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PURPOSE Constitutional mismatch repair deficiency syndrome (CMMRD) is a lethal cancer predisposition syndrome characterized by early-onset synchronous and metachronous multiorgan tumors. We designed a surveillance protocol for early tumor detection in these individuals. PATIENTS AND METHODS Data were collected from patients with confirmed CMMRD who were registered in the International Replication Repair Deficiency Consortium. Tumor spectrum, efficacy of the surveillance protocol, and malignant transformation of low-grade lesions were examined for the entire cohort. Survival outcomes were analyzed for patients followed prospectively from the time of surveillance implementation. RESULTS A total of 193 malignant tumors in 110 patients were identified. Median age of first cancer diagnosis was 9.2 years (range: 1.7-39.5 years). For patients undergoing surveillance, all GI and other solid tumors, and 75% of brain cancers were detected asymptomatically. By contrast, only 16% of hematologic malignancies were detected asymptomatically ( P < .001). Eighty-nine patients were followed prospectively and used for survival analysis. Five-year overall survival (OS) was 90% (95% CI, 78.6 to 100) and 50% (95% CI, 39.2 to 63.7) when cancer was detected asymptomatically and symptomatically, respectively ( P = .001). Patient outcome measured by adherence to the surveillance protocol revealed 4-year OS of 79% (95% CI, 54.8 to 90.9) for patients undergoing full surveillance, 55% (95% CI, 28.5 to 74.5) for partial surveillance, and 15% (95% CI, 5.2 to 28.8) for those not under surveillance ( P < .0001). Of the 64 low-grade tumors detected, the cumulative likelihood of transformation from low-to high-grade was 81% for GI cancers within 8 years and 100% for gliomas in 6 years. CONCLUSION Surveillance and early cancer detection are associated with improved OS for individuals with CMMRD.

Background/Aim: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. Patient and Methods: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. Results: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3’ end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. Conclusion: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

Background The elapsed time taken to diagnose tumors of the central nervous system in children and adolescents varies widely. The aim of the present study was to investigate such diagnostic time intervals at a national level in Sweden as they correlate with clinical features. Methods Data prospectively accumulated over a 4-year period in the Swedish Childhood Cancer Registry from patients aged 0-18 years were pooled, and diagnostic time intervals were analyzed considering tumor location, tumor type, patient age and sex, initial symptoms, and clinical timelines. All six pediatric oncology centers in Sweden contributed to collection of data. Time points for calculating the total diagnostic interval (TDI) defined as the time from symptom onset to diagnosis were reported in 257 of 319 patients (81%). Results The time from symptom onset to the first healthcare consultation, median 2.6 weeks, did not vary significantly between patients categorized according to tumor type or location. The median TDI was 8.3 weeks for the 4-year study period. Patients with optic pathway glioma (TDI 26.6 weeks), those with tumors of the spinal cord (TDI 25.9 weeks), and those with midline tumors (TDI 24.6 weeks) had the longest lead times. Additionally, older age, too few initial symptoms, and seeking initial redress outside an emergency ward were factors associated with a longer time to diagnosis. Conclusion This study identified several factors associated with delayed diagnosis of central nervous system tumors among Swedish children and adolescents. These novel data ought to help direct future efforts toward clinical improvement. Funding Agencies|Swedish Childhood Cancer Foundation

Aims: Paediatric brain tumours are rare, and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify central nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation-based classification to standard paediatric brain tumour diagnostics in an unselected cohort. Methods: All CNS tumours diagnosed in children (0-18 years) during 2017-2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared with the DNA methylation-based classification. For incongruent results, a blinded re-evaluation was performed by an experienced neuropathologist. Results: Two hundred forty tumours with a histopathology-based diagnosis were profiled. A high-confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed, and for some of these also refined, 6% were incongruent, and the re-evaluation favoured the methylation-based classification. In the remaining 3% of cases, the methylation class was non-contributory. The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients. Conclusions: Integrating DNA methylation-based tumour classification into routine clinical analysis improves diagnostics and provides molecular information that is important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information. Funding Agencies|Swedish government; ALF-agreement; Lions Cancer Research Fund of Western Sweden; Swedish Research Council; Swedish Cancer Society; Swedish Childhood Cancer Fund

BACKGROUND/AIM: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. PATIENT AND METHODS: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. RESULTS: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. CONCLUSION: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse. CC BY-NC-ND 4.0Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.Correspondence to: Frida AbelThis work was supported by the Swedish Cancer Society (www.cancerfonden.se, grant number 2018/825 to FA and grant number 2018/652 to JAN), the Swedish Children’s Cancer Foundation (www.barncancerfonden.se, grant number PR2017-0029 to FA, PR2019-0079 to KE, and KP2019-0010 to HC), and the ALF-agreement (www.researchweb.org/is/alfgbg, ALFGBG-716231 to FA, ALFGBG-965828 to HC, and ALFGBG-719301 to JAN).

AIMS: Paediatric brain tumours are rare and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify central nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation-based classification to standard paediatric brain tumour diagnostics in an unselected cohort. Methods: All CNS tumours diagnosed in children (0-18 years) during 2017-2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared to the DNA methylation-based classification. For incongruent results, a blinded re-evaluation was performed by an experienced neuropathologist. Results: 240 tumours with a histopathology-based diagnosis were profiled. A high-confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed and for some of these also refined, 6% were incongruent and the re-evaluation favoured the methylation-based classification. In the remaining 3% of cases, the methylation class was non-contributory or could not be predicted. The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients.Conclusions: Integrating DNA methylation-based tumour classification into routine clinical analysis improves diagnostics and molecular information important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information.

BACKGROUND: High-grade gliomas (HGG) with replication-repair deficiency (RRD) harbour high mutation burden (TMB) and are rapidly fatal following chemo-radiation approaches. Although hypermutation results in objective responses and prolonged survival in >30% of patients undergoing PD1-blockade, salvage following failure of PD1-inhibition remains a challenge. METHODS: We performed a real-world study of Ipilimumab (anti-CTLA4) in combination with Nivolumab/Pembrolizumab for patients failing single-agent PD1-inhibition. RESULTS: Among 68 consortium patients with relapsed HGG treated with single-agent PD1-inhibitors, progression was observed in 43 (63%). Ipilimumab was added to 20/43 (46.5%), 14 (32.5%) received best supportive care (BSC), and 9 (21%) received miscellaneous therapies. For patients receiving CTLA4/PD1-inhibition, median age at progression was 12.3-years (IQR: 9; 15.6). Time from anti-PD1 initiation to progression was 8-months (IQR: 3.8; 18.5). Germline predisposition was observed in all patients (CMMRD: 70%, Lynch: 25%, polymerase-proofreading deficiency: 5%). All HGG were hypermutant (median TMB: 182 mutations/Mb; IQR: 15.6; 369.4). Centralized radiology review revealed objective responses in 3/20 (15%, all ultra-hypermutant: 320, 496, 834 mutations/Mb), stable disease in 5 (25%), and 12 (60%) eventually progressed (iRANO). Following failure of PD1-blockade, estimated progression-free and overall survival at 18-months for patients receiving CTLA4/PD1-inhibition were 11% and 25%, respectively. Importantly, survival was superior to patients receiving BSC (median OS 1-year on the anti-CTLA4/PD1combination (range:1-48 months). The combinational immunotherapy resulted in significant autoimmune toxicity in 11/20 (55%), warranting immunosuppressive therapy in all, and treatment abandonment in 6 patients. CONCLUSION: Combined CTLA4/PD1-blockade after failure of single-agent PD1-inhibition revealed objective responses and prolonged survival in an otherwise rapidly-fatal disease. This needs to be assessed in the context of significant autoimmunity, supporting the need for the current prospective trial (NCT04500548), and novel strategies to limit treatment-related toxicity.

BACKGROUND: The heterogeneous group of tumors, primitive neuro-ectodermal tumors of the central nervous system (CNS-PNETs), is a group of rare childhood embryonal tumors associated with a poor prognosis. In recent years, molecular analyzes have shown that CNS-PNETs consist of high-grade gliomas (HGG), ependymomas, different embryonal entities like atypical teratoid /rhabdoid tumors (AT/RT), CNS neuroblastoma FOXR2 and embryonal tumor with multi-layered rosettes (ETMR). Each of these tumor types is unusual and long-term clinical follow-up data are sparse. METHODS: We retrospectively re-evaluated all children (0-18 years old) diagnosed with a CNS-PNET in Sweden during 1984-2015. In total, 88 supratentorial CNS-PNETs were identified in the Swedish Childhood Cancer Registry and from these formalin-fixed paraffin-embedded tumor material was available for 69 patients. All tumors were reviewed histopathologically by an experienced neuropathologist and were analyzed using genome-wide DNA methylation profiling and classified by the MNP brain tumor classifier. RESULTS: The largest entities, after re-evaluation, were HGG (30%), CNS NB-FOXR2 (12%), AT/RT (10%) and ETMR (8%). Some tumors were difficult to classify and will be further evaluated molecularly. Some examples: Best treatment results were seen for patients with CNS-NB FOXR2 (5-year PFS: 100%) where all patients had received craniospinal radiotherapy (CSI). Patients with ETMR were all very young and survival data show early progression and poor survival (5-year OS 34%). CONCLUSIONS: Although the patient material is relatively small, it is population-based with long follow-up times. Our findings are in line with other studies and shows that CSI is important for cure for CNS-NB FOXR2 and that intensive multi-modal therapies needs to be evaluated in up-front studies for these rare embryonal tumors.

Cancers arising from germline DNA mismatch-repair or polymerase-proofreading deficiencies (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion/deletion (MS-indel) burden in humans and are lethal due to inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICI) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI in these patients. ICI treatment of 45 progressive/recurrent tumours from 38 patients revealed durable objective responses in the majority, culminating in 3-year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations/Mb) enriched for combined MMRD+PPD, while MS-indels predicted response in MMRD tumours with lower mutation burden (10-100 mutations/Mb). Further, both mechanisms were associated with increased immune infiltration even in “immunologically-cold” tumours such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and associated with immune activation in both the tumour microenvironment and systemically. Further, patients with flare continuing ICI treatment achieved durable responses. Our study demonstrates improved survival for patients with tumours not previously known to respond to ICI, including CNS and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained responses to immunotherapy.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion–deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10–100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in ‘immunologically cold’ tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy., Hypermutation and microsatellite burden determine responses and long-term survival following PD-1 blockade in children and young adults with refractory cancers resulting from germline DNA replication repair deficiency.

Here we report a case of an 11-year-old girl with an inoperable tumor in the optic chiasm/hypothalamus, who experienced several tumor progressions despite three lines of chemotherapy treatment. Routine clinical examination classified the tumor as a BRAF-negative pilocytic astrocytoma. Copy-number variation profiling of fresh frozen tumor material identified two duplications in 9q21.32–33 leading to breakpoints within the GKAP1 and NTRK2 genes. RT-PCR Sanger sequencing revealed a GKAP1-NTRK2 exon 10–16 in-frame fusion, generating a putative fusion protein of 658 amino acids with a retained tyrosine kinase (TK) domain. Functional analysis by transient transfection of HEK293 cells showed the GKAP1-NTRK2 fusion protein to be activated through phosphorylation of the TK domain (Tyr705). Subsequently, downstream mediators of the MAPK- and PI3K-signaling pathways were upregulated in GKAP1-NTRK2 cells compared to NTRK2 wild-type; phosphorylated (p)ERK (3.6-fold), pAKT (1.8- fold), and pS6 ribosomal protein (1.4-fold). Following these findings, the patient was enrolled in a clinical trial and treated with the specific TRK-inhibitor larotrectinib, resulting in the arrest of tumor growth. The patient’s condition is currently stable and the quality of life has improved significantly. Our findings highlight the value of comprehensive clinical molecular screening of BRAF-negative pediatric low-grade gliomas, to reveal rare fusions serving as targets for precision therapy. CC BY-NC-ND 4.0© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.

PURPOSE Children with pediatric gliomas harboring a BRAF V600E mutation have poor outcomes with current chemoradiotherapy strategies. Our aim was to study the role of targeted BRAF inhibition in these tumors. PATIENTS AND METHODS We collected clinical, imaging, molecular, and outcome information from patients with BRAF V600E–mutated glioma treated with BRAF inhibition across 29 centers from multiple countries. RESULTS Sixty-seven patients were treated with BRAF inhibition (pediatric low-grade gliomas [PLGGs], n = 56; pediatric high-grade gliomas [PHGGs], n = 11) for up to 5.6 years. Objective responses were observed in 80% of PLGGs, compared with 28% observed with conventional chemotherapy ( P < .001). These responses were rapid (median, 4 months) and sustained in 86% of tumors up to 5 years while receiving therapy. After discontinuation of BRAF inhibition, 76.5% (13 of 17) of patients with PLGG experienced rapid progression (median, 2.3 months). However, upon rechallenge with BRAF inhibition, 90% achieved an objective response. Poor prognostic factors in conventional therapies, such as concomitant homozygous deletion of CDKN2A, were not associated with lack of response to BRAF inhibition. In contrast, only 36% of those with PHGG responded to BRAF inhibition, with all but one tumor progressing within 18 months. In PLGG, responses translated to 3-year progression-free survival of 49.6% (95% CI, 35.3% to 69.5%) versus 29.8% (95% CI, 20% to 44.4%) for BRAF inhibition versus chemotherapy, respectively ( P = .02). CONCLUSION Use of BRAF inhibition results in robust and durable responses in BRAF V600E–mutated PLGG. Prospective studies are required to determine long-term survival and functional outcomes with BRAF inhibitor therapy in childhood gliomas.

BACKGROUND Constitutional mismatch repair deficiency syndrome (CMMRD) is a severe cancer predisposition syndrome resulting in early onset central nervous system (CNS) and other cancers. International guidelines for surveillance exist but no study has systematically evaluated the efficacy of this protocol. METHODS We surveyed all confirmed CMMRD patients in the International Replication Repair Deficiency Consortium. A surveillance protocol consisting of frequent biochemical, endoscopic and imaging (CNS and total body MRI) studies were employed. Survival analyses and efficacy of each method were assessed. RESULTS Surveillance data were collected from 105 CMMRD individuals from 41 countries. Of the 193 malignant tumors, CNS malignancies were the most common (44%). The surveillance protocol uncovered 49 asymptomatic tumors including 16 glioblastomas and medulloblastomas. Five-year overall survival was 89% for tumors discovered by surveillance, and 61% for symptomatic tumors (p6 months as per protocol. Finally, of the low grade tumors identified asymptomatically, 5 were low grade gliomas. All of the low grade gliomas, which were not resected transformed to high grade tumors at a median of 1.6 ± 0.9 years. CONCLUSION These data support a survival benefit in CMMRD patients undergoing a surveillance protocol. Adherence to protocol and resection of lower grade lesions may improve survival for patients with CNS tumors.

Background. Physical activity can enhance cognitive functions in both animals and humans. We hypothesized that physically active video gaming could: i) improve cognitive functions and ii) improve the execution of activities of daily living among survivors of childhood brain tumors. Methods. Children 7 to 17 years old who completed treatment, including radiotherapy, for a brain tumor 1 to 5 years earlier were randomized to either intervention or waiting list. After 10 to 12 weeks the groups crossed over. The intervention consisted of active video gaming, using a motion-controlled video console (Nintendo Wii), for a minimum of 30 minutes a day, 5 days a week and weekly Internet-based coaching sessions. Evaluations before and after each period included tests of the execution of activities of daily living, using the Assessment of Motor and Process Skills (AMPS) and cognitive tests. Test scores before and after the intervention were compared. A parallel group comparison was performed as a sensitivity analysis. Results. All 13 children enrolled completed the program. Compared to baseline, the motor (P= .012) and process (P=.002) parts of AMPS improved significantly after active video gaming. In the parallel group analysis the improvement in the process part of AMPS remained statistically significant (P= .029), but not the change in AMPS motor score (P= .059). No significant change was found in cognitive tests although there were trends for improvement in sustained attention (P = .090) and selective attention (P = .078). Conclusion. In this pilot study, active video gaming used as a home-based intervention for childhood brain tumor survivors improved motor and process skills in activities of daily living.

Pediatric brain tumors with replication repair deficiency (RRD) are hypermutant and may respond favorably to immune checkpoint inhibition (ICI). We are collecting ongoing clinical and molecular data from patients with RRD hypermutant cancers treated with ICI as a part of our consortium registry study. Companion biomarkers include tumor mutational burden (TMB), neoantigens and genetic signatures obtained from whole genome and exome sequencing. Immune inference is obtained from RNAseq. Additionally, T-cell receptor rearrangement data are collected from the tumor and blood throughout treatment. From 2015–2018, 53 patients were treated with ICI and combination therapies. Of these 39 had brain tumors, with 93% having high grade gliomas. Two-year overall survival for the entire cohort is 47+/-8%, which compares favorably with historical controls. Tumor location had major impact on outcome. Non-CNS solid tumor patients have 2-year OS of 78+/-11%, all failures occurring within the first 2 months, and sustained responses are observed for 3 years. In contrast, OS for brain tumors is 39+/-10% with late recurrences observed even after 2 years of therapy (p=0.02). Large tumor size and total tumor burden are associated with higher rates of “flare” and poor outcome throughout all cancers. While all tumors are hypermutant, TMB and predicted neoantigens do not correlate with response. However, specific signatures extracted from SNVs and indels are significantly associated with response to ICI and favorable outcome (p=0.005). Notably, RRD IDH1 mutant glioblastomas have particularly poor response to ICI. Interestingly, glioblastomas (n=8) which failed single agent ICI had favorable responses to combination immunotherapies with prolonged survival of 65%+/-8% one year after failure vs 0 for untreated patients (p=0.01). The favorable outcome and responses to ICI are encouraging. Since brain tumors respond less favorably to ICI than other solid tumors, combination therapies based on tumor and immune signatures of these cancers may be beneficial.

Children with pediatric gliomas harboring BRAF V600E mutation have a poor outcome with current chemoradiation strategies. Our aim was to study the role of targeted BRAF inhibition in these tumors. We collected clinical, imaging, molecular and outcome information from BRAF V600E glioma patients treated with BRAFi across 29 centers from multiple countries. Sixty-seven patients were treated with BRAFi (56 pediatric low grade gliomas, PLGG and 11 pediatric high grade gliomas, PHGG) for up to 5.6 years. Objective responses were observed in 80% of PLGGs compared to 28% with conventional chemotherapy (p

Pediatric brain tumors with replication repair deficiency (RRD) are hypermutant and may respond to immune checkpoint inhibition (ICI). We performed a consortium registry study of ICI in recurrent RRD cancers. Clinical and companion biomarkers were collected longitudinally on all patients. Biomarkers included tumor mutational burden (TMB), neoantigens and genetic signatures obtained from whole genome and exome sequencing. Immune inference was obtained by RNAseq and T cell rearrangement was collected in the tumor and in blood throughout treatment. Of the 46 tumors on the study, 32 were brain tumors with glioblastoma in 96%. Rapid, objective responses (>50%) were observed in 50% of glioblastomas. Three year overall survival for the whole cohort was 48+/-8% which compares favorably with historical controls. Brain tumors fared worse with OS of 39+/-10% and late recurrences observed even after 2 years of therapy (p=0.02). Tumor size and acute “flare” constitute poor outcome throughout all cancers. While all tumors are hypermutant, TMB and predicted neoantigens correlated with response to ICI (p=0.02). Specific signatures extracted from SNVs and total mutations predicted response to ICI and favorable outcome (p=0.005). RNA inference and TCR reveal that the FLARE phenotype is mostly acute nonspecific immune response and not true progression. Finally, glioblastomas (n=8) which failed single agent ICI had favorable responses to combinational immunotherapies with prolonged survival of 65%+/-8% at one year after failure vs 0 for other patients (p=0.01). RRD glioblastomas exhibit favorable outcome and responses to ICI. Combinational therapies based on tumor and immune signatures of these cancers are necessary.

BACKGROUND: Hypermutation constitute an important subgroup of cancers and confers sensitivity to immune checkpoint inhibition (ICI). Glioblastoma arising in children with Constitutional Mismatch Repair Deficiency Syndrome (CMMRD) are ultrahypermutant. Our objective was to quantify the frequency of hypermutant tumors in children, determine whether mutational signatures can predict germline mutations, and treat hypermutant tumors with ICIs. METHODS: Deep panel sequencing of 2984 pediatric tumors (585 brain tumors). Tumor mutation burden was correlated to mutation burden from exome and genome sequencing (R(2) = 0.94). Mutational signatures were analyzed to predict source of hypermutation. Data on 36 patients with hypermutant tumors identified by sequencing were enrolled on an ICI registry trial. RESULTS: Hypermutant tumors (>10 mut/MB) comprised 5% of all pediatric tumors (n=143). These were highly enriched for replication repair mutations (p

BACKGROUND: The leading cause of cancer-related mortality among children is brain tumours and glioblastoma multiforme (GBM) has the worst prognosis. Epigenetic deregulation is believed to be a driver as these patients have few mutations compared to adults. New treatments are urgently needed but few pre-clinical in vivo models exist. One approach of generating these is to transplant tumour tissue into mice, but it yields highly variable results and requires serial passaging in mice, which is time-consuming, expensive and ethically questionable. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. METHODS: Patient-derived cancer stem cell (CSC) lines from paediatric GBM were orthotopically transplanted into immunodeficient mice. The xenograft tumours were evaluated by histology for glioma features. Genome-wide DNA methylation analysis was performed on the CSC, xenograft and patient tumours. RESULTS: All CSC lines initiated tumours and the survival of the mice correlated with the survival of their respective patient. The xenograft tumours had key glioma features and were classified as GBM by histology and methylation profiling. The methylation profile of the xenograft tumour clustered together with the patient tumour (

We have established a robust and reliable cell line-based glioma stem cell xenograft mouse model, genetically and epigenetically similar to its originating patient tumours. The survival of patients and injected mice correlate significantly, demonstrating that the transplanted mice mimic the clinical course of the patient., The leading cause of cancer-related mortality among children is brain tumour, and glioblastoma multiforme (GBM) has the worst prognosis. New treatments are urgently needed, but with few cases and clinical trials in children, pre-clinical models such as patient-derived tumour xenografts (PDTX) are important. To generate these, tumour tissue is transplanted into mice, but this yields highly variable results and requires serial passaging in mice, which is time-consuming and expensive. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. Glioma stem cell (GSC) lines derived from paediatric GBM were orthotopically transplanted into immunodeficient mice. Overall survival data were collected and histological analysis of the resulting neoplasias was performed. Genome-wide DNA methylation arrays were used for methylation and copy-number alterations (CNA) profiling. All GSC lines initiated tumours on transplantation and the survival of the mice correlated well with the survival of the patients. Xenograft tumours presented histological hallmarks of GBM, and were also classified as GBM by methylation profiling. Each xenograft tumour clustered together with its respective injected GSC line and patient tumour based on the methylation data. We have established a robust and reproducible cell line-based xenograft paediatric GBM model. The xenograft tumours accurately reflected the patient tumours and mirrored the clinical course of the patient. This model can therefore be used to assess patient response in pre-clinical studies.

BACKGROUND Children with pediatric low grade glioma’s (PLGG) harboring BRAF V600E mutation have poor outcome due to relative resistance to chemo-radiation and higher risk of malignant transformation. However, the role of targeted BRAF inhibition in these tumors is poorly defined. METHODS We assembled an international cohort of children with BRAF V600E mutant gliomas treated with BRAF inhibition, from 29 centers participating in the PLGG taskforce, and collected response, survival and molecular parameters. RESULTS Sixty-seven patients were treated with BRAFi (56 PLGG and 11 high grade gliomas) for a median time of 17.4 months (6 – 61 months), with 13 PLGG treated upfront. Objective responses was observed in 80% of PLGG patients compared to 28% with conventional chemotherapy (p< 0.001). Rapid responses were observed in most PLGG patients (median of 4 months), sustained in 86% of tumors up to 5 years while on therapy. In contrast, only 36% of PHGG responded to BRAFi with all but one tumor progressing within 18 months. Seventeen patients with PLGG discontinued BRAFi and 76.5% (13/17) progressed rapidly after discontinuation (median time 2.3 months). However, upon re-challenge with BRAFi therapy, 90% achieved an objective response. Poor prognostic factors to conventional therapies such as concomitant homozygous deletion of CDKN2A or H3K27M mutation were not associated with lack of response to BRAFi. Overall these responses translated to 2-year progression-free survival of 0.636 (95%CI 0.505–0.802) and 0.43 (95% CI 0.32–0.57) for BRAFi and chemotherapy treated BRAF V600E PLGG respectively (p=0.003). CONCLUSION The use of BRAFi results in objective, robust and durable responses in BRAF V600E PLGG and is associated with favorable survival. Larger prospective studies will be required to determine appropriate regiments, and long-term functional outcomes with BRAFi therapy in childhood gliomas.

BACKGROUND: A significant amount of childhood brain tumors emerge from cancer predisposition syndromes. Although tumor sequencing is common practice, it is currently impossible to infer germline mutations from tumor data. Glioblastoma Multiforme arising in children with Biallelic Mismatch Repair Deficiency Syndrome (bMMRD) are ultrahypermutant which is highly specific to this syndrome and confers eligibility for immune checkpoint inhibition therapy (ICI). Our objective was to quantify the frequency of hypermutant tumors in children, determine whether mutational signatures can predict germline mutations, and treat hypermutant tumors with ICIs. METHODS: Deep panel sequencing (1000x) of 315 genes in 2984 pediatric tumors (585 brain tumors) and 79 842 adult tumors (3910 brain tumors) was performed. An algorithm was devised to determine mutation burden from 2 MB of panel sequencing data and results correlated strongly with mutation burden from exome sequencing (R2 = 0.94). Mutational signatures were analyzed to predict source of hypermutation. Patients with hypermutant brain tumors identified by panel sequencing were treated with ICI. RESULTS: Hypermutant tumors (>10 mut/MB) comprised 5% of all pediatric tumors (n=143). These were highly enriched for replication repair mutations (p=100 Mut/MB harbored MMR/polymerase mutations suggesting germline bMMRD (p =10–7). Clinical data collected on 15 ultrahypermutant tumors revealed germline mutations in replication repair genes in all patients. Of the 22 patients with hypermutant cancers treated with ICI, 16 had brain tumors, and favorable sustained responses are observed. CONCLUSION: High mutation burden is a sensitive predictor of germline bMMRD. Hypermutant tumors are more common in the pediatric setting than previously appreciated, opening novel therapeutic avenues. ICI shows promise for hypermutant pediatric cancers.

[This corrects the article DOI: 10.1371/journal.pone.0175638.].

The BRAF-V600E mutation is found in 15–20% of pediatric low grade gliomas (PLGG) and result in worse outcome and higher risk of transformation to high grade gliomas (PHGG). Although ongoing trials are assessing the role of BRAF inhibitors (BRAFi) in these children, data are still limited. We aimed to report overall response rates and predictors of outcome in childhood BRAF-V600E gliomas. We collected clinical, imaging and molecular information of patients treated with BRAFi outside trials from centers participating in the PLGG taskforce. Response was calculated by RANO criteria and follow up data were collected for all patients. Sixty-six patients were treated with BRAFi (55 PLGG and 11 PHGG); median follow-up time was 1.5 years (0.1-5y). In PLGG, objective response (tumor reduction of >25%) was observed in 77% compared to 15% in a cohort treated with conventional chemotherapy (pCDKN2A deletion was not associated with lack of response, while specific enhancing patterns correlated strongly with response to BRAFi. Two-year PFS for the BRAF-V600E PLGG was 74% vs 47% for BRAFi vs chemotherapy, respectively (p=0.02). Our data reveal rapid, dramatic and sustained response of BRAF-V600E PLGG to BRAFi. These are in contrast to BRAF-V600E PHGG and non-enhancing PLGG. Additional molecular analyses are being performed to identify poor responders and emerging mechanisms of resistance in these tumors.

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics., BACKGROUND:Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics.METHODS:A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes.RESULTS:Analyses revealed a mutation detection sensitivity of 97.3%, an average coverage per gene of 98.0%, and adequate coverage over 98.6% of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38%). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals.CONCLUSIONS:We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3′-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

Activation of the MAPK pathway represents a hallmark of pediatric low-grade glioma (pLGG) and is frequently caused by BRAF alterations. BRAF-V600E represent an aggressive type of pLGG with less than optimal response to conventional chemo-radiation approaches. While clinical trials using BRAF-V600E inhibitors are ongoing, these data are not yet available. We have assembled an international cohort of BRAF-V600E glioma patients treated off-label with BRAF inhibitors as a monotherapy. Complete molecular, clinical and imaging data is being collected and compared to previous chemo-radiation therapies. Ongoing data form the taskforce on 40 BRAF-V600E gliomas from 25 international institutions is summarized below. The most prevalent histologies were ganglioglioma, pilocytic astrocytoma and pleomorphic xanthoastrocytoma, located mainly in the chiasm, brainstem and temporal lobes. Strikingly, 66% of BRAF V600E pLGG patients achieved partial response (PR) to targeted inhibitors versus only 6.6% response to conventional chemotherapy (p

Background Classification of pediatric tumors into biologically defined subtypes is challenging, and multifaceted approaches are needed. For this aim, we developed a diagnostic classifier based on DNA methylation profiles. Results Methylation data generated by the Illumina Infinium HumanMethylation 450 BeadChip arrays were downloaded from the Gene Expression Omnibus (n = 472). Using the data, we built MethPed, which is a multiclass random forest algorithm, based on DNA methylation profiles from nine subgroups of pediatric brain tumors. DNA from 18 regional samples was used to validate MethPed. MethPed was additionally applied to a set of 28 publically available tumors with the heterogeneous diagnosis PNET. MethPed could successfully separate individual histology tumor types at a very high accuracy (κ = 0.98). Analysis of a regional cohort demonstrated the clinical benefit of MethPed, as confirmation of diagnosis of tumors with clear histology but also identified possible differential diagnoses in tumors with complicated and mixed type morphology. Conclusions We demonstrate the utility of methylation profiling of pediatric brain tumors and offer MethPed as an easy-to-use toolbox that allows researchers and clinical diagnosticians to test single samples as well as large cohorts for subclass prediction of pediatric brain tumors. This will immediately aid clinical practice and importantly increase our molecular knowledge of these tumors for further therapeutic development. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0103-3) contains supplementary material, which is available to authorized users.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management ofpatients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis., Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through release of perforin-containing lytic granules. Here, we first performed probability state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTL with NK cells. Analysis identified CD57(bright)-expression as a reliable phenotype of granule marker-containing CTL. We then compared CD3(+)CD8(+)CD57(bright) CTL with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTL expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. Upon stimulation, such CTL degranulated more readily than other T cell subsets, but had a similar propensity to degranulate as NK cells. Remarkably, the CTL produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistocytosis (FHL), CTL and NK cell degranulation was similarly impaired. Thus, cytotoxic lymphocyte subsets have similar requirements for Munc13-4,syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTL and NK cells,and suggest that analysis of CD57(bright) CTL-function may prove useful in the diagnosis of primary immunodeficiencies including FHL.