Conventional therapies deplete Brain-Infiltrating adaptive immune cells in a Mouse Model of Group 3 Medulloblastoma implicating Myeloid Cells as favorable immunotherapy targets

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments

Outcome of infants younger than 1 year with acute lymphoblastic leukemia treated with the interfant-06 protocol: Results from an international phase III randomized study

PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by KMT2A (MLL) gene rearrangements and coexpression of myeloid markers. The Interfant-06 study, comprising 18 national and international study groups, tested whether myeloid-style consolidation chemotherapy is superior to lymphoid style, the role of stemcell transplantation (SCT), and which factors had independent prognostic value. MATERIALS AND METHODS Three risk groups were defined: low risk (LR): KMT2A germline; high risk (HR): KMT2A-rearranged and older than 6 months with WBC count 300 3 109/L or more or a poor prednisone response; and medium risk (MR): all other KMT2A-rearranged cases. Patients in the MR and HR groups were randomly assigned to receive the lymphoid course low-dose cytosine arabinoside [araC], 6-mercaptopurine, cyclophosphamide (IB) or experimental myeloid courses, namely araC, daunorubicin, etoposide (ADE) and mitoxantrone, araC, etoposide (MAE). RESULTS A total of 651 infants were included, with 6-year event-free survival (EFS) and overall survival of 46.1% (SE, 2.1) and 58.2% (SE, 2.0). In West European/North American groups, 6-year EFS and overall survival were 49.4% (SE, 2.5) and 62.1% (SE, 2.4), which were 10% to 12% higher than in other countries. The 6-year probability of disease-free survival was comparable for the randomized arms (ADE1MAE 39.3% [SE 4.0; n = 169] v IB 36.8% [SE, 3.9; n = 161]; log-rank P = .47). The 6-year EFS rate of patients in the HR group was 20.9% (SE, 3.4) with the intention to undergo SCT; only 46% of them received SCT, because many had early events. KMT2A rearrangement was the strongest prognostic factor for EFS, followed by age, WBC count, and prednisone response. CONCLUSION Early intensification with postinduction myeloid-type chemotherapy courses did not significantly improve outcome for infant ALL compared with the lymphoid-type course IB. Outcome for infant ALL in Interfant- 06 did not improve compared with that in Interfant-99

The combination of intravitreal triamcinolone and phacoemulsification surgery in patients with diabeticfoveal oedema and cataract

BACKGROUND: The management of diabetic patients with refractory macular oedema or patients with no adequate pre-operative view to administer laser treatment provide a challenge to the ophthalmologist. We wished to assess the use, safety and effect of intravitreal triamcinolone injection at the time of cataract surgery in patients with diabetic foveal oedema and sight limiting lens opacities. METHOD: This was a longitudinal non-randomised prospective pilot study in 18 eyes (12 patients). All patients had visually significant lens opacities and either persistent diabetic foveal oedema unresponsive to laser treatment-group A, or foveal oedema with no adequate pre-operative view for laser treatment- group B. The cataract surgery was carried out under full aseptic technique using a self-sealing temporal incision and a foldable acrylic lens. Intravitreal triamcinolone was given infratemporally pars plana at the completion of the cataract surgery. The patients were reviewed at day 5, 2 weeks, 2 months and then every 3 months as required. The Wilcoxin matched-pairs test was used to assess the significance of the improvement in visual acuity at 2 months. RESULTS: Twelve patients with a total of 18 eyes were included in the study. There were 10 patients (15 eyes) in group A and 3 patients (3 eyes) in group B. Preoperatively 16 of the 18 eyes had a visual acuity of 6/24 or worse. Postoperatively 83% of patients had completely dry foveae at 2 weeks. Best-corrected visual acuities at two months review ranged from 6/6 to CF with 9 eyes (50%) achieving 6/12 or better (7 eyes (47%) in group A and 2 eyes (67%) in group B). Three eyes had no recorded improvement in visual acuity, but no eyes had deterioration in acuity. The improvement in visual acuity was significant at p = 0.001. There were no significant sight threatening complications. CONCLUSION: Intravitreal triamcinolone has been shown to lead to an improvement in macular oedema and visual improvement in diabetic patients not undergoing cataract surgery but has not, to our knowledge, been previously used in a study like this one. We suggest that intravitreal injection at the time of cataract surgery could be carried out safely with encouraging visual outcomes in patients with diabetic foveal oedema and cataract

Assessment of cannabidiol and Δ9-Tetrahydrocannabiol in mouse models of Medulloblastoma and Ependymoma

Children with medulloblastoma and ependymoma are treated with a multidisciplinary approach that incorporates surgery, radiotherapy, and chemotherapy; however, overall survival rates for patients with high-risk disease remain unsatisfactory. Data indicate that plant-derived cannabinoids are effective against adult glioblastoma; however, preclinical evidence supporting their use in pediatric brain cancers is lacking. Here we investigated the potential role for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in medulloblastoma and ependymoma. Dose-dependent cytotoxicity of medulloblastoma and ependymoma cells was induced by THC and CBD in vitro, and a synergistic reduction in viability was observed when both drugs were combined. Mechanistically, cannabinoids induced cell cycle arrest, in part by the production of reactive oxygen species, autophagy, and apoptosis; however, this did not translate to increased survival in orthotopic transplant models despite being well tolerated. We also tested the combination of cannabinoids with the medulloblastoma drug cyclophosphamide, and despite some in vitro synergism, no survival advantage was observed in vivo. Consequently, clinical benefit from the use of cannabinoids in the treatment of high-grade medulloblastoma and ependymoma is expected to be limited. This study emphasizes the importance of preclinical models in validating therapeutic agent efficacy prior to clinical trials, ensuring that enrolled patients are afforded the most promising therapies available

JS04.5.A Enhancing the activity of anti-CD47 antibody therapy with radiotherapy in preclinical models of medulloblastoma

Background Brain cancers are the most common solid cancer in children and the leading cause of cancer-related deaths in children. Medulloblastoma is the most common paediatric brain tumour. Treatment for medulloblastoma involves surgery, craniospinal irradiation (CSI) and chemotherapy. These therapies are extremely damaging to the developing brain and have not changed in decades, resulting in stagnation in the survival outcomes for children with medulloblastoma, and poor quality of life for children who survive their treatment. Immunotherapy has become a focus of novel treatment development. While there are multiple clinical trials aiming to increase immune recognition of medulloblastoma, none have been successful to date. Anti-CD47 is an immune-modulating therapeutic antibody which blocks the anti-phagocytic signal, CD47, expressed by brain cancer cells. Anti-CD47 has shown promising preliminary efficacy in brain cancer models. Material and Methods Using a small animal radiotherapy platform, we have developed a preclinical CSI protocol which mimics clinical radiotherapy. Using an orthotopic xenograft model of medulloblastoma, mice were treated with either anti-CD47 antibody therapy, CSI, or the combination of both anti-CD47 and CSI. Results CSI was found to deplete adaptive immune cells in the brain, while myeloid cells remained the dominant populations. Anti-CD47 antibody therapy was ineffective as a single agent against a patient derived xenograft model of Group 3 medulloblastoma, and CSI as a monotherapy resulted in temporary tumour regression. We found that the combination of anti-CD47 with CSI resulted in marked and persistent tumour regression. Conclusion This preclinical work has shown promising efficacy of anti-CD47 in combination with CSI, which we are currently testing in additional models. Our work is currently employing a range of techniques such as high dimensional flow cytometry and single cell sequencing to elucidate the mechanisms by which radiotherapy enhances the anti-tumour activity of myeloid cells. This work will enable the rational design and translation of optimal combination therapies for medulloblastoma clinical trials

MODL-18. Enhancing anti-CD47 mAb efficacy with radiotherapy for Group 3 paediatric medulloblastoma in preclinical models

Medulloblastoma is the most common paediatric brain cancer. Standard treatment approaches, including craniospinal irradiation (CSI), can result in severe lifelong side effects and have not changed for decades resulting in a stagnation of survival outcomes for children with aggressive medulloblastoma. Despite successes in other cancers, no immunotherapies have been approved for use in paediatric medulloblastoma. Unlike other solid tumours, medulloblastomas are myeloid dominant, and immunotherapies must be rationally designed with the tumour microenvironment in mind. Anti-CD47 antibody therapy activates macrophages against cancer cells by blocking anti-phagocytic signalling mediated by CD47-SIRPa ligation and has shown preclinical efficacy in brain cancer models. We have developed preclinical CSI protocols that mimic clinical treatment response using a small animal radiotherapy platform. We show that CSI depletes adaptive immune cells in the brain, increasing the proportional abundance of myeloid cells, suggesting an opportunity to combine radiation with myeloid-targeted immunotherapy. We show that anti-CD47 therapy is ineffective as a single agent against a patient-derived xenograft model of Group 3 medulloblastoma (SJ_MB002), and that while the CSI protocol causes temporary tumour regression, the combination of anti-CD47 with CSI results in marked and persistent tumour regression. To enhance our preclinical evaluation of CSI and anti-CD47, we have developed new mouse models that more accurately reflect the developing microenvironment of children and show that immune populations in paediatric brain are distinct from adult mouse brain. Future work will elucidate the mechanisms by which radiotherapy alters the medulloblastoma microenvironment to enhance the anti-tumour activity of myeloid immune cells in the brain. By evaluating this novel combination of immunotherapy with standard medulloblastoma treatments, in age-appropriate models, our research should facilitate the rational selection and rapid translation of optimised treatment combinations for future medulloblastoma clinical trials

Exploring threats to generalisability in a large international rehabilitation trial (AVERT)

Objective The purpose of this paper is to examine potential threats to generalisability of the results of a multicentre randomised controlled trial using data from A Very Early Rehabilitation Trial (AVERT). Design AVERT is a prospective, parallel group, assessor-blinded randomised clinical trial. This paper presents data assessing the generalisability of AVERT. Setting Acute stroke units at 44 hospitals in 8 countries. Participants The first 20 000 patients screened for AVERT, of whom 1158 were recruited and randomised. Model We use the Proximal Similarity Model, which considers the person, place, and setting and practice, as a framework for considering generalisability. As well as comparing the recruited patients with the target population, we also performed an exploratory analysis of the demographic, clinical, site and process factors associated with recruitment. Results The demographics and stroke characteristics of the included patients in the trial were broadly similar to population-based norms, with the exception that AVERT had a greater proportion of men. The most common reason for non-recruitment was late arrival to hospital (ie, 24 h). Overall, being older and female reduced the odds of recruitment to the trial. More women than men were excluded for most of the reasons, including refusal. The odds of exclusion due to early deterioration were particularly high for those with severe stroke (OR=10.4, p<0.001, 95% CI 9.27 to 11.65). Conclusions A model which explores person, place, and setting and practice factors can provide important information about the external validity of a trial, and could be applied to other clinical trials

Minimal residual disease and outcome characteristics in infant KMT2A-germline acute lymphoblastic leukaemia treated on the Interfant-06 protocol

Background: The outcome of infants with KMT2A-germline acute lymphoblastic leukaemia (ALL) is superior to that of infants with KMT2A-rearranged ALL but has been inferior to non-infant ALL patients. Here, we describe the outcome and prognostic factors for 167 infants with KMT2A-germline ALL enrolled in the Interfant-06 study. Methods: Univariate analysis on prognostic factors (age, white blood cell count at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI; n = 163). Bone marrow minimal residual disease (MRD) was measured in 73 patients by real-time quantitative polymerase chain reaction at various time points (EOI, n = 68; end of consolidation, n = 56; and before OCTADAD, n = 57). MRD results were classified as negative, intermediate (<5∗10−4), and high (≥5∗10−4). Results: The 6-year event-free and overall survival was 73.9% (standard error [SE] = 3.6) and 87.2% (SE = 2.7). Relapses occurred early, within 36 months from diagnosis in 28 of 31 (90%) infants. Treatment-related mortality was 3.6%. Age <6 months was a favourable prognostic factor with a 6-year disease-free survival (DFS) of 91% (SE = 9.0) compared with 71.7% (SE = 4.2) in infants >6 months of age (P = 0.04). Patients with high EOI MRD ≥5 × 10−4 had a worse outcome (6-year DFS 61.4% [SE = 12.4], n = 16), compared with patients with undetectable EOI MRD (6-year DFS 87.9% [SE = 6.6], n = 28) or intermediate EOI MRD <5 × 10−4 (6-year DFS 76.4% [SE = 11.3], n = 24; P = 0.02). Conclusion: We conclude that young age at diagnosis and low EOI MRD seem favourable prognostic factors in infants with KMT2A-germline ALL and should be considered for risk stratification in future clinical trials

Minimal residual disease and outcome characteristics in infant KMT2A-germline acute lymphoblastic leukaemia treated on the Interfant-06 protocol

Background: The outcome of infants with KMT2A-germline acute lymphoblastic leukaemia (ALL) is superior to that of infants with KMT2A-rearranged ALL but has been inferior to non-infant ALL patients. Here, we describe the outcome and prognostic factors for 167 infants with KMT2A-germline ALL enrolled in the Interfant-06 study. Methods: Univariate analysis on prognostic factors (age, white blood cell count at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI; n = 163). Bone marrow minimal residual disease (MRD) was measured in 73 patients by real-time quantitative polymerase chain reaction at various time points (EOI, n = 68; end of consolidation, n = 56; and before OCTADAD, n = 57). MRD results were classified as negative, intermediate (6 months of age (P = 0.04). Patients with high EOI MRD ≥5 × 10−4 had a worse outcome (6-year DFS 61.4% [SE = 12.4], n = 16), compared with patients with undetectable EOI MRD (6-year DFS 87.9% [SE = 6.6], n = 28) or intermediate EOI MRD <5 × 10−4 (6-year DFS 76.4% [SE = 11.3], n = 24; P = 0.02). Conclusion: We conclude that young age at diagnosis and low EOI MRD seem favourable prognostic factors in infants with KMT2A-germline ALL and should be considered for risk stratification in future clinical trials

Clinical Implications of Minimal Residual Disease Detection in Infants With KMT2A-Rearranged Acute Lymphoblastic Leukemia Treated on the Interfant-06 Protocol

PURPOSE: Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide). MATERIALS AND METHODS: MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10-4), and high (≥ 5 × 10-4). RESULTS: EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively (P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively (P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively (P < .0001), while for myeloid-style-treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9). CONCLUSION: This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol