Nada Jabado's research while affiliated with SickKids and other places

Pediatric low-grade gliomas (pLGG) are the most common brain tumors in children and are associated with significant morbidity. Therefore, there is an urgent need for novel therapeutic strategies. While some studies have described pLGG’s tumor microenvironment (TME) from bulk RNAseq and scRNAseq, little is known about the spatial architecture of pLGG and its association with clinico-molecular features. Our study utilized imaging mass cytometry and a panel of 35 metal-labelled antibodies to unravel the spatial organization of key TME cell populations in 120 primary pLGG samples from the LOGGIC Core BioClinical DataBank. Cellular neighborhood analysis was performed to map the spatial organization of pLGG TME. Several clinic-molecular features (entity, tumor location, genetic driver alteration, disease progression status, age and sex) were used to measure their putative association with the enrichment of key cell populations and cellular structures to identify diagnosis and prognosis markers. Here, we identified a predominant presence of myeloid cells in the TME, particularly notable in optic pathway tumors, which exhibited unique immune profiles. Optic pathway tumors demonstrated elevated immune subsets, delineating distinct architectural features in the TME of this brain region. Spatial analysis defined cellular neighborhoods and specific interactions thereof, including myeloid interaction and macrophage-abundant regions. Clinically, these myeloid cell populations, associated with an increased expression of the immune checkpoint protein TIM3, suggesting the presence of an immuno-suppressive environment, were associated with inferior survival outcomes. Importantly, we identified an immunophenotype signature based on the presence of 4 myeloid cell populations significantly associated with progression free survival in our cohort. Our study underscored the need for accurate identification of immune cell populations influencing tumor progression, offering valuable insights for the identification of prognostic markers, and for the development of effective therapeutic strategies, such as immune checkpoint inhibitors, for the treatment of pLGG.

BACKGROUND Embryonal tumors with PLAGL1 and PLAGL2 amplification (ET, PLAGL) display substantial clinical heterogeneity regarding applied treatment and outcomes. As a recently-defined entity, the spectrum of tumor-driving PLAG-family alterations is not yet fully elucidated. METHODS We analyzed clinical and MRI data from patients with ET, PLAGL. Second, we identified and analyzed tumors with epigenetic similarities. RESULTS 18 patients with ET, PLAGL revealed diverse clinical behavior. Patients with PLAGL1-amplified tumors (ET, PLAGL1) were older (median 8 years), had a higher rate of complete resections (6/9) and fewer relapses (3/9). Patients with PLAGL2-amplified tumors (ET, PLAGL2) were younger (median 1.9 years), often incompletely resected (6/9) and prone to earlier relapse/progression. Five-year PFS was 89% and 17% (ET, PLAGL1/ET, PLAGL2), with no predictive value of initial surgical extent on PFS/OS. Postoperative treatment included chemotherapy (17/18, various protocols) alone (n=8) or combined with RT (n=9). The three survivors with ET, PLAGL2 underwent induction and high-dose chemotherapy. All five patients with less intensive chemotherapy relapsed. Most first and all subsequent ET, PLAGL2 relapses were distant, questioning the value of initial local radiotherapy. Two ET, PLAGL1 relapses occurred >8 years after diagnosis (one after, one without previous RT). Through methylation-based t-SNE we identified 143 tumors with epigenetic similarities to ET, PLAGL. Filtering revealed a core set of 27 tumors, of which seven displayed evidence of PLAG1-fusion events and one PLAG1-amplification. RNAseq analysis (n=4) revealed distinct gene fusions involving PLAG1, with similar genes upregulated (RET, CYP2W1 and imprinted genes) as in ET, PLAGL. Like ET, PLAGL, PLAG1-fused tumors, occurred in young children with different diagnoses and localizations. CONCLUSION PLAG1-fused tumors are epigenetically similar to ET, PLAGL, show similarities in gene expression, and need to be differentiated from neuroepithelial tumor with PLAGL1-fusion. Future investigations will examine differences in clinical behavior and the utility of PLAG1/L1/L2 IHC for diagnosis.

BACKGROUND: Tovorafenib is an investigational, selective, CNS-penetrant, type II RAF inhibitor. The ongoing FIREFLY-1 (NCT04775485) phase 2 study (Kilburn LK, et al. Nat Med. 2023) of tovorafenib in BRAF-altered pLGG resulted in antitumor activity and manageable safety. Decreased growth velocity (GV) was observed; this is an update on GV changes in skeletally immature children receiving tovorafenib. Methods A planned safety analysis was completed on August 8, 2023 on 137 patients (Arm 1: 77 & Arm 2: 60). Additional follow-up on all cases of decreased GV (an AESI) reported to the global safety database (GSDB) as of January 19, 2024 is provided. Results Overall, 29% had decreased GV from baseline (BL); 19% had ≥50% decrease. Of the 40 with this AESI, 75% had pre-existing neuromuscular or endocrine comorbidities potentially affecting normal growth, including 6 on GnRH-analogues for precocious puberty and 9 with BL heights 2 SDs above/below average for age and sex. Nineteen had on-treatment bone age assessments; none showed bone age advancement from BL or premature growth plate closure. No osteopenia or abnormal fractures reported. All 10 who discontinued or interrupted tovorafenib for ≥3 months for any reason (mean follow-up: 5.8 months), with off-treatment growth measurements available, showed post-treatment annualized GV (AGV) recovery (average AGV: on-treatment, 1.1 cm/y; off-treatment, 8 cm/y), with some exceeding expected average AGV for age. A 4-year-old boy with 1.2 cm/y AGV on-treatment had 12.3 cm/y AGV off-treatment (follow-up: 2 months). Five additional events of decreased GV in patients not on study FIREFLY-1 were reported to the GSDB; 4 of 5 had ≥3 months of off-treatment follow-up, all 4 recovered GV. Conclusions Decreased GV has been observed in patients on tovorafenib. Preliminary follow-up data in those who interrupted treatment show consistent evidence of GV recovery and preservation of growth potential on bone age studies.

BACKGROUND Diffuse hemispheric glioma, H3 G34-mutant (G34-DHG) is generally associated with very poor outcome. Little is known, however, regarding long-term survivors and associated prognostic factors. METHODS This retrospective, multicentre study was designed to investigate clinical, molecular, and imaging variables that may impact the survival of patients with G34-DHG. Patients of any age diagnosed with G34-DHG after January 1, 1995, with at least one measure of survival or progression were eligible. RESULTS 69 patients with G34-DHG (62 G34R, 5 G34V, 2 diagnosed by methylation profiling only) have been included to date. Median age at diagnosis was 16.0 years (range: 2–38). 75% of patients were under the age of 18. 52% of patients were male. 8% of patients had multifocal/disseminated disease at diagnosis. Upfront therapy included gross/near total resection in 47% of patients, radiation therapy in 88% (89% focal, 6% CSI), and maintenance chemotherapy in 85% (62% TMZ-based, 33% TMZ/CCNU, 5% non-TMZ). Median follow-up time was 20.0 months (IQR=12.0–30.0). 86% of patients had progressive disease with a median time-to-progression of 14.0 months (IQR=6.6-22.5). At last follow-up, 70% of patients had died from disease, 23% were alive with disease, and 7% were alive without disease. Median overall survival was 24.7 months (IQR=18.8–30.6). Median time from progression to death was 6.0 months (IQR=2.0–10.8). Six patients were found to be long-term survivors > 5 years (four dead of disease, one alive with disease, one alive without disease). Log-rank assessment revealed improved survival to be associated with upfront gross/near-total resection (p=0.002) and age at diagnosis ≥ 18 years (p=0.012). CONCLUSIONS Surprisingly, we found that nearly 10% of G34-DHG patients survive 5 years or longer. Radical surgical resection and older age at diagnosis appear to be associated with longer survival. Imaging and molecular prognostic factors are being further investigated.

BACKGROUND Metastatic infant medulloblastoma (MB) is a major challenge given the devastating long-term effects of craniospinal irradiation to the developing brain. However large cohorts of metastatic infant medulloblastoma have not been analysed to date. METHODS A multicentre molecularly informed international cohort of children <6 years old with metastatic medulloblastoma was assembled and analysed. RESULTS Eighty-four patients with a median age of 2.95 (0.66-6.58) years were identified. 48.8% were Group3-MB, 23.8% Group4-MB and 27.4% SHH-MB. 78% of Group3-MB were subtype II (40.4%) and IV (38.1%), 50% of Group 4 were subtype VII, and within SHH, SHHß and SHHγ were 52.2% and 43.5% respectively. 28.5% of all Group3-MB harboured MYC amplification. A multivariable analysis incorporating subgroup, MYC amplification and upfront craniospinal irradiation revealed that survival was superior for SHH patients compared to Group 3 irrespective of MYC status (Group3-veMYCamp: HR 3.05 95%-CI 1.05-8.87 p=0.04, Group3+veMYCamp HR 7.87 95%-CI 2.49-24.87 p=0.00044). Group 4 had a trend to a poor outcome (HR 2.63 95%-CI 0.82-8.39 p=0.1). SHH subjects had a superior outcome with 5-year PFS of 0.657 (95%-CI 0.47-0.92), without a significant difference between SHHß and SHHγ. High-dose chemotherapy regimens portended to a superior outcome for both subtypes of SHH-MB. 5-year PFS for non-radiated SHH-MB was 61% (95%-CI 40.8-91.4), compared to non-radiated Group3-MB (18.5%; 95%-CI 5.4-62.8) and Group4-MB (20%; 95%-CI 3.4-100). SHH-MB patients had more local than metastatic relapses compared to non-SHH-MB (p=0.01). CONCLUSIONS In the largest study dedicated to metastatic infant medulloblastoma assembled to date, outcomes for infants with Group 3- and 4-MB with metastatic disease treated with radiation sparing approaches is dismal despite intensified regimens. MYC amplification portends to a near uniformly fatal prognosis with current treatment irrespective of upfront radiotherapy. Novel radiation sparing approaches are urgently needed for this group. Infants with both metastatic SHHß and SHHγ benefit from intensified therapy.

BACKGROUND Pediatric low-grade gliomas (pLGGs) are the most frequent brain tumors in children and comprise a heterogeneous group of tumors with different locations, histologic subtypes, ages at presentation, and clinical behavior. Tumors frequently respond to treatment with chemotherapy or radiation therapy, but they can regrow after a period of quiescence, requiring further therapy. Thus, a deeper understanding of the molecular processes involved in these tumors is required to develop therapeutic strategies that are effective against their disease mechanisms. METHODS To better understand the cellular behaviors of this heterogenous group of tumors, we have employed single-cell and single-nuclei RNA sequencing technologies to analyze a large-scale dataset (>250,000 cells) of 55 pLGGs across many histological subtypes (pilocytic astrocytoma, pleomorphic xanthoastrocytoma, pilomyxoid astrocytoma, DNET, ganglioglioma, RGNT, diffuse astrocytoma, SEGA, glioneuronal, oligodendroglioma). RESULTS Analysis of this data identified a heterogenous population of cell types and cell states, detecting mature and progenitor-like astrocytes and oligodendrocytes, as well as cells exhibiting senescence or cycling programs. Moreover, we identify a significant immune infiltrate, comprised primarily of microglia. In addition to heterogeneity within pLGG tumors, heterogeneity between LGG subtypes represents another layer that stratifies pLGG biology. We performed a compositional analysis of the cell types present in these tumors and compared transcription signatures and gene expression programs across shared cellular populations of histologically and genetically distinct pLGGs. Finally, we used spatial transcriptomic data to investigate and annotate the cellular architecture of multiple pLGGs with BRAF mutations and draw comparisons to findings from our single-cell and single-nuclei transcriptomic analysis. CONCLUSIONS Our analysis of human pLGGs at the single-cell and single-nuclei resolution provides critical insight into the heterogenous biological activities that constitute these tumors.

BACKGROUND Pediatric patients with Diffuse Midline Gliomas (DMG), H3K27M altered have a dismal prognosis and novel therapeutic approaches are urgently needed. Factors that drive development of pediatric DMG are unknown. METHODS To determine the prevalence of germline pathogenic/likely pathogenic variants (P/LPV) in DMG, we assembled an international cohort of 252 patients with germline whole genome or whole exome sequencing data, including diffuse intrinsic pontine glioma (DIPG; n=153), from Australian, European and North American centres. RESULTS We identified germline P/LPV in cancer predisposition genes in 7.5% (19/252) of patients, mainly homologous recombination (n=9; BRCA1, BRCA2, PALB2) and Fanconi anemia genes (n=4). Germline P/LPV in mismatch repair genes (MSH2, PMS2) were found in two patients. Two patients each had two separate germline P/LPV. The prevalence of germline P/LPV was not significantly different according to age, location of DMG nor H3K27M mutational status. Furthermore, tumor profiles differed, with absence of somatic drivers in the PI3K/mTOR pathway in patients with germline P/LPV compared to those without (P = 0.023). Knockdown of BRCA1 in DMG cell cultures sensitized tumor cells to PARP inhibition. Reflecting the potential therapeutic relevance of these findings, we describe one H3.3 K27M-mutant DMG patient with a pathogenic germline BRCA2 and FANCE variant and multiple recurrences, who was treated with a PARP inhibitor (olaparib) and immune checkpoint inhibitor, leading to a near complete radiological response after 4 months. CONCLUSION Our study is the largest series to date investigating germline P/LPV in cancer predisposition genes in DMG and provides new therapeutic insights. It is expected that these germline findings will also guide cascade testing for a proband’s relatives. Our data highlight the importance of germline testing in H3K27-altered DMG patients at diagnosis.

Background. Cancer is the leading cause of disease-related death among children of more than 1 year of age. However, childhood cancer risk factors and etiology are yet to be fully understood. The goal of this study is to identify geographic variation among children and adolescents diagnosed with pediatric tumors between 2001 and 2018 in the province of Quebec. Methods. We analyzed pediatric patients less than 15 years of age from the Cancer in Young People in Canada (CYP-C) surveillance system who were diagnosed between 2001 and 2018 with cancer in the province of Quebec. The age-standardized age-adjusted incidence rates (AAIR) per 100,000 person years were calculated for all childhood cancers by cancer subgroups, Quebec Health regions, and age groups. Results. Overall, 3904 pediatric patients less than 15 years old were diagnosed with cancer in the province of Quebec in 2001–2018. The overall incidence rate (IR) in the province of Quebec was 16.14 (95%CL [15.56–16.73]) per 100,000 person years. For childhood cancers, regions that presented a higher AAIR were Chaudière-Appalaches and Capitale-Nationale with 18.2 and 17.5 per 100,000 person years, respectively. The incidence rates (IRs) in Chaudière-Appalaches (95% CI 1.0439–1.3532) and in Capitale-Nationale (95% CI 1.0124–1.2942) were statistically higher than the incidence in the province of Quebec (p = 0.0090 and p = 0.0310, respectively). When comparing the AAIR of the CNS tumor subgroup in Chaudière-Appalaches and in Capitale-Nationale, with the provincial average, we noticed a statistically higher incidence in Chaudière-Appalaches and a trend for Capitale-Nationale (p < 0.0001 and p = 0.0602, respectively). Conclusion. There is evidence of spatial clusters in Chaudière-Appalaches and Capitale-Nationale as areas for all childhood cancers. Further studies should be performed to investigate potential risk factors in these regions.

Background Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain. Methods We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT. Results DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo. Conclusions Our results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

Diffuse intrinsic pontine gliomas are lethal tumors with a prognosis generally less than 1 year. Few cases of survivors of 5 years or more have been reported. This case report highlights the journey of a 9.5-year survivor who underwent 3 rounds of focal radiotherapy; she experienced 6 years of progression-free survival following the first round but ultimately succumbed to her disease. An autopsy revealed a favorable IDH1 mutation and the absence of H3K27M. This case reiterates the importance of extensive molecular analyses in diffuse intrinsic pontine gliomas and explores the potential benefit of re-irradiation in patients with positive responses and long periods of remission.