Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development.

Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

The tidyomics ecosystem: enhancing omic data analyses.

The growth of omic data presents evolving challenges in data manipulation, analysis and integration. Addressing these challenges, Bioconductor provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming offers a revolutionary data organization and manipulation standard. Here we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analyzing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas, spanning six data frameworks and ten analysis tools.

Plasma microbial cell-free DNA following chimeric antigen receptor T cell therapy in pediatric patients with relapsed/refractory leukemia.

Chimeric antigen receptor (CAR) T cell therapy is a promising treatment for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia (R/R B ALL). Cytokine release syndrome (CRS) is a common toxicity after CAR T cell therapy and fever is often the first symptom. Differentiating CRS from infection after CAR T cell therapy can be challenging. Plasma microbial cell free DNA (mcfDNA) is a novel diagnostic tool which allows for qualitative and quantitative assessment of over 1000 organisms. This pilot study sought to characterize mcfDNA results in pediatric patients with R/R B ALL in the first 2 months after CAR T cell therapy.

CD22-directed CAR T-cell therapy induces complete remissions in CD19-directed CAR-refractory large B-cell lymphoma.

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.

BACKGROUND: In a single-center phase 1-2a study, the anti-CD19 chimeric antigen receptor (CAR) T-cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). METHODS: We conducted a phase 2, single-cohort, 25-center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B-cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. RESULTS: For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event-free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. CONCLUSIONS: In this global study of CAR T-cell therapy, a single infusion of tisagenlecleucel provided durable remission with long-term persistence in pediatric and young adult patients with relapsed or refractory B-cell ALL, with transient high-grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849 .).

Use of cardiac radiation therapy as bridging therapy to CAR-T for relapsed pediatric B-cell acute lymphoblastic leukemia.

The use of radiotherapy as bridging therapy to chimeric antigen receptor T-cell therapy (CAR-T) in pre-B acute lymphoblastic leukemia (B-ALL) has been minimally explored. Here, we present a boy with B-ALL who relapsed after allogeneic bone marrow transplant with disseminated disease, including significant symptomatic cardiovascular and gastrointestinal (GI) involvement. The cardiac and GI leukemic infiltrates were successfully treated with bridging radiation therapy (BRT) prior to CAR-T infusion. Using this approach, he successfully tolerated CAR-T with no evidence of disease or sequelae on 3-month follow-up. This is the first reported case of safe and effective delivery of cardiac BRT in B-ALL.

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity.

An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.

Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): a multicentre, open-label, single-arm, phase 1-2 trial.

BACKGROUND: Immune checkpoint inhibitors targeting PD-1 have shown clinical benefit in adults with cancer, but data on these drugs in children are scarce. We did a phase 1-2 study of nivolumab, a PD-1 blocking monoclonal antibody, to determine its safety, pharmacokinetics, and antitumour activity in children and young adults with recurrent or refractory non-CNS solid tumours or lymphoma. METHODS: We did a multicentre, open-label, single-arm, dose-confirmation and dose-expansion, phase 1-2 trial in 23 hospitals in the USA. Eligible patients for part A (dose-confirmation phase) of the study were aged 1-18 years with solid tumours with measurable or evaluable disease (by Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1) regardless of histology. Eligible patients for part B (dose-expansion phase) were aged 1-30 years with measurable disease (by RECIST criteria) in the following disease cohorts: rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, neuroblastoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and melanoma. Patients in part A and were given nivolumab 3 mg/kg intravenously over 60 min on days 1 and 15 of a 28-day cycle in a rolling 6 study design with de-escalation upon dose-limiting toxicities to establish the recommended phase 2 dose. Patients in part B were given the recommended phase 2 dose. The primary outcomes were the tolerability, systemic exposure, maximum tolerated dose, and the antitumour activity of nivolumab at the adult recommended dose in children and young adults. This trial is registered with ClinicalTrials.gov, NCT02304458, with follow-up ongoing and is closed to new participants. FINDINGS: 85 patients were enrolled between Feb 22, 2015, and Dec 31, 2018, and 75 patients were fully evaluable for toxicity. Median follow-up was 30 days (IQR 27-83). In part A, 13 patients were enrolled and 12 were evaluable for toxicity. There were no dose de-escalations or dose-limiting toxicities and nivolumab 3 mg/kg was confirmed as the paediatric recommended phase 2. 72 patients were enrolled in part B and 63 were evaluable for toxicity. Five (7%) patients in part B had dose-limiting toxicities. The most common overall toxicity was anaemia (35 [47%] of 75 patients; five patients had grade 3 or grade 4) and non-haematological toxicity was fatigue (28 [37%] patients; none had grade 3 or grade 4). Responses were observed in patients with lymphoma (three [30%] of ten with Hodgkin lymphoma and one [10%] of ten with non-Hodgkin lymphoma; all responders had PD-L1 expression). Objective responses were not observed in other tumour types. INTERPRETATION: Nivolumab was safe and well tolerated in children and young adults and showed clinical activity in lymphoma. Nivolumab showed no significant single-agent activity in the common paediatric solid tumours. This study defines the recommended phase 2 dose and establishes a favourable safety profile for nivolumab in children and young adults, which can serve as the basis for its potential study in combinatorial regimens for childhood cancer. FUNDING: Bristol-Myers Squibb, Children's Oncology Group, National Institutes of Health, Cookies for Kids Cancer Foundation.

Progressive B Cell Loss in Revertant X-SCID.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.

A Cancer Biologist's Primer on Machine Learning Applications in High-Dimensional Cytometry.

The application of machine learning and artificial intelligence to high-dimensional cytometry data sets has increasingly become a staple of bioinformatic data analysis over the past decade. This is especially true in the field of cancer biology, where protocols for collecting multiparameter single-cell data in a high-throughput fashion are rapidly developed. As the use of machine learning methodology in cytometry becomes increasingly common, there is a need for cancer biologists to understand the basic theory and applications of a variety of algorithmic tools for analyzing and interpreting cytometry data. We introduce the reader to several keystone machine learning-based analytic approaches with an emphasis on defining key terms and introducing a conceptual framework for making translational or clinically relevant discoveries. The target audience consists of cancer cell biologists and physician-scientists interested in applying these tools to their own data, but who may have limited training in bioinformatics. © 2020 International Society for Advancement of Cytometry.

Immunotherapy for the Treatment of Acute Lymphoblastic Leukemia.

PURPOSE OF REVIEW: Immunotherapy for the treatment of acute lymphoblastic leukemia (ALL) broadens therapeutic options beyond chemotherapy and targeted therapy. Here, we review the use of monoclonal antibody-based drugs and cellular therapies to treat ALL. We discuss the challenges facing the field regarding the optimal timing and sequencing of these therapies in relation to other treatment options as well as considerations of cost effectiveness. RECENT FINDINGS: By early identification of patients at risk for leukemic relapse, monoclonal antibody and cellular immunotherapies can be brought to the forefront of treatment options. Novel CAR design and manufacturing approaches may enhance durable patient response. Multiple clinical trials are now underway to evaluate the sequence and timing of monoclonal antibody, cellular therapy, and/or stem cell transplantation. The biologic and clinical contexts in which immunotherapies have advanced the treatment of ALL confer optimism that more patients will achieve durable remissions. Immunotherapy treatments in ALL will expand through rationally targeted approaches alongside advances in CAR T cell therapy design and clinical experience.

Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.

Patient-reported quality of life after tisagenlecleucel infusion in children and young adults with relapsed or refractory B-cell acute lymphoblastic leukaemia: a global, single-arm, phase 2 trial.

BACKGROUND: The ELIANA trial showed that 61 (81%) of 75 paediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia achieved overall remission after treatment with tisagenlecleucel, a chimeric antigen receptor targeted against the CD19 antigen. We aimed to evaluate patient-reported quality of life in these patients before and after tisagenlecleucel infusion. METHODS: ELIANA, a global, single-arm, open-label, phase 2 trial, was done in 25 hospitals across Australia, Austria, Belgium, Canada, France, Germany, Italy, Japan, Norway, Spain, and the USA. Patients with B-cell acute lymphoblastic leukaemia aged at least 3 years at the time of screening and 21 years or younger at the time of initial diagnosis who were in second or greater bone marrow relapse, chemorefractory, relapsed after allogeneic stem-cell transplantation, or were otherwise ineligible for allogeneic stem-cell transplantation were enrolled. Patients received a single intravenous administration of a target dose of 0·2-5 × 106 transduced viable T cells per kg for patients weighing 50 kg or less or 0·1-2·5 × 108 transduced viable T cells for patients weighing more than 50 kg. The primary outcome, reported previously, was the proportion of patients who achieved remission. A prespecified secondary endpoint, reported here, was patient-reported quality of life measured with the Pediatric Quality of Life Inventory (PedsQL) and European Quality of Life-5 Dimensions questionnaire (EQ-5D). Patients completed the questionnaires at baseline, day 28, and months 3, 6, 9, and 12 after treatment. The data collected were summarised using descriptive statistics and post-hoc mixed models for repeated measures. Change from baseline response profiles were illustrated with cumulative distribution function plots. The proportion of patients achieving the minimal clinically important difference and normative mean value were reported. Analysis was per protocol. This study is registered with ClinicalTrials.gov, NCT02435849. FINDINGS: Between April 8, 2015, and April 25, 2017, 107 patients were screened, 92 were enrolled, and 75 received tisagenlecleucel. 58 patients aged 8-23 years were included in the analysis of quality of life. At baseline, 50 (86%) patients had completed the PedsQL questionnaire and 48 (83%) had completed the EQ-5D VAS. Improvements in patient-reported quality-of-life scores were observed for all measures at month 3 after tisagenlecleucel infusion (mean change from baseline to month 3 was 13·3 [95% CI 8·9-17·6] for the PedsQL total score and 16·8 [9·4-24·3] for the EQ-5D visual analogue scale). 30 (81%) of 37 patients achieved the minimal clinically important difference at month 3 for the PedsQL total score and 24 (67%) of 36 patients achieved this for the EQ-5D visual analogue scale. INTERPRETATION: These findings, along with the activity and safety results of ELIANA, suggest a favourable benefit-risk profile of tisagenlecleucel in the treatment of paediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. FUNDING: Novartis.

Automated mapping of phenotype space with single-cell data.

Accurate identification of cell subsets in complex populations is key to discovering novelty in multidimensional single-cell experiments. We present X-shift (http://web.stanford.edu/~samusik/vortex/), an algorithm that processes data sets using fast k-nearest-neighbor estimation of cell event density and arranges populations by marker-based classification. X-shift enables automated cell-subset clustering and access to biological insights that 'prior knowledge' might prevent the researcher from discovering.

New developments in immunotherapy for pediatric solid tumors.

PURPOSE OF REVIEW: Building upon preclinical advances, we are uncovering immunotherapy strategies that are translating into improved outcomes in tumor subsets. Advanced pediatric solid tumors carry poor prognoses and resultant robust efforts to apply immunotherapy advances to pediatric solid tumors are in progress. Here, we discuss recent developments in the field using mAb and mAb-based therapies including checkpoint blockade and chimeric antigen receptors (CARs). RECENT FINDINGS: The pediatric solid tumor mAb experience targeting the diganglioside, GD2, for patients with neuroblastoma has been the most compelling to date. GD2 and alternative antigen-specific mAbs are now being incorporated into antibody-drug conjugates, bispecific antibodies and CARs for treatment of solid tumors. CARs in pediatric solid tumors have not yet achieved comparative responses to the hematologic CAR experience; however, novel strategies such as bispecific targeting, intratumoral administration and improved understanding of T-cell biology may yield enhanced CAR-efficacy. Therapeutic effect using single-agent checkpoint blocking antibodies in pediatric solid tumors also remains limited to date. Combinatorial strategies continue to hold promise and the clinical effect in tumor subsets with high antigenic burden is being explored. SUMMARY: Pediatric immunotherapy remains at early stages of translation, yet we anticipate that with advanced technology, we will achieve widespread, efficacious use of immunotherapy for pediatric solid tumors.

Checkpoint inhibition in pediatric hematologic malignancies.

Immune surveillance comprising of adaptive and innate immune systems is naturally designed to eliminate cancer development; overexpression of inhibitory receptors and their ligands prevent this check and lead to evasion and hence cancer progression and metastasis. The use of tumor-specific monoclonal antibodies (MAbs) targeting these checkpoint regulators is promising and has led to this novel field of cancer immunotherapy. The first antibody directed against cytotoxic T-lymphocyte associated protein 4 (CTLA-4), ipilimumab, showed promising results in clinical trials and was approved by the US Food and Drug Administration (FDA) for the treatment of metastatic melanoma in 2011. Since then, various other immune checkpoint inhibitors are being studied in preclinical and clinical trial phases, targeting programmed-death-1 (PD-1) and its ligand programmed death ligand 1 (PD-L1), T cell lymphocyte activation gene-3 (LAG-3), and others. Results from clinical trials are promising, and currently this approach has proven effective and safe in patients with solid tumors and some hematological malignancies in adults. In general, CTLA-4 and PD-1 inhibitors are well tolerated; however, the augmented immune response enabled by this class of agents is associated with a unique group of side effects called immune-related adverse events (irAEs). Experience in pediatrics using immune checkpoint inhibitors for hematological malignancies is limited to Hodgkin's disease and non-Hodgkin's lymphoma as in the ongoing Children's Oncology Group (COG) protocol ADVL1412. Therapeutic advances in childhood leukemia and lymphoma (TACL) consortium will initiate an early phase clinical trial with PD-1 inhibitor nivolumab in relapsed/refractory acute myeloid leukemia (AML) in the next few months.