TGF-ß signalling limits effector function capacity of NK cell anti-tumour immunity in human bladder cancer.

BACKGROUND: Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of antibody-opsonization and antibody-dependant cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to therapeutic antibodies are typically immunosuppressed, and these immunosuppressive mechanisms are largely unknown. METHODS: Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumour-infiltrating NK cells in patients with bladder cancer. Further, in vitro, and in vivo models of this disease were used to validate these findings. FINDINGS: NK cells within bladder tumours displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-ß-signalling, a pleiotropic cytokine known for its immunosuppressive and tissue residency-inducing effects, were upregulated in tumour-infiltrating NK cells. TGF-ß mediated CD16 downregulation on NK cells, was further validated in vitro, which was accompanied by a transition into a tissue residency phenotype. This CD16 downregulation was also abrogated by TGF-ßR signalling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-ß effects. In a humanized mouse model of bladder cancer, mice treated with a TGF-ß inhibitor exhibited increased ADCC activity compared to mice treated only with antibodies. INTERPRETATION: This study highlights how TGF-ß-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-ß inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells in bladder cancer. FUNDING: The Guimaraes Laboratory is funded by a US Department of Defense-Breast Cancer Research Program-Breakthrough Award Level 1 (#BC200025), a grant (#2019485) awarded through the Medical Research Future Fund (MRFF, with the support of the Queensland Children's Hospital Foundation, Microba Life Sciences, Richie's Rainbow Foundation, Translational Research Institute (TRI) and UQ), and a grant (#RSS_2023_085) funded by a Metro South Health Research Support Scheme. J.K.M.W. is funded by a UQ Research Training Program PhD Scholarship and N.O. is funded by a NHMRC Postgraduate Scholarship (#2021932).

High-dose chemotherapy for Ewing sarcoma and Rhabdomyosarcoma: A systematic review by the Australia and New Zealand sarcoma association clinical practice guidelines working party.

INTRODUCTION: Patients with high-risk or metastatic Ewing sarcoma (ES) and rhabdomyosarcoma (RMS) have a guarded prognosis. High-dose chemotherapy (HDT) with autologous stem cell transplant (ASCT) has been evaluated as a treatment option to improve outcomes. However, survival benefits remain unclear, and treatment is associated with severe toxicities. METHODS: A systematic review was conducted, using the population, intervention, comparison outcome (PICO) model, to evaluate whether utilization of HDT/ASCT impacts the outcome of patients with ES and RMS compared to standard chemotherapy alone, as part of first line treatment or in the relapse setting. Medline, Embase and Cochrane Central were queried for publications from 1990 to October 2022 that evaluated event-free survival (EFS), overall survival (OS), and toxicities. Each study was screened by two independent reviewers for suitability. A qualitative synthesis of the results was performed. RESULTS: Of 1,172 unique studies screened, 41 studies were eligible for inclusion with 29 studies considering ES, 10 studies considering RMS and 2 studies considering both. In ES patients with high-risk localised disease who received HDT/ASCT after VIDE chemotherapy, consolidation with melphalan-based HDT/ASCT as first line therapy conveyed an EFS and OS benefit over standard chemotherapy consolidation. Efficacy of HDT/ASCT using a VDC/IE backbone, which is now standard care, has not been established. Survival benefits are not confirmed for ES patients with metastatic disease at initial diagnosis. For relapsed/refractory ES, four retrospective studies report improvement in outcomes with HDT/ASCT with the greatest evidence in patients who demonstrate a treatment response before HDT, and in patients under the age of 14. In RMS, there is no proven survival benefit of HDT/ASCT in primary localised, metastatic or relapsed disease. CONCLUSION: Prospective randomised trials are required to determine the utility of HDT/ASCT in ES and RMS. Selected patients with relapsed ES could be considered for HDT/ASCT.

Precision-guided treatment in high-risk pediatric cancers.

Recent research showed that precision medicine can identify new treatment strategies for patients with childhood cancers. However, it is unclear which patients will benefit most from precision-guided treatment (PGT). Here we report consecutive data from 384 patients with high-risk pediatric cancer (with an expected cure rate of less than 30%) who had at least 18 months of follow-up on the ZERO Childhood Cancer Precision Medicine Program PRecISion Medicine for Children with Cancer (PRISM) trial. A total of 256 (67%) patients received PGT recommendations and 110 (29%) received a recommended treatment. PGT resulted in a 36% objective response rate and improved 2-year progression-free survival compared with standard of care (26% versus 12%; P = 0.049) or targeted agents not guided by molecular findings (26% versus 5.2%; P = 0.003). PGT based on tier 1 evidence, PGT targeting fusions or commenced before disease progression had the greatest clinical benefit. Our data show that PGT informed by comprehensive molecular profiling significantly improves outcomes for children with high-risk cancers. ClinicalTrials.gov registration: NCT03336931.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Enhancing Natural Killer Cell Targeting of Pediatric Sarcoma.

Osteosarcoma, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) are the most common pediatric sarcomas. Conventional therapy for these sarcomas comprises neoadjuvant and adjuvant chemotherapy, surgical resection of the primary tumor and/or radiation therapy. Patients with metastatic, relapsed, or refractory tumors have a dismal prognosis due to resistance to these conventional therapies. Therefore, innovative therapeutic interventions, such as immunotherapy, are urgently needed. Recently, cancer research has focused attention on natural killer (NK) cells due their innate ability to recognize and kill tumor cells. Osteosarcoma, EWS and RMS, are known to be sensitive to NK cell cytotoxicity in vitro. In the clinical setting however, NK cell cytotoxicity against sarcoma cells has been mainly studied in the context of allogeneic stem cell transplantation, where a rapid immune reconstitution of NK cells plays a key role in the control of the disease, known as graft-versus-tumor effect. In this review, we discuss the evidence for the current and future strategies to enhance the NK cell-versus-pediatric sarcoma effect, with a clinical focus. The different approaches encompass enhancing antibody-dependent NK cell cytotoxicity, counteracting the NK cell mechanisms of self-tolerance, and developing adoptive NK cell therapy including chimeric antigen receptor-expressing NK cells.

Enhancing the Potential of Immunotherapy in Paediatric Sarcomas: Breaking the Immunosuppressive Barrier with Receptor Tyrosine Kinase Inhibitors.

Despite aggressive surgery, chemotherapy, and radiotherapy, survival of children and adolescents and young adults (AYAs) with sarcoma has not improved significantly in the past four decades. Immune checkpoint inhibitors (ICIs) are an exciting type of immunotherapy that offer new opportunities for the treatment of paediatric and AYA sarcomas. However, to date, most children do not derive a benefit from this type of treatment as a monotherapy. The immunosuppressive tumour microenvironment is a major barrier limiting their efficacy. Combinations of ICIs, such as anti-PD-1 therapy, with targeted molecular therapies that have immunomodulatory properties may be the key to breaking through immunosuppressive barriers and improving patient outcomes. Preclinical studies have indicated that several receptor tyrosine kinase inhibitors (RTKi) can alter the tumour microenvironment and boost the efficacy of anti-PD-1 therapy. A number of these combinations have entered phase-1/2 clinical trials, mostly in adults, and in most instances have shown efficacy with manageable side-effects. In this review, we discuss the status of ICI therapy in paediatric and AYA sarcomas and the rationale for co-treatment with RTKis. We highlight new opportunities for the integration of ICI therapy with RTK inhibitors, to improve outcomes for children with sarcoma.

Chimeric Antigen Receptor T cell Therapy and the Immunosuppressive Tumor Microenvironment in Pediatric Sarcoma.

Sarcomas are a diverse group of bone and soft tissue tumors that account for over 10% of childhood cancers. Outcomes are particularly poor for children with refractory, relapsed, or metastatic disease. Chimeric antigen receptor T (CAR T) cells are an exciting form of adoptive cell therapy that potentially offers new hope for these children. In early trials, promising outcomes have been achieved in some pediatric patients with sarcoma. However, many children do not derive benefit despite significant expression of the targeted tumor antigen. The success of CAR T cell therapy in sarcomas and other solid tumors is limited by the immunosuppressive tumor microenvironment (TME). In this review, we provide an update of the CAR T cell therapies that are currently being tested in pediatric sarcoma clinical trials, including those targeting tumors that express HER2, NY-ESO, GD2, EGFR, GPC3, B7-H3, and MAGE-A4. We also outline promising new CAR T cells that are in pre-clinical development. Finally, we discuss strategies that are being used to overcome tumor-mediated immunosuppression in solid tumors; these strategies have the potential to improve clinical outcomes of CAR T cell therapy for children with sarcoma.

Phase-II trials in osteosarcoma recurrences: A systematic review of past experience.

BACKGROUND: The most appropriate design of Phase-II trials evaluating new therapies in osteosarcoma remains poorly defined. OBJECTIVE: To study consistency in phase-II clinical trials evaluating new therapies for osteosarcoma recurrences with respect to eligibility criteria, response assessment, end-points, statistical design and reported results. METHODS: Systematic review of clinical trials registered on clinicaltrials.gov, clinicaltrialsregister.eu and French National Cancer Institute website or referenced in PubMed and American Society of Clinical Oncology websites, between 2003 and 2016, using the following criteria: (osteosarcoma OR bone sarcoma) AND (Phase-II). RESULTS: Among the 99 trials identified, 80 were Phase-II, 17 I/II and 2 II/III, evaluating mostly targeted therapy (n = 40), and chemotherapy alone (n = 26). Results were fully (n = 28) or partially (abstract, n = 6) published. Twenty-four trials were dedicated to osteosarcoma, 22 had an osteosarcoma stratum. Twenty-eight out of 99 trials refer to the age range observed at recurrence (28%). Overall, 65 trials were run in multicentre settings, including 17 international trials. Only 9 trials were randomised. The primary end-point was tumour response in 71 trials (response rate, n = 40 or best response, n = 31), with various definitions (complete + partial ± minor response and stable disease), mainly evaluated with RECIST criteria (n = 69); it was progression-free survival in 24 trials and OS in 3. In single-arm trials evaluating response rate, the null hypothesis tested (when available, n = 12) varied from 5% to 25%. CONCLUSION: No robust historical data can currently be derived from past efficacy Phase-II trials. There is an urgent need to develop international randomised Phase-II trials across all age ranges with standardised primary end-point.