Molecular classification and outcome of children with rare CNS embryonal tumors: results from St. Jude Children's Research Hospital including the multi-center SJYC07 and SJMB03 clinical trials.

Methylation profiling has radically transformed our understanding of tumors previously called central nervous system primitive neuro-ectodermal tumors (CNS-PNET). While this marks a momentous step toward defining key differences, reclassification has thrown treatment into disarray. To shed light on response to therapy and guide clinical decision-making, we report outcomes and molecular features of children with CNS-PNETs from two multi-center risk-adapted studies (SJMB03 for patients ≥ 3 years; SJYC07 for patients < 3 years) complemented by a non-protocol institutional cohort. Seventy patients who had a histological diagnosis of CNS-PNET or CNS embryonal tumor from one of the new categories that has supplanted CNS-PNET were included. This cohort was molecularly characterized by DNA methylation profiling (n = 70), whole-exome sequencing (n = 53), RNA sequencing (n = 20), and germline sequencing (n = 28). Clinical characteristics were detailed, and treatment was divided into craniospinal irradiation (CSI)-containing (SJMB03 and SJMB03-like) and CSI-sparing therapy (SJYC07 and SJYC07-like). When the cohort was analyzed in its entirety, no differences were observed in the 5-year survival rates even when CSI-containing therapy was compared to CSI-sparing therapy. However, when analyzed by DNA methylation molecular grouping, significant survival differences were observed, and treatment particulars provided suggestions of therapeutic response. Patients with CNS neuroblastoma with FOXR2 activation (CNS-NB-FOXR2) had a 5-year event-free survival (EFS)/overall survival (OS) of 66.7% ± 19.2%/83.3% ± 15.2%, and CIC rearranged sarcoma (CNS-SARC-CIC) had a 5-year EFS/OS both of 57.1% ± 18.7% with most receiving regimens that contained radiation (focal or CSI) and multidrug chemotherapy. Patients with high-grade neuroepithelial tumor with BCOR alteration (HGNET-BCOR) had abysmal responses to upfront chemotherapy-only regimens (5-year EFS = 0%), but survival extended with salvage radiation after progression [5-year OS = 53.6% ± 20.1%]. Patients with embryonal tumor with multilayered rosettes (ETMR) or high-grade glioma/glioblastoma multiforme (HGG/GBM) did not respond favorably to any modality (5-year EFS/OS = 10.7 ± 5.8%/17.9 ± 7.2%, and 10% ± 9.0%/10% ± 9.0%, respectively). As an accompaniment, we have assembled this data onto an interactive website to allow users to probe and query the cases. By reporting on a carefully matched clinical and molecular cohort, we provide the needed insight for future clinical management.

Medulloblastoma Down Under 2013: a report from the third annual meeting of the International Medulloblastoma Working Group.

Medulloblastoma is curable in approximately 70% of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5-10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organization's classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Children's Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

A feasibility and efficacy study of rapamycin and erlotinib for recurrent pediatric low-grade glioma (LGG).

BACKGROUND: To determine the toxicity and efficacy of rapamycin and erlotinib for the treatment of recurrent pediatric low-grade gliomas (LGGs). METHODS: Patients <21 years of age with recurrent LGGs who had failed conventional treatment were eligible, including those with NF1. The treatment consisted of two phases, a feasibility portion which assessed the toxicity of erlotinib at 65 mg/m(2) /day once daily and rapamycin at 0.8 mg/m(2) /dose twice daily for 28 consecutive days. RESULTS: Nineteen (19) patients, median age of 8 years, with recurrent LGGs received the two-drug regimen. Eight (8) of the patients had NF1. The combination of erlotinib and rapamycin was well tolerated and no patient was removed from study due to toxicity. All 19 patients were evaluable for response and one child, with NF1, had a partial response to treatment. Six (6) patients received the planned 12 courses of treatment. The reasons for stoppage of therapy before 1 year of treatment were poor compliance (1), parental desire for withdrawal (1), persistent vomiting which pre-dated initiation of therapy (1), and radiographic progression (10). In those patients with stabilization of disease for 12 months or greater, 3 stayed on therapy and ultimately developed progressive disease, and one patient stopped therapy at 12 months and progressed. Two (2) patients, both with NF1, have had >1 year disease control. CONCLUSIONS: The combination of rapamycin and erlotinib is well tolerated in children with LGGs. Objective responses were infrequent, although there was prolonged disease stabilization in some patients with LGGs, especially in two children with NF1.

From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

Genome-wide analyses of platinum-induced ototoxicity in childhood cancer patients: Results of GO-CAT and United Kingdom MAGIC consortia.

Hearing loss (ototoxicity) is a major adverse effect of cisplatin and carboplatin chemotherapy. The aim of this study is to identify novel genetic variants that play a role in platinum-induced ototoxicity. Therefore, a genome-wide association study was performed in the Genetics of Childhood Cancer Treatment (GO-CAT) cohort (n = 261) and the United Kingdom Molecular Genetics of Adverse Drug Reactions in Children Study (United Kingdom MAGIC) cohort (n = 248). Results of both cohorts were combined in a meta-analysis. In primary analysis, patients with SIOP Boston Ototoxicity Scale grade ≥1 were considered cases, and patients with grade 0 were controls. Variants with a p-value <10-5 were replicated in previously published data by the PanCareLIFE cohort (n = 390). No genome-wide significant associations were found, but variants in TSPAN5, RBBP4P5, AC010090.1 and RNU6-38P were suggestively associated with platinum-induced ototoxicity. The lowest p-value was found for rs7671702 in TSPAN5 (odds ratio 2.0 (95% confidence interval 1.5-2.7), p-value 5.0 × 10-7). None of the associations were significant in the replication cohort, although the effect directions were consistent among all cohorts. Validation and functional understanding of these genetic variants could lead to more insights in the development of platinum-induced ototoxicity.

SLC7A8 coding for LAT2 is associated with early disease progression in osteosarcoma and transports doxorubicin.

Background: Despite (neo) adjuvant chemotherapy with cisplatin, doxorubicin and methotrexate, some patients with primary osteosarcoma progress during first-line systemic treatment and have a poor prognosis. In this study, we investigated whether patients with early disease progression (EDP), are characterized by a distinctive pharmacogenetic profile. Methods and Findings: Germline DNA from 287 Dutch high-grade osteosarcoma patients was genotyped using the DMET Plus array (containing 1,936 genetic markers in 231 drug metabolism and transporter genes). Associations between genetic variants and EDP were assessed using logistic regression models and associated variants (p <0.05) were validated in independent cohorts of 146 (Spain and United Kingdom) and 28 patients (Australia). In the association analyses, EDP was significantly associated with an SLC7A8 locus and was independently validated (meta-analysis validation cohorts: OR 0.19 [0.06-0.55], p = 0.002). The functional relevance of the top hits was explored by immunohistochemistry staining and an in vitro transport models. SLC7A8 encodes for the L-type amino acid transporter 2 (LAT2). Transport assays in HEK293 cells overexpressing LAT2 showed that doxorubicin, but not cisplatin and methotrexate, is a substrate for LAT2 (p < 0.0001). Finally, SLC7A8 mRNA expression analysis and LAT2 immunohistochemistry of osteosarcoma tissue showed that the lack of LAT2 expression is a prognostic factor of poor prognosis and reduced overall survival in patients without metastases (p = 0.0099 and p = 0.14, resp.). Conclusion: This study identified a novel locus in SLC7A8 to be associated with EDP in osteosarcoma. Functional studies indicate LAT2-mediates uptake of doxorubicin, which could give new opportunities to personalize treatment of osteosarcoma patients.

PD-1 blockade using pembrolizumab in adolescent and young adult patients with advanced bone and soft tissue sarcoma.

BACKGROUND: Sarcomas represent 10%-15% of cancers in adolescent and young adult (AYA) patients, and survival for those with metastatic disease or relapse is poor. Immunotherapy with checkpoint inhibition has improved outcomes in multiple tumor types, but data in advanced sarcomas, particularly within the AYA population, are limited. AIM: We aim to evaluate response and toxicity for AYA patients with sarcoma treated with pembrolizumab. METHODS AND RESULTS: We retrospectively reviewed AYA patients with advanced bone and soft tissue sarcoma who received self-funded pembrolizumab between May 2015 and January 2019. Eighteen patients were identified. One patient with Ewing sarcoma had a sustained complete response to therapy. Two patients with alveolar soft part sarcoma received a clinical benefit from pembrolizumab: one had a radiological partial response with an excellent clinical response and one patient achieved stable disease. Four patients died of disease prior to first scheduled assessment and thus were not evaluable. The remaining eleven patients had progressive disease. CONCLUSION: The role of immunotherapy in AYA sarcoma warrants further investigation. Biomarkers of response need to be further evaluated in order to guide patient selection.

Diffuse leptomeningeal glioneuronal tumour (DLGNT) in children: the emerging role of genomic analysis.

Diffuse leptomeningeal glioneuronal tumours (DLGNT) represent rare enigmatic CNS tumours of childhood. Most patients with this disease share common radiological and histopathological features but the clinical course of this disease is variable. A radiological hallmark of this disease is widespread leptomeningeal enhancement that may involve the entire neuroaxis with predilection for the posterior fossa and spine. The classic pathologic features include low- to moderate-density cellular lesions with OLIG2 expression and evidence of 'oligodendroglioma-like' appearance. The MAPK/ERK signaling pathway has recently been reported as a potential driver of tumourigenesis in up to 80% of DLGNT with KIAA1549:BRAF fusions being the most common event seen. Until now, limited analysis of the biological drivers of tumourigenesis has been undertaken via targeted profiling, chromosomal analysis and immunohistochemistry. Our study represents the first examples of comprehensive genomic sequencing in DLGNT and shows that it is not only feasible but crucial to our understanding of this rare disease. Moreover, we demonstrate that DLGNT may be more genomically complex than single-event MAPK/ERK signaling pathway tumours.

Malignant Melanoma in Children and Adolescents Treated in Pediatric Oncology Centers: An Australian and New Zealand Children's Oncology Group (ANZCHOG) Study.

OBJECTIVES: Unlike adults, malignant melanoma in children and adolescents is rare. In adult melanoma, significant progress in understanding tumor biology and new treatments, including targeted therapies and immunotherapy have markedly improved overall survival. In sharp contrast, there is a paucity of data on the biology and clinical behavior of pediatric melanoma. We report a national case series of all pediatric and adolescent malignant melanoma presenting to ANZCHOG Childhood Cancer Centers in Australia and New Zealand. METHODS: A retrospective, descriptive, multi-center study was undertaken to identify patients less than 18 years of age treated for cutaneous malignant melanoma over a twenty-year period (1994 to 2014). Data on clinical characteristics, histopathology, and extent of disease, treatment and follow-up are described. RESULTS: A total of 37 cases of malignant melanoma were identified from all of the Australasian tertiary Childhood Cancer Centers. The median age was 10 years (range 1 month - 17 years). Clinically, the most common type of lesion was pigmented, occurring in sixteen (57%) patients, whilst amelanotic was seen in 7 patients (25%). In 11 (27.9%) the Breslow thickness was greater than 4mm. A total of 11 (29.7%) patients relapsed and 90% of these died of disease. Five-year event free survival (EFS) and overall survival were 63.2 (95% CI: 40.6 - 79.1) and 67.7% (95% CI: 45.1 - 82.6) respectively. CONCLUSION: Our data confirms that melanoma is a rare presentation of cancer to tertiary Australasian Childhood Cancer Centers with only 37 cases identified over two decades. Notably, melanoma managed in Childhood Cancer Centers is frequently at an advanced stage, with a high percentage of patients relapsing and the majority of these patients who relapsed died of disease. This study confirms previous clinical and prognostic information to support the early multidisciplinary management in Childhood Cancer Centers, in conjunction with expert adult melanoma centers, of this rare and challenging patient group.

Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle.

Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O(6)-methylguanine-DNA methyltransferase MGMT (P140K) gene-mediated drug resistance-based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%-100% donor cell engraftment in the host's hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)-mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)-expressing male CD34(+ve) donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative-polymerase chain reaction using Y-chromosome specific primers. Fluorescent in situ hybridization analysis using a Y-chromosome paint probe revealed donor-derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo-protected donor CD34(+ve) cells induced substantial endogenous regeneration of the chemo-ablated host muscle that is otherwise unable to self-regenerate. We conclude that the MGMT (P140K)-mediated enrichment strategy can be successfully implemented in muscle.

Addition of Vincristine and Irinotecan to Vincristine, Dactinomycin, and Cyclophosphamide Does Not Improve Outcome for Intermediate-Risk Rhabdomyosarcoma: A Report From the Children's Oncology Group.

Purpose Intermediate-risk rhabdomyosarcoma (RMS) includes patients with either nonmetastatic, unresected embryonal RMS (ERMS) with an unfavorable primary site or nonmetastatic alveolar RMS (ARMS). The primary aim of this study was to improve the outcome of patients with intermediate-risk RMS by substituting vincristine and irinotecan (VI) for half of vincristine, dactinomycin, and cyclophosphamide (VAC) courses. All patients received a lower dose of cyclophosphamide and earlier radiation therapy than in previous trials. Patients and Methods Patients were randomly assigned at study entry to either VAC (cumulative cyclophosphamide dose, 16.8 g/m2) or VAC/VI (cumulative cyclophosphamide dose, 8.4 g/m2) for 42 weeks of therapy. Radiation therapy started at week 4, with individualized local control plans permitted for patients younger than 24 months. The primary study end point was event-free survival (EFS). The study design had an 80% power (5% one-sided α-level) to detect an improved long-term EFS from 65% (with VAC) to 76% (with VAC/VI). Results A total of 448 eligible patients were enrolled in the study. At a median follow-up of 4.8 years, the 4-year EFS was 63% with VAC and 59% with VAC/VI ( P = .51), and 4-year overall survival was 73% for VAC and 72% for VAC/VI ( P = .80). Within the ARMS and ERMS subgroups, no difference in outcome by treatment arm was found. Severe hematologic toxicity was less common with VAC/VI therapy. Conclusion The addition of VI to VAC did not improve EFS or OS for patients with intermediate-risk RMS. VAC/VI had less hematologic toxicity and a lower cumulative cyclophosphamide dose, making VAC/VI an alternative standard therapy for intermediate-risk RMS.

Methyl-Guanine-Methyl-Transferase Transgenic Bone Marrow Transplantation Allows N,N-bis(2-chloroethyl)-Nitrosourea Driven Donor Mixed-Chimerism Without Graft-Versus-Host Disease, and With Donor-Specific Allograft Tolerance.

BACKGROUND: Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed-chimerism strategies were limited either by loss of long-term mixed chimerism or risk of graft-versus-host disease (GVHD). Selective bone marrow (BM) engraftment using marrow protective strategies are currently reaching clinical use. In this study, we tested the utility of methyl-guanine-methyl-transferase (MGMT)-transgenic-C57BL/6 BM into a major histocompatibility complex mismatched-BALB/c model followed by N,N-bis(2-chloroethyl)-nitrosourea (BCNU) treatment to enhance donor-cell engraftment and then evaluated transplant tolerance induction. METHODS: A single-dose of anti-CD8 antibody and busulfan was administered into BALB/c-host-mice at day 1. The BALB/c-mice also received costimulatory blockade through multiple-doses of anti-CD40L antibody. 10 × 10(6) BM-cells from MGMT-transgenic-mice were transplanted into host BALB/c mice at day 0. The BCNU was administered at 4 time points after BM transplantation (BMT). Heterotopic donor C57BL/6 cardiac allografts were performed at day 243 after BMT. Skin transplantation with third-party CBA, host BALB/c and donor C57BL/6 grafts was performed at day 358 after BMT. RESULTS: The BALB/c-mice showed long-term stable and high-level donor-cell engraftment with MGMT transgenic C57BL/6 BMT after BCNU treatment, demonstrating full reconstitution and donor cardiac-allograft tolerance and no GVHD with expanded donor and host Foxp3 T regulatory cells. Further, skin grafts from donor, host, and third party showed good immune function with rejection of third-party grafts from all mice and benefit from enhanced chimerism after BCNU with less cell infiltrate and no chronic rejection in the donor skin grafts of BCNU treated mice compared no BCNU treated mice. CONCLUSIONS: High-level mixed chimerism without GVHD can be achieved using MGMT transgenic BM in a mixed-chimerism model receiving BCNU across a major histocompatibility complex mismatch. Enhanced mixed chimerism leads to long-term donor-specific allograft tolerance.