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Purpose: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Background Children ≤36 months with diffuse intrinsic pontine glioma (DIPG) have increased long-term survival (LTS, overall survival (OS) ≥24 months). Understanding distinguishing characteristics in this population is critical to improving outcomes. Methods Patients ≤36 months at diagnosis enrolled on the International DIPG Registry (IDIPGR) with central imaging confirmation were included. Presentation, clinical course, imaging, pathology and molecular findings were analyzed. Results Among 1183 patients in IDIPGR, 40 were eligible (median age: 29 months). Median OS was 15 months. Twelve patients (30%) were LTS, 3 (7.5%) very long-term survivors ≥5 years. Among 8 untreated patients, median OS was 2 months. Patients enrolled in the registry but excluded from our study by central radiology review or tissue diagnosis had median OS of 7 months. All but 1 LTS received radiation. Among 32 treated patients, 1-, 2-, 3-, and 5-year OS rates were 68.8%, 31.2%, 15.6% and 12.5%, respectively. LTS had longer duration of presenting symptoms (P = .018). No imaging features were predictive of outcome. Tissue and genomic data were available in 18 (45%) and 10 patients, respectively. Among 9 with known H3K27M status, 6 had a mutation. Conclusions Children ≤36 months demonstrated significantly more LTS, with an improved median OS of 15 months; 92% of LTS received radiation. Median OS in untreated children was 2 months, compared to 17 months for treated children. LTS had longer duration of symptoms. Excluded patients demonstrated a lower OS, contradicting the hypothesis that children ≤36 months with DIPG show improved outcomes due to misdiagnosis.

Supplementary Figure 3: PPP1CB-ALK fusion is oncogenic and sensitive to targeted therapy. A. Immunoblot analysis of ALK downstream signaling proteins in cortical mNSC (CTX#6) and brainstem mNSC (BS#3) expressing PPP1CB-ALK. Cells were treated for 4 hours with ceritinib and lorlatinib at the indicated concentrations.B. Left panel: Soft agar colony-forming assay using stable mNSC PPP1CB-ALK demonstrated colony formation with dose-dependent inhibition by ceritinib. mNSC KRAS did not respond to ceritinib validating the drug specificity. Right panel: Quantification of colony formation under targeted drug inhibition using CellProfiler. Values represent colony counts relative to the untreated group ± s.d. The mean of three independent replicates is shown. Significance between treatments determined by the Mann-Whitney test. *P < 0.05, **P < 0.01.C. Cell viability of ceritinib-treated PPP1CB-ALK NIH-3T3 relative to eGFP-positive cells by CellTiterGlo.D. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 ceritinib-treated cells versus DMSO control.F. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.G. Pharmacodynamic analysis of NIH 3T3-PPP1CB-ALK s.c. tumors treated with vehicle or ceritinib at 30mg/kg for 5 days. Representative H&E stain, ALK, pSTAT3, pAKT S473 and Ki-67 IHC.H. Quantification of Ki-67-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.I. Quantification of pSTAT3-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.J. Tumor growth following subcutaneous implantation of PPP1CB-ALK and eGFP-expressing fibroblasts.K. Kaplan-Meier survival curves of SCID mice injected subcutaneously with mNSC CTX-PPP1CB-ALK (red, n = 5) or mNSC CTX-eGFP (black, n = 4) and treated with ceritinib at 30mg/kg/d for 15 days. Mice were euthanized at endpoint (tumor volume of 2000mm3). The grey area represents the treatment period.L. Vehicle or ceritinib-treated mouse weights at start and end of treatment.

Supplementary Figure 5: Single-cell RNA Sequencing and IHC examination of ALK in human neural development and immature neural cells. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (cortex-dev.cells.ucsc.edu) shows that ALK transcript appears to be enriched in a certain cell type. Color-coded by ALK gene expression (beige to red) and age in weeks (rainbow).B. ALK-stained sagittal section of normal human cerebellum at 15 postconceptional weeks (pcw).C. ALK-stained sagittal section of human fetal neural stem cell pellets at 13 postconceptional weeks (pcw).D. ALK-stained sagittal section of human fetal neural stem cell pellets at 15 postconceptional weeks (pcw).

Purpose:Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established.Experimental Design:We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations.Results:ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib.Conclusions:These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Supplementary Figure 6: Single cell RNA sequencing comparison of ALK expression and other infant/pediatric glioma kinase drivers in developing human and mouse brain. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (http://cells.ucsc.edu/?ds=cortex-dev) shows that ALK positive cells make up less than 1% of all cells in the dataset and aren't localized within a specific cluster.B. Cluster-level summary of the human scRNA dataset shows that ALK expression (TPM) is decreased compared to EGFR, NTRK2, and NTRK3, and ALK positive cells make up a much lower percentage of each cluster compared to those 3 genes.C. 2D representation of developmental mouse forebrain (E12.5 – P6) in a single cell RNA-seq atlas (http://cc-shiny-01.functionalgenomics.ca/braindex/clusters) shows similar results as seen in the human scRNA Nowakowski dataset.D. Cluster-level summary of the mouse scRNA dataset corroborates results seen in the Nowkowski scRNA dataset; that Alk expression (log-normalized) is decreased, and less prevalent than EGFR, NTRK2, and NTRK3.E. 2D representation of RNA sequencing data for NTRK1-3 genes in the Nowakowski developing human cortex dataset. Compared to ALK expression, the NTRK genes have increased expression levels, increased prevalence in the dataset, and show increased localization to specific cluster.F. Similar to (E), the 2D representation of Ntrk genes in the developing mouse scRNA dataset shows increased expression and prevalence compared to Alk.

Supplementary Figure 2: T1 post-contrast axial and sagittal images of ALK-fused and non ALK-altered GBMs and ALK staining in ALK-fused congenital GBMs. A. Example images from patients with ALK aberrations (76, 89, 116, 197)B. In contrast to those without ALK aberrations (right columns). Contrast is gadolinium for those where applicable and show enhancement within both the tumor categories.C. Representative H&E and ALK IHCs of congenital GBM harboring an ALK fusion.

Supplementary Figure 4: Characterization of the lung cancer recurrent DCTN1-ALK fusion identified in cGBM (case ALK.232). A. Integrative Genomic Viewer (IGV) screenshot of the DCTN1-ALK variant.B. Predicted sequence of DCTN1-ALK fusion (DCTN1 exon 1-26, ALK exon 20-29 conserved).C. PDX DCTN1-ALK sub-renal capsule engraftment confimed by IHC.C. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 ceritinib-treated cells versus DMSO control.D. Relative viability of BT164 and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.F. PDX DCTN1-ALK sub-renal capsule engraftment confirmed by IHC.

Supplementary Figure 1: Characterization of the novel LRRFIP1-ALK fusion (case ALK.223) A. Visualization of the read coverage at the fusion sites in ALK and LRRFIP1 genes using Integrative Genomic Viewer.B. Pile-up plot generated by SvABA using whole-genome sequencing data shows the breakpoint coordinates.C. Copy number ratio profile by WGS showing no somatic copy number alterations anywhere in the genome. The x-axis represents the chromosomes.D. Copy number ratio profile on chromosome 2 showing no copy number change of ALK and LRRFIP1 genes.E. Two RT-PCR products of 186bp (variant 1) and 189bp (variant 3) were detected by agarose gel electrophoresis with total RNA from tumor specimen ALK.223.F. Primer sequences of ALK-LRRFIP1 (variants 1 and 2) and LRRFIP1-ALK (variant 3) and the predicted DNA product size.G. Western Blot analysis of ALK signaling pathway in subcutaneous tumors treated with vehicle or lorlatinib. Normal brain was used as control.H. Representative 40x images and CellProfiler quantification of Cleaved Caspase 3- and Ki-67- stained subcutaneous tumors.

Additional information tables 1 to 8.

Purpose Pure germinomas account for 40% of pineal tumors and are characterized by the lack of appreciable tumor markers, thus requiring a tumor biopsy for diagnosis. MicroRNAs (miRNA) have emerged as potential non-invasive biomarkers for germ cell tumors and may facilitate the non-invasive diagnosis of pure pineal germinomas. Methods A retrospective chart review was performed of all patients treated at Children’s Cancer Hospital Egypt diagnosed with a pineal region tumor between June 2013 and March 2021 for whom a research blood sample was available. Plasma samples were profiled for miRNA expression and DESeq2 was used to compare between pure germinoma and other tumor types. Differentially expressed miRNAs were identified. The area under the curve of the receiver operating characteristic curve was constructed to evaluate diagnostic performance. Results Samples from 39 pediatric patients were available including 12 pure germinomas and 27 pineal region tumors of other pathologies, including pineal origin tumors [n = 17; pineoblastoma (n = 13), and pineal parenchymal tumors of intermediate differentiation (n = 4)], and others [n = 10; low grade glioma (n = 6) and atypical teratoid rhabdoid tumor (n = 4)]. Using an adjusted p value p = 0.0058, p = 0.0478 and p = 0.0366, respectively), and good discriminatory power between the two groups (AUC 90.7%, p

PURPOSE For decades, academic clinical trials consortia have collaborated to optimize outcomes for childhood cancers through evaluating incremental improvements in conventional mutimodality treatment regimes. There are now increasing opportunities to partner with industry to test new medicines in academic-sponsored trials, but these collaborative studies rarely contribute to marketing authorizations. We addressed why this is the case and sought solutions to enable academic-sponsored trials to directly contribute to the licensing of new medicines. METHODS Under the auspices of the multistakeholder platform ACCELERATE, we convened a working group of representatives from clinical academia, pharmaceutical industry, European Medicines Agency, US Food and Drug Administration, and patient advocacy to define the challenges and propose recommendations to facilitate academic-sponsored trial design and conduct to be aligned to both the needs of the pharmaceutical company who own the asset and the expectations of the regulatory (licensing) authorities. RESULTS We identified that although academic consortia have long-standing expertise to conduct robust clinical trials, there were critical gaps in knowledge, standard procedures, and resources that hindered the trial data directly contributing to marketing authorization applications. We propose a suite of recommendations focused on (1) essential documents, (2) essential data, (3) data management, and (4) trial resources, specifically aimed at enabling academic-industry partnerships to deliver an academic-sponsored trial that meets the requirements for a marketing authorization submission. These recommendations pivot around transparency in academic-industry partnerships and early engagement with regulators. CONCLUSION Academic sponsors and industry partners need to prospectively recognize when the planned collaborative trial could contribute to an application to marketing authorization and plan accordingly. Transparent collaboration and knowledge sharing between the partners opens an important pathway for accelerating new treatments into clinical practice for children with cancer.

Purpose: Anaplastic Lymphoma Kinase (ALK) aberrations have been identified in pediatric type infant gliomas, but their occurrence across age groups, functional effects, and treatment response have not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly-generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs, and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages and no gross effects on perinatal brain development was seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support expanded evaluation of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. Citation Format: Anne-Florence Blandin, Ross Giglio, Maya Srikanth Graham, Guadalupe Garcia, Seth Malinowski, Jared K. Woods, Shakti Ramkissoon, Lori Ramkissoon, Frank Dubois, Kate Schoolcraft, Jessica W. Tsai, Dayle K. Wang, Robert Jones, Jayne Vogelzang, Kristine Pelton, Sarah Becker, Fiona Watkinson, Claire Sinai, Elizabeth Cohen, Matthew Booker, Michael Tolstorukov, Veerle Haemels, Liliana Goumnerova, Karen Wright, Mark Kieran, Katie Fehnel, David Reardon, Arnault Tauziede-Espariat, Rishi Lulla, Benjamin Carcamo, Stanley Chaleff, Alain Charest, Frederik De Smet, Azra H. Ligon, Adrian Dubuc, Melanie Pagès, Pascale Varlet, Patrick Wen, Brian Alexander, Susan Chi, Sanda Alexandrescu, Ralf Kittler, Robert Bachoo, Rameen Beroukhim, Pratiti Bandopadhayay, Keith L. Ligon. ALK amplification and rearrangements are recurrent targetable events in congenital and adult glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1201.

Rapid evaluation and subsequent regulatory approval of new drugs are critical to improving survival and reducing long-term side-effects for children and adolescents with cancer. The international multi-stakeholder organisation ACCELERATE was created to advance the timely investigation of new anti-cancer drugs. ACCELERATE has enhanced communication and understanding between academia, industry, patient advocates and regulators. It has promoted a mechanism-of-action driven drug development approach and developed Paediatric Strategy Forums. These initiatives have facilitated prioritisation of medicinal products and a focused and sequential strategy for drug development where there are multiple potential agents. ACCELERATE has championed the early assessment of promising drugs in adolescents through their inclusion in adult early phase trials. ACCELERATE has strongly supported alignment between the European Medicines Agency and the US Food and Drug Administration and identification of unmet medical needs through multi-stakeholder collaboration. Early engagement between all stakeholders in the development of new drugs is critical. Innovative clinical trial designs are required, necessitating early discussion with sponsors and regulators. Amplifying the patient advocate voice through inclusion across the drug development continuum will lead to better, patient-centric trials. By these means, children and adolescents with cancer can maximally and rapidly benefit from innovative products to improve outcomes and reduce burdensome sequelae.(c) 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

BACKGROUND RAS/RAF/MEK/ERK pathway activation is the primary driver for most pediatric low-grade gliomas (pLGG). MEK162 (binimetinib) is an orally bioavailable MEK1/2 inhibitor with superior brain penetration in a preclinical model. The primary objective of this multi-institutional phase II and target validation study was to assess stratum-specific efficacy of binimetinib in progressive pLGG. METHODS Eligible children aged 1-18 years with previously treated radiographically progressive pLGG were enrolled and treated with binimetinib, starting dose 32mg/m2/dose twice daily. Stratum 1 included patients with pLGG with documented BRAF fusion; stratum 2, neurofibromatosis 1 (NF1)-associated pLGG; stratum 3, sporadic pLGG without documented BRAF fusion; and stratum 4, patients undergoing planned tumor biopsy who began binimetinib preoperatively. Partial and minor responses (PR and MR) were defined as ≥50% and ≥25% decrease in maximal two-dimensional measurements. RESULTS Of 86 patients enrolled, 85 were evaluable for response. Of these, 48 (56%) showed a radiographic response (30 PR and 18 MR) in the first year of treatment. Response rate for stratum 1 (n=28) was 50% (12 PR and 2 MR); 12 (43%) had stable disease (SD) and 2 (7%) progressive disease (PD). Stratum 2 (n=21) response rate was 43% (5 PR, 4 MR), with 12 (57%) SD and no PD. Stratum 3 (n=29) response rate was 69% (10 PR, 10 MR), 4 (14%) SD and 5 (17%) PD. Stratum 4 (n=7) include 3 PR, 2 MR, 2 SD. Nineteen (22%) discontinued treatment for toxicity (most commonly dermatologic), and an additional 42 (49%) required dose reduction. Median dose at the time of PR/MR was 28mg/m2; responses were seen at doses as low 16mg/m2. CONCLUSION Binimetinib is highly effective in the treatment of both NF1-associated and sporadic pLGG, with or without documented BRAF fusion. Modified dosing strategies to improve tolerability may be considered in future trials.

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.

The fifth multistakeholder Paediatric Strategy Forum focussed on epigenetic modifier therapies for children and adolescents with cancer. As most mutations in paediatric malignancies influence chromatin-associated proteins or transcription and paediatric cancers are driven by developmental gene expression programs, targeting epigenetic mechanisms is predicted to be a very important therapeutic approach in paediatric cancer. The Research to Accelerate Cures and Equity (RACE) for Children Act FDARA amendments to section 505B of the FD&C Act was implemented in August 2020, and as there are many epigenetic targets on the FDA Paediatric Molecular Targets List, clinical evaluation of epigenetic modifiers in paediatric cancers should be considered early in drug development. Companies are also required to submit to the EMA paediatric investigation plans aiming to ensure that the necessary data to support the authorisation of a medicine for children in EU are of high quality and ethically researched. The specific aims of the forum were i) to identify epigenetic targets or mechanisms of action associated with epigenetic modification relevant to paediatric cancers and ii) to define the landscape for paediatric drug development of epigenetic modifier therapies. DNA methyltransferase inhibitors/hypomethylating agents and histone deacetylase inhibitors were largely excluded from discussion as the aim was to discuss those targets for which therapeutic agents are currently in early paediatric and adult development. Epigenetics is an evolving field and could be highly relevant to many paediatric cancers; the biology is multifaceted and new targets are frequently emerging. Targeting epigenetic mechanisms in paediatric malignancy has in most circumstances yet to reach or extend beyond clinical proof of concept, as many targets do not yet have available investigational drugs developed. Eight classes of medicinal products were discussed and prioritised based on the existing level of science to support early evaluation in children: inhibitors of menin, DOT1L, EZH2, EED, BET, PRMT5 and LSD1 and a retinoic acid receptor alpha agonist. Menin inhibitors should be moved rapidly into paediatric development, in view of their biological rationale, strong preclinical activity and ability to fulfil an unmet clinical need. A combination approach is critical for successful utilisation of any epigenetic modifiers (e.g. EZH2 and EED) and exploration of the optimum combination(s) should be supported by preclinical research and, where possible, molecular biomarker validation in advance of clinical translation. A follow-up multistakeholder meeting focussing on BET inhibitors will be held to define how to prioritise the multiple compounds in clinical development that could be evaluated in children with cancer. As epigenetic modifiers are relatively early in development in paediatrics, there is a clear opportunity to shape the landscape of therapies targeting the epigenome in order that efficient and optimum plans for their evaluation in children and adolescents are developed in a timely manner. (C) 2020 The Authors. Published by Elsevier Ltd.

While human tumors are shaped by the genetic evolution of cancer cells, evidence also suggests that they display superimposed hierarchies reminiscent of normal development. Yet, relating the genetic and hierarchical models of cancer organization has been limited by technical challenges. Here, studying H3 K27M-mutant diffuse midline gliomas we profiled thousands of single cells from fresh tumors by RNA-seq and reconstructed the genetic and epigenetic cellular architecture of tumors in patients. We identified a subset of cancer cells that are differentiated along specialized glial programs while a distinct subset expresses oligodendrocyte precursor expression programs. By identifying distinct genetic clones, we show that these cellular architectures evolve during the progression of the tumors, suggesting that developmental programs and genetic influences coordinately determine tumor architecture. These results provide unprecedented insight into the cellular composition of midline brain tumors at single-cell resolution and may help harmonize the cancer stem cell and the genetic models of cancer, with critical implications for disease management.

There is little known regarding the immune infiltrate present in pediatric brain tumors and how this compares to what is known about histologically similar adult tumors and its correlation with survival. Here, we provide a descriptive analysis of the immune infiltrate of 22 fresh pediatric brain tumor tissue samples of mixed diagnoses and 40 peripheral blood samples. Samples were analyzed using a flow cytometry panel containing markers for immune cell subtypes, costimulatory markers, inhibitory signals, and markers of activation. This was compared to the standard method of immunohistochemistry (IHC) for immune markers for 89 primary pediatric brain tumors. Both flow cytometry and IHC data did not correlate with the grade of tumor and IHC data was not significantly associated with survival for either low grade or high grade gliomas. There is a trend towards a more immunosuppressive phenotype in higher grade tumors including more regulatory T cells and B cells. Both PD1 and PDL1 were present in only a small percentage of the tumor infiltrate. Elisa assays for NKG2D soluble ligands was also negative for all tumor types. T cell receptor sequencing revealed up to 10% clonality of T cells in tumor infiltrates and no significant difference in clonality between low and high grade gliomas. We have shown the immune infiltrate of pediatric brain tumors does not appear to correlate with grade or survival for a small sample of patients. Further research and larger studies are needed to fully understand the interaction of pediatric brain tumors and the immune system.

INTRODUCTION: Patients ≥10 years of age with a DIPG are reported to have a higher rate of long-term survival (≥24 months). The purpose of this study was to compare clinical, radiological, pathological and molecular characteristics between patients ≥10 years old with DIPG who were long-term survivors (LTS) vs short-term survivors (STS) and assess biological differences in this age group compared to patients

Introduction: Intraluminal pulmonary vein stenosis (PVS) can occur in isolation, with congenital heart disease (CHD), chronic lung disease, or both. PVS can progress rapidly with vessel atresia, pulmonary hypertension, and RV failure, and can be fatal within months (mos) of diagnosis. Previous research has demonstrated that intraluminal obstruction occurs due to neointimal proliferation of myofibroblast-like cells, which express VEGF and PDGFR. Hypothesis: Adjunct targeted inhibition of VEGF (bevacizumab) and PDGFR (imatinib mesylate) may reduce disease activity in patients (pts) with multivessel intraluminal PVS and prolong survival. Methods: This is a single-arm, prospective, open-label FDA approved study using bevacizumab and imatinib mesylate to treat progressive multivessel intraluminal PVS. Pts with obstruction of ≥ 2 veins (echocardiographic (Echo) mean gradient ≥ 4 mmHg) were included after aggressive surgical or catheter based relief of obstruction. All but 3 pts had ≥ 1 PVS surgery prior to initiation. Pts with CHD received imatinib mesylate only, those without CHD or who progressed on study also received bevacizumab. Follow-up included lung scan and Echo q 4-8 weeks (wks); CT scan or angiography q 24 wks. We report initial (31 of 44 enrolled pts) who have reached 48 wks or study endpoint. Results: Among 31 pts, 17 were treated for 48 wks; 3 transplanted; 11 discontinued drug < 48 wks. Mean age at diagnosis was 4 mos; drug initiation 9 mos. Two had isolated disease, 16 CHD, 3 lung disease, 10 both. Twenty-four pts received imatinib mesylate only; 7 both drugs. Median number of pulmonary veins involved was 4 (range 2-5); 12 pts had ≥ 1 atretic vessel at initiation. Pts received mean of 85% planned doses of drug. At 48 wks, 26/31 (84%) were alive, 2 post-transplant. Eight of 31 (26%) stabilized, 6/31 (19%) did not require any subsequent pulmonary vein intervention. Conclusions: Multi-modal treatment including aggressive surgical and catheter based intervention and targeted inhibition of myofibroblast proliferation resulted in a 48 wk survival of 84% among pts with multivessel intraluminal PVS. These results suggest that targeted inhibition may play an important role in managing aggressive pulmonary vein disease.

Maintenance biology-based therapy following HDCT/AHSCR in pediatric brain tumors has not been tested as yet. Failure of the HDCT/AHSCR might be due to tumor-immunity dysregulation, reactivation of the angiogenic switch and other mechanisms. Angiogenesis has been shown to be reactivated following chemotherapy. The angiogenic factors engaged in this process in childhood brain tumors following HDCT/AHSCR have not been tested in the clinic. Metronomic chemotherapy has been found to be safe and angiogenesis inhibitors are currently tested in children. Other good possible candidates for clinical trials in this setting include retinoic acid and immunotherapy.

The phenomenon of "homing" is discussed with respect to patterns of lymphocyte circulation and the molecules on the surface of both endothelium and lymphocytes that mediate this process. In addition, the data are analysed in the context of a model for lymphocyte homing.