Case Report: Immune dysregulation associated with long-lasting regression of a (pre)leukemic clone.

Regression of leukemia in the absence of disease-modifying therapy remains poorly understood, although immunological mechanisms are thought to play a role. Here, we present a unique case of a 17-year-old boy with immune dysregulation and long-lasting regression of a (pre)leukemic clone in the absence of disease-modifying therapy. Using molecular and immunological analyses, we identified bone marrow features associated with disease control and loss thereof. In addition, our case reveals that detection of certain fusion genes with hardly any blasts in the bone marrow may be indicative of an accompanying oncogenic fusion gene, with implications for disease surveillance- and management in future patients.

A multidimensional analysis reveals distinct immune phenotypes and tertiary lymphoid structure-like aggregates in the bone marrow of pediatric acute myeloid leukemia.

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a 'cold' tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with response to ipilimumab-based therapy. Altogether, our study provides immune correlates of response to T cell-directed immunotherapies and indicates starting points for further investigations into immunomodulatory mechanisms in AML.

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox.

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

Panhematopoietic RNA barcoding enables kinetic measurements of nucleate and anucleate lineages and the activation of myeloid clones following acute platelet depletion.

BACKGROUND: Platelets and erythrocytes constitute over 95% of all hematopoietic stem cell output. However, the clonal dynamics of HSC contribution to these lineages remains largely unexplored. RESULTS: We use lentiviral genetic labeling of mouse hematopoietic stem cells to quantify output from all lineages, nucleate, and anucleate, simultaneously linking these with stem and progenitor cell transcriptomic phenotypes using single-cell RNA-sequencing. We observe dynamic shifts of clonal behaviors through time in same-animal peripheral blood and demonstrate that acute platelet depletion shifts the output of multipotent hematopoietic stem cells to the exclusive production of platelets. Additionally, we observe the emergence of new myeloid-biased clones, which support short- and long-term production of blood cells. CONCLUSIONS: Our approach enables kinetic studies of multi-lineage output in the peripheral blood and transcriptional heterogeneity of individual hematopoietic stem cells. Our results give a unique insight into hematopoietic stem cell reactivation upon platelet depletion and of clonal dynamics in both steady state and under stress.

Definitions, incidence and outcome of poor graft function after hematopoietic cell transplantation: A systematic review and meta-analysis.

Poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation (HCT) is a serious complication with high morbidity and mortality. The reported incidence of PGF, its risk factors and outcome vary substantially between studies. This variability may be explained by heterogeneity in patient cohorts and HCT strategies, differences in the underlying causes of cytopenia, as well as by differences in PGF definition. In this systematic review and meta-analysis, we provide an overview of the various PGF definitions used and determined the impact of this variability on the reported incidence and outcome. We searched MEDLINE, EMBASE and Web of Science up to July 2022, for any study on PGF in HCT recipients. We performed random-effect meta-analyses for incidence and outcome and subgroup analyses based on different PGF criteria. Among 69 included studies (14.265 HCT recipients), we found 63 different PGF definitions, using various combinations of 11 common criteria. The median incidence of PGF was 7% (IQR: 5-11%, 22 cohorts). The pooled survival of PGF patients was 53% (95% CI: 45-61%, 23 cohorts). The most commonly reported risk factors associated with PGF were history of cytomegalovirus infection and prior graft-versus-host disease. Incidence was lower in studies with strict cytopenic cutoffs, while survival was lower for primary compared to secondary PGF. This work indicates that a standardized, quantitative definition of PGF is needed to facilitate clinical guideline development and to advance scientific progress.

The cellular composition and function of the bone marrow niche after allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for patients with a variety of malignant and non-malignant diseases. Despite its life-saving potential, HCT is associated with significant morbidity and mortality. Reciprocal interactions between hematopoietic stem cells (HSCs) and their surrounding bone marrow (BM) niche regulate HSC function during homeostatic hematopoiesis as well as regeneration. However, current pre-HCT conditioning regimens, which consist of high-dose chemotherapy and/or irradiation, cause substantial short- and long-term toxicity to the BM niche. This damage may negatively affect HSC function, impair hematopoietic regeneration after HCT and predispose to HCT-related morbidity and mortality. In this review, we summarize current knowledge on the cellular composition of the human BM niche after HCT. We describe how pre-HCT conditioning affects the cell types in the niche, including endothelial cells, mesenchymal stromal cells, osteoblasts, adipocytes, and neurons. Finally, we discuss therapeutic strategies to prevent or repair conditioning-induced niche damage, which may promote hematopoietic recovery and improve HCT outcome.

Measures of Clonal Hematopoiesis: Are We Missing Something?

Clonal Hematopoiesis (CH) is a common, age-related phenomenon of growing scientific interest, due to its association with hematologic malignancy, cardiovascular disease and decreased overall survival. CH is commonly attributed to the preferential outgrowth of a mutant hematopoietic stem cell (HSC) with enhanced fitness, resulting in clonal imbalance. In-depth understanding of the relation between HSC clonal dynamics, CH and hematologic malignancy requires integration of fundamental lineage tracing studies with clinical data. However, this is hampered by lack of a uniform definition of CH and by inconsistency in the analytical methods used for its quantification. Here, we propose a conceptual and analytical framework for the definition and measurement of CH. First, we transformed the conceptual definition of CH into the CH index, which provides a quantitative measure of clone numbers and sizes. Next, we generated a set of synthetic data, based on the beta-distribution, to simulate clonal populations with different degrees of imbalance. Using these clonal distributions and the CH index as a reference, we tested several established indices of clonal diversity and (in-)equality for their ability to detect and quantify CH. We found that the CH index was distinct from any of the other tested indices. Nonetheless, the diversity indices (Shannon, Simpson) more closely resembled the CH index than the inequality indices (Gini, Pielou). Notably, whereas the inequality indices mainly responded to changes in clone sizes, the CH index and the tested diversity indices also responded to changes in the number of clones in a sample. Accordingly, these simulations indicate that CH can result not only by skewing clonal abundancies, but also by variation in their overall numbers. Altogether, our model-based approach illustrates how a formalized definition and quantification of CH can provide insights into its pathogenesis. In the future, use of the CH index or Shannon index to quantify clonal diversity in fundamental as well as clinical clone-tracing studies will promote cross-disciplinary discussion and progress in the field.

Mutation signatures of pediatric acute myeloid leukemia and normal blood progenitors associated with differential patient outcomes.

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.

Antiviral treatment causes a unique mutational signature in cancers of transplantation recipients.

Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.

KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

Hematopoietic Dysfunction during Graft-Versus-Host Disease: A Self-Destructive Process?

Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic (stem) cell transplantation (HCT). Clinically, GvHD is associated with severe and long-lasting hematopoietic dysfunction, which may contribute to the high mortality of GvHD after HCT. During GvHD, excessive immune activation damages both hematopoietic stem and progenitor cells and their surrounding bone marrow niche, leading to a reduction in cell number and functionality of both compartments. Hematopoietic dysfunction can be further aggravated by the occurrence-and treatment-of HCT-associated complications. These include immune suppressive therapy, coinciding infections and their treatment, and changes in the microbiome. In this review, we provide a structured overview of GvHD-mediated hematopoietic dysfunction, including the targets in the bone marrow, the mechanisms of action and the effect of GvHD-related complications and their treatment. This information may aid in the identification of treatment options to improve hematopoietic function in patients, during and after GvHD.

CD4+ T-cell reconstitution predicts survival outcomes after acute graft-versus-host-disease: a dual-center validation.

Acute graft-versus-host-Disease (aGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). We previously showed that early CD4+ T-cell immune reconstitution (IR; CD4+ IR) predicts survival after HCT. Here, we studied the relation between CD4+ IR and survival in patients developing aGVHD. Pediatric patients undergoing first allogeneic HCT at University Medical Center Utrecht (UMC)/Princess Máxima Center (PMC) or Memorial Sloan Kettering Cancer Center (MSK) were included. Primary outcomes were nonrelapse mortality (NRM) and overall survival (OS), stratified for aGVHD and CD4+ IR, defined as ≥50 CD4+ T cells per µL within 100 days after HCT or before aGVHD onset. Multivariate and time-to-event Cox proportional hazards models were applied, and 591 patients (UMC/PMC, n = 276; MSK, n = 315) were included. NRM in patients with grade 3 to 4 aGVHD with or without CD4+ IR within 100 days after HCT was 30% vs 80% (P = .02) at UMC/PMC and 5% vs 67% (P = .02) at MSK. This was associated with lower OS without CD4+ IR (UMC/PMC, 61% vs 20%; P = .04; MSK, 75% vs 33%; P = .12). Inadequate CD4+ IR before aGVHD onset was associated with significantly higher NRM (74% vs 12%; P < .001) and inferior OS (24% vs 78%; P < .001). In this retrospective analysis, we demonstrate that early CD4+ IR, a simple and robust marker predictive of outcomes after HCT, is associated with survival after moderate to severe aGVHD. This association must be confirmed prospectively but suggests strategies to improve T-cell recovery after HCT may influence survival in patients developing aGVHD.

Clonal Analysis of Patient-Derived Samples Using Cellular Barcodes.

Cellular barcoding is a relatively simple method that allows quantitative assessment of the clonal dynamics of normal, nonmalignant hematopoietic stem cells and of leukemia. Cellular barcodes are (semi-)random synthetic DNA sequences of a fixed length, which are used to uniquely mark and track cells over time. A successful barcoding experiment consists of several essential steps, including library production, transfection, transduction, barcode retrieval, and barcode data analysis. Key challenges are to obtain sufficient number of barcoded cells to conduct experiments and reliable barcode data analysis. This is especially relevant for experiments using primary leukemia cells (which are of limited availability and difficult to transduce), when studying low levels of chimerism, or when the barcoded cell population is sorted in different smaller subpopulations (e.g., lineage contribution of normal hematopoietic stem cells in murine xenografts). In these settings, retrieving accurate barcode data from low input material using standard PCR amplification techniques might be challenging and more sophisticated approaches are required. In this chapter we describe the procedures to transfect and transduce patient-derived leukemia cells, to retrieve barcoded data from both high and low input material, and to filter barcode data from sequencing noise prior to quantitative clonal analysis.

Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes.

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-resistant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rγ-/- (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patient-derived xenografts had significantly fewer clones compared with placebo-treated mice (100 ± 10 vs. 160 ± 15 clones, p = 0.0005), while clonal complexity in vincristine- and dexamethasone-treated xenografts was unaffected (115 ± 33 and 150 ± 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapy-mediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse.

C/EBPα and GATA-2 Mutations Induce Bilineage Acute Erythroid Leukemia through Transformation of a Neomorphic Neutrophil-Erythroid Progenitor.

Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.

Correction: Quantitative distribution of patient-derived leukemia clones in murine xenografts revealed by cellular barcodes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Donor-to-Donor Heterogeneity in the Clonal Dynamics of Transplanted Human Cord Blood Stem Cells in Murine Xenografts.

Umbilical cord blood (UCB) provides an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Administration of sufficient donor HSCs is critical to restore recipient hematopoiesis and to maintain long-term polyclonal blood formation. However, due to lack of unique markers, the frequency of HSCs among UCB CD34+ cells is the subject of ongoing debate, urging for reproducible strategies for their counting. Here, we used cellular barcoding to determine the frequency and clonal dynamics of human UCB HSCs and to determine how data analysis methods affect these parameters. We transplanted lentivirally barcoded CD34+ cells from 20 UCB donors into Nod/Scid/IL2Ry-/- (NSG) mice (n = 30). Twelve recipients (of 8 UCB donors) engrafted with >1% GFP+ cells, allowing for clonal analysis by multiplexed barcode deep sequencing. Using multiple definitions of clonal diversity and strategies for data filtering, we demonstrate that differences in data analysis can change clonal counts by several orders of magnitude and propose methods to improve their consistency. Using these methods, we show that the frequency of NSG-repopulating cells was low (median ∼1 HSC/104 CD34+ UCB cells) and could vary up to 10-fold between donors. Clonal patterns in blood became increasingly consistent over time, likely reflecting initial output of transient progenitors, followed by long-term HSCs with stable hierarchies. The majority of long-term clones displayed multilineage output, yet clones with lymphoid- or myeloid-biased output were also observed. Altogether, this study uncovers substantial interdonor and analysis-induced variability in the frequency of UCB CD34+ clones that contribute to post-transplant hematopoiesis. As clone tracing is increasingly relevant, we urge for universal and transparent methods to count HSC clones during normal aging and upon transplantation.