutic resistance develops. It also furthers the toolkits available to us by extending standard single cell RNA sequencing with targeted genotyping and explores the ability to utilize mitochondrial DNA mutations as an alternative natural barcoding system for the study of leukemic subclones. Together, this sets the stage for more stream-lined single cell studies that foreshadow the adoption of these technologies into clinical hematologic practice.

While most lymphomas are mature B-cell neoplasms deriving from germinal center B-cells, a stem cell origin has been debated for indolent NHL (iNHL). Lymphoma-initiating stem cell pools may serve as reservoirs for further clonal evolution, contributing to frequent relapses in iNHL such as follicular lymphoma (FL). Thus, identifying and characterizing lymphoma-initiating hematopoietic progenitors is crucial. Here, we map the molecular ontogeny of NHLs and profile genetic precursor lesions potentially contributing to lymphomagenesis.First, we obtained genetic profiles of tumor/normal pairs of 25 aggressive NHLs (aNHL: 18 DLBCL, 4 MCL, 2 PMBCL, 1 BL) and 13 iNHLs (12 FL, 1 MZL) by whole exome sequencing (274x). iNHLs exhibited lower mutation numbers than aNHLs (41 vs 63), fewer chromosomal aberrations (3 vs 7) and a higher prevalence of mutations in chromatin modifying genes, particularly KMT2D …

IntroductionAlthough immune checkpoint inhibitors (ICI) in combination with hypomethylating agents (HMA) have had limited activity in acute myeloid leukemia (AML), a subset of patients has achieved durable responses. This emphasizes the need to define the immunomodulatory effects and immune phenotypes that associate with response to different ICIs. Further, understanding individual responses can point towards new immunotherapeutic strategies in AML.MethodsWe combined single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) data from AML patients (N=37) treated on clinical trials with the combination of HMA with either anti-CTLA4 (responders CR/CRi/PR: 10/18; Penter et al., Blood 2023), anti-PD1 (CR/CRi/PR: 5/12; Abbas Hu et al., Nat Comm 2021, Goswami et al., JITC 2022) or anti-TIM3 (CR/CRi/PR: 3/7; Huuhtanen et al., ASH 2021). Overall, we pooled together 122 longitudinal …

Graft-versus-leukemia (GvL) responses enable successful allogeneic hematopoietic cell transplantation (HCT), a cornerstone treatment for high-risk blood cancers such as acute myeloid leukemia (AML) or advanced chronic lymphocytic leukemia (CLL). Yet, monitoring of GvL effects remains challenging, including early detection of emerging immune escape or tracking of clonal evolution during phases of incomplete leukemia control. We hypothesized that mitochondrial DNA (mtDNA) mutation analysis would be an effective tool for sensitive monitoring of clonal evolution and measurable residual disease (MRD) that could yield new insights into the dynamics and cellular phenotypes of GvL response or resistance.To establish mtDNA mutations as natural genetic barcodes capable of resolving leukemic evolution, we analyzed 126 serially collected bulk RNA and whole-exome sequencing (WES) datasets of CD19 …

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations coevolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Furthermore, detection of mtDNA mutations via single-cell assay for transposase-accessible chromatin with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells but also their phenotype at frequencies of 0.1% to 1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh …

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations co-evolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Further, detection of mtDNA mutations via single-cell ATAC with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells, but also their phenotype, at frequencies of 0.1-1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh insights into residual donor-derived …

Patients with CD37-positive (CD37pos) HL and B-and T-cell non-Hodgkin lymphoma were treated with autologous anti-CD37 CAR T cells coexpressing a tEGFR. Median vein-to-vein time from apheresis to infusion of CAR T cells was 10 days. The CAR T cells expanded after infusion and showed clinical efficacy in heavily pretreated patients. A prolonged CRS and prolonged cytopenia were also observed. B-NHL, B-cell non-Hodgkin lymphoma; CAR, chimeric antigen receptor; CRS, cytokine release syndrome; HL, Hodgkin lymphoma; tEGFR, truncated epithelial growth factor receptor; T-NHL, T-cell non-Hodgkin lymphoma.

Patients with CD37-positive (CD37pos) HL and B-and T-cell non-Hodgkin lymphoma were treated with autologous anti-CD37 CAR T cells coexpressing a tEGFR. Median vein-to-vein time from apheresis to infusion of CAR T cells was 10 days. The CAR T cells expanded after infusion and showed clinical efficacy in heavily pretreated patients. A prolonged CRS and prolonged cytopenia were also observed. B-NHL, B-cell non-Hodgkin lymphoma; CAR, chimeric antigen receptor; CRS, cytokine release syndrome; HL, Hodgkin lymphoma; tEGFR, truncated epithelial growth factor receptor; T-NHL, T-cell non-Hodgkin lymphoma.

Because of the low mutational burden and consequently, fewer potential neoantigens, children with acute myeloid leukemia (AML) are thought to have a T cell-depleted or ‘cold’ tumor microenvironment and may have a low likelihood of response to T cell-directed immunotherapies. Understanding the composition, phenotype, and spatial organization of T cells and other microenvironmental populations in the pediatric AML bone marrow (BM) is essential for informing future immunotherapeutic trials about targetable immune-evasion mechanisms specific to pediatric AML. Here, we conducted a multidimensional analysis of the tumor immune microenvironment in pediatric AML and non-leukemic controls. We demonstrated that nearly one-third of pediatric AML cases has an immune-infiltrated BM, which is characterized by a decreased ratio of M2- to M1-like macrophages. Furthermore, we detected the presence of …

T cell alloreactivity against minor histocompatibility antigens (mHAgs)—polymorphic peptides resulting from donor–recipient (D–R) disparity at sites of genetic polymorphisms—is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D–R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D–R pairs …

T cell alloreactivity against minor histocompatibility antigens (mHAgs)—polymorphic peptides resulting from donor–recipient (D–R) disparity at sites of genetic polymorphisms—is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D–R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D–R pairs …

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti–TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti–TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and …