The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

Transcriptional signals of transformation in human cancer.

BACKGROUND: As normal cells transform into cancers, their cell state changes, which may drive cancer cells into a stem-like or more primordial, foetal, or embryonic cell state. The transcriptomic profile of this final state may encode information about cancer's origin and how cancers relate to their normal cell counterparts. METHODS: Here, we used single-cell atlases to study cancer transformation in transcriptional terms. We utilised bulk transcriptomes across a wide spectrum of adult and childhood cancers, using a previously established method to interrogate their relationship to normal cell states. We extend and validate these findings using single-cell cancer transcriptomes and organ-specific atlases of colorectal and liver cancer. RESULTS: Our bulk transcriptomic data reveals that adult cancers rarely return to an embryonic state, but that a foetal state is a near-universal feature of childhood cancers. This finding was confirmed with single-cell cancer transcriptomes. CONCLUSIONS: Our findings provide a nuanced picture of transformation in human cancer, indicating cancer-specific rather than universal patterns of transformation pervade adult epithelial cancers.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Evaluating preclinical evidence for clinical translation in childhood brain tumours: Guidelines from the CONNECT, PNOC, and ITCC brain networks.

Clinical outcomes for many childhood brain tumours remain poor, despite our increasing understanding of the underlying disease biology. Advances in molecular diagnostics have refined our ability to classify tumour types and subtypes, and efforts are underway across multiple international paediatric neuro-oncology consortia to take novel biological insights in the worst prognosis entities into innovative clinical trials. Whilst for the first time we are designing such studies on the basis of disease-specific biological data, the levels of preclincial evidence in appropriate model systems on which these trials are initiated is still widely variable. We have considered these issues between CONNECT, PNOC and ITCC-Brain, and developed a framework in which we can assess novel concepts being brought forward for possible clinical translation. Whilst not intended to be proscriptive for every possible circumstance, these criteria provide a basis for self-assessment of evidence by laboratory scientists, and a platform for discussion and rational decision-making prior to moving forward clinically.

Tumor inflammation-associated neurotoxicity.

Cancer immunotherapies have unique toxicities. Establishment of grading scales and standardized grade-based treatment algorithms for toxicity syndromes can improve the safety of these treatments, as observed for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) in patients with B cell malignancies treated with chimeric antigen receptor (CAR) T cell therapy. We have observed a toxicity syndrome, distinct from CRS and ICANS, in patients treated with cell therapies for tumors in the central nervous system (CNS), which we term tumor inflammation-associated neurotoxicity (TIAN). Encompassing the concept of 'pseudoprogression,' but broader than inflammation-induced edema alone, TIAN is relevant not only to cellular therapies, but also to other immunotherapies for CNS tumors. To facilitate the safe administration of cell therapies for patients with CNS tumors, we define TIAN, propose a toxicity grading scale for TIAN syndrome and discuss the potential management of this entity, with the goal of standardizing both reporting and management.

Precise identification of cancer cells from allelic imbalances in single cell transcriptomes.

A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.

Single-cell transcriptomics reveals a distinct developmental state of KMT2A-rearranged infant B-cell acute lymphoblastic leukemia.

KMT2A-rearranged infant ALL is an aggressive childhood leukemia with poor prognosis. Here, we investigated the developmental state of KMT2A-rearranged infant B-cell acute lymphoblastic leukemia (B-ALL) using bulk messenger RNA (mRNA) meta-analysis and examination of single lymphoblast transcriptomes against a developing bone marrow reference. KMT2A-rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state, whereas less adverse NUTM1-rearranged infant ALL demonstrated signals of later developing B cells, in line with most other childhood B-ALLs. We compared infant lymphoblasts with ELP cells and revealed that the cancer harbored hybrid myeloid-lymphoid features, including nonphysiological antigen combinations potentially targetable to achieve cancer specificity. We validated surface coexpression of exemplar combinations by flow cytometry. Through analysis of shared mutations in separate leukemias from a child with infant KMT2A-rearranged B-ALL relapsing as AML, we established that KMT2A rearrangement occurred in very early development, before hematopoietic specification, emphasizing that cell of origin cannot be inferred from the transcriptional state.

Fine-tuning CARs for best performance.

Chimeric antigen receptors (CARs) allow redirection of T cells against any surface antigen. However, CARs require optimization to achieve activity against low-density antigens. Heitzeneder et al. perform an iterative adjustment of CAR components to reach a design for targeting cerebroglycan (GPC2) that shows potent pre-clinical activity in neuroblastoma models.

Single cell derived mRNA signals across human kidney tumors.

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.

Tumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell.

Neuroblastoma is a childhood cancer that resembles developmental stages of the neural crest. It is not established what developmental processes neuroblastoma cancer cells represent. Here, we sought to reveal the phenotype of neuroblastoma cancer cells by comparing cancer (n = 19,723) with normal fetal adrenal single-cell transcriptomes (n = 57,972). Our principal finding was that the neuroblastoma cancer cell resembled fetal sympathoblasts, but no other fetal adrenal cell type. The sympathoblastic state was a universal feature of neuroblastoma cells, transcending cell cluster diversity, individual patients, and clinical phenotypes. We substantiated our findings in 650 neuroblastoma bulk transcriptomes and by integrating canonical features of the neuroblastoma genome with transcriptional signals. Overall, our observations indicate that a pan-neuroblastoma cancer cell state exists, which may be attractive for novel immunotherapeutic and targeted avenues.

Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4+ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM.

Antitumor activity without on-target off-tumor toxicity of GD2-chimeric antigen receptor T cells in patients with neuroblastoma.

The reprogramming of a patient's immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity.

Pediatric pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of antitumor immunity.

Immune-therapy is an attractive alternative therapeutic approach for targeting central nervous system (CNS) tumors and the constituency of the Tumor Immune Microenvironment (TIME) likely to predict patient response. Here, we describe the TIME of >6000 primarily pediatric CNS tumors using a deconvolution approach (methylCIBERSORT). We produce and validate a custom reference signature defining 11 non-cancer cell types to estimate relative proportions of infiltration in a panCNS tumor cohort spanning 80 subtypes. We group patients into three broad immune clusters associated with CNS tumor types/subtypes. In cohorts of medulloblastomas (n = 2325), malignant rhabdoid tumors (n = 229) and pediatric high-grade gliomas (n = 401), we show significant associations with molecular subgroups/subtypes, mutations, and prognosis. We further identify tumor-specific immune clusters with phenotypic characteristics relevant to immunotherapy response (i.e. Cytolytic score, PDL1 expression). Our analysis provides an indication of the potential future therapeutic and prognostic possibilities of immuno-methylomic profiling in pediatric CNS tumor patients that may ultimately inform approach to immune-therapy.

Embryonal precursors of Wilms tumor.

Adult cancers often arise from premalignant clonal expansions. Whether the same is true of childhood tumors has been unclear. To investigate whether Wilms tumor (nephroblastoma; a childhood kidney cancer) develops from a premalignant background, we examined the phylogenetic relationship between tumors and corresponding normal tissues. In 14 of 23 cases studied (61%), we found premalignant clonal expansions in morphologically normal kidney tissues that preceded tumor development. These clonal expansions were defined by somatic mutations shared between tumor and normal tissues but absent from blood cells. We also found hypermethylation of the H19 locus, a known driver of Wilms tumor development, in 58% of the expansions. Phylogenetic analyses of bilateral tumors indicated that clonal expansions can evolve before the divergence of left and right kidney primordia. These findings reveal embryonal precursors from which unilateral and multifocal cancers develop.

Establishment and phenotyping of neurosphere cultures from primary neuroblastoma samples.

Background: Primary cell culture using serum free media supplemented with growth factors has been used in a number of cancers to propagate primary cells with stem like properties, which form as spherical cellular aggregates. Methods: We systematically evaluated the capacity of freshly disaggregated neuroblastoma tumors to become established as neurospheres in stem cell media using a uniform protocol. 67 primary neuroblastoma samples from patients treated at a single institution were prospectively evaluated for their ability to become established in culture. Samples, either solid tissue or cells from surgical transit fluid both post chemotherapy and chemotherapy naïve, were evaluated from diagnostic needle biopsies or surgical resections. Results: Overall 37 neurosphere cultures were successfully established from 67 samples. In 11 out of 14 cases investigated by flow cytometry, uniform staining for neuroblastoma markers CD56 and GD2 was demonstrated in CD45 negative non-hemopoietic cells, confirming neuroblastoma origin. Conclusion: We present a simple and reproducible approach for producing primary neurospheres from neuroblastoma samples, which provides a reliable resource for future work including genetic analysis, stem cell research and models for therapeutics.

An Optimized GD2-Targeting Retroviral Cassette for More Potent and Safer Cellular Therapy of Neuroblastoma and Other Cancers.

Neuroblastoma is the commonest extra cranial solid cancer of childhood. Despite escalation of treatment regimens, a significant minority of patients die of their disease. Disialoganglioside (GD2) is consistently expressed at high-levels in neuroblastoma tumors, which have been targeted with some success using therapeutic monoclonal antibodies. GD2 is also expressed in a range of other cancer but with the exception of some peripheral nerves is largely absent from non-transformed tissues. Chimeric Antigen Receptors (CARs) are artificial type I proteins which graft the specificity of a monoclonal antibody onto a T-cell. Clinical data with early CAR designs directed against GD2 have shown some promise in Neuroblastoma. Here, we describe a GD2-targeting CAR retroviral cassette, which has been optimized for CAR T-cell persistence, efficacy and safety.

Highlights of Children with Cancer UK's Workshop on Drug Delivery in Paediatric Brain Tumours.

The first Workshop on Drug Delivery in Paediatric Brain Tumours was hosted in London by the charity Children with Cancer UK. The goals of the workshop were to break down the barriers to treating central nervous system (CNS) tumours in children, leading to new collaborations and further innovations in this under-represented and emotive field. These barriers include the physical delivery challenges presented by the blood-brain barrier, the underpinning reasons for the intractability of CNS cancers, and the practical difficulties of delivering cancer treatment to the brains of children. Novel techniques for overcoming these problems were discussed, new models brought forth, and experiences compared.

Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging.

BACKGROUND: In vivo imaging using genetic reporters is a central supporting tool in the development of cell and gene therapies affording us the ability to selectively track the therapeutic indefinitely. Previous studies have demonstrated the utility of the human norepinephrine transporter (hNET) as a positron emission tomography/single photon emission computed tomography (PET/SPECT) genetic reporter for in vivo cellular imaging. Here, our aim was to extend on this work and construct a tricistronic vector with dual optical (firefly luciferase) and nuclear (hNET) in vivo imaging and ex vivo histochemical capabilities. Guiding this development, we describe how a fluorescent substrate for hNET, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)), can be used to optimise vector design and serve as an in vitro functional screen. METHODS: Vectors were designed to co-express a bright red-shifted firefly luciferase (FLuc), hNET and a small marker gene RQR8. Genes were co-expressed using 2A peptide linkage, and vectors were transduced into a T cell line, SupT1. Two vectors were constructed with different gene orientations; FLuc.2A.RQR8.2A.hNET and hNET.2A.FLuc.2A.RQR8. hNET function was assessed using ASP(+)-guided flow cytometry. In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice. RESULTS: SupT1/FLuc.2A.RQR8.2A.hNET cells resulted in >4-fold higher ASP(+) uptake compared to SupT1/hNET.2A.FLuc.2A.RQR8, suggesting that 2A orientation effected hNET function. SupT1/FLuc.2A.RQR8.2A.hNET cells were readily visualised with both BLI and SPECT, demonstrating high signal to noise at 24 h post (123)I-meta-iodobenzylguanidine (MIBG) administration. CONCLUSIONS: In this study, a pre-clinical tricistronic vector with flow cytometry, BLI, SPECT and histochemical capabilities was constructed, which can be widely applied in cell tracking studies supporting the development of cell therapies. The study further demonstrates that hNET function in engineered cells can be assessed using ASP(+)-guided flow cytometry in place of costly radiosubstrate methodologies. This fluorogenic approach is unique to the hNET PET/SPECT reporter and may prove valuable when screening large numbers of cell lines or vector/mutant constructs.

A highly compact epitope-based marker/suicide gene for easier and safer T-cell therapy.

A compact marker/suicide gene that utilizes established clinical-grade reagents and pharmaceuticals would be of considerable practical utility to T-cell cancer gene therapy. Marker genes enable measurement of transduction and allow selection of transduced cells, whereas suicide genes allow selective deletion of administered T cells in the face of toxicity. We have created a highly compact marker/suicide gene for T cells combining target epitopes from both CD34 and CD20 antigens (RQR8). This construct allows selection with the clinically approved CliniMACS CD34 system (Miltenyi). Further, the construct binds the widely used pharmaceutical antibody rituximab, resulting in selective deletion of transgene-expressing cells. We have tested the functionality of RQR8 in vitro and in vivo as well as in combination with T-cell engineering components. We predict that RQR8 will make T-cell gene therapy both safer and cheaper.