Abhijit Joshi

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease

Summary We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study

Summary Background International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. Methods In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Childrens Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. Findings Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82–100]); standard risk (50 [23%] patients; 81% survival [70–94]); high-risk (82 [38%] patients; 42% survival [31–56]); and very high-risk (29 [13%] patients; 28% survival [14–56]). Interpretation The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. Funding Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Childrens Charity, and Children with Cancer UK).

Fatty acid oxidation is required for the respiration and proliferation of malignant glioma cells

Background. Glioma is the most common form of primary malignant brain tumor in adults, with approximately 4 cases per 100 000 people each year. Gliomas, like many tumors, are thought to primarily metabolize glucose for energy production; however, the reliance upon glycolysis has recently been called into question. In this study, we aimed to identify the metabolic fuel requirements of human glioma cells. Methods. We used database searches and tissue culture resources to evaluate genotype and protein expression, tracked oxygen consumption rates to study metabolic responses to various substrates, performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates, and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. Results. We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition, we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover, inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. Conclusions. Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition, the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice.

Imaging Invasion: Micro-CT imaging of adamantinomatous craniopharyngioma highlights cell type specific spatial relationships of tissue invasion

Tissue invasion and infiltration by brain tumours poses a clinical challenge, with destruction of structures leading to morbidity. We assessed whether micro-CT could be used to map tumour invasion in adamantinomatous craniopharyngioma (ACP), and whether it could delineate ACPs and their intrinsic components from surrounding tissue.Three anonymised archival frozen ACP samples were fixed, iodinated and imaged using a micro-CT scanner prior to the use of standard histological processing and immunohistochemical techniques.We demonstrate that micro-CT imaging can non-destructively give detailed 3D structural information of tumours in volumes with isotropic voxel sizes of 4–6 microns, which can be correlated with traditional histology and immunohistochemistry.Such information complements classical histology by facilitating virtual slicing of the tissue in any plane and providing unique detail of the three dimensional relationships of tissue compartments.

Audit of practice in SUDEP post mortems and neuropathological findings.

Sudden unexpected death in epilepsy (SUDEP) is one of the leading causes of death in people with epilepsy. For classification of definite SUDEP, a post mortem (PM), including anatomical and toxicological examination, is mandatory to exclude other causes of death. We audited PM practice as well as the value of brain examination in SUDEP.

Audit of practice in sudden unexpected death in epilepsy (SUDEP) post mortems and neuropathological findings.

Sudden unexpected death in epilepsy (SUDEP) is one of the leading causes of death in people with epilepsy. For classification of definite SUDEP, a post mortem (PM), including anatomical and toxicological examination, is mandatory to exclude other causes of death. We audited PM practice as well as the value of brain examination in SUDEP.

Exome Sequencing of an Adult Pituitary Atypical Teratoid Rhabdoid Tumor

Atypical teratoid rhabdoid tumors (AT/RTs) are rare pediatric brain tumors characterized by bialleic loss of the SMARCB1 tumor suppressor gene. In contrast to pediatric AT/RT that has a simple genome, very little is known about the adult AT/RT genomic landscape. Using a combination of whole-exome sequencing and high-resolution SNP array in a single adult pituitary AT/RT, we identified a total of 47 non-synonymous mutations, of which 20 were predicted to cause non-conservative amino acid substitutions, in addition to a subclone of cells with trisomy 8. We suggest that adult AT/RT may not be markedly dissimilar to other adult brain tumors where mutations in a range of genes, reflecting the functional specialization of different brain regions, but including SMARCB1 inactivation, may be required for its pathogenesis.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Spontaneous and tetracosactide-induced anti-ACTH antibodies in man

During a clinical trial of regular tetracosactide depot injections, four of 13 patients with autoimmune Addisons disease (AAD) developed adverse reactions immediately following tetracosactide injections. We wished to investigate whether these adverse effects could be due to the production of circulating antitetracosactide (ACTH1–24) antibodies.

Abstract 5082: Whole-exome sequencing of an adult pituitary atypical teratoid rhabdoid tumor (AT/RT) - A not so simple genome as pediatric AT/RT

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction Atypical teratoid rhabdoid tumors (AT/RT) are rare pediatric embryonal brain tumors. AT/RT are genomically characterized by a very simple genomic landscape caused by inactivation of the SMARCB1 tumor suppressor, chromatin remodelling, gene. Adult AT/RT is a super-rare disease with only 33 cases previously reported. For the first time, we report an adult pituitary AT/RT which has undergone whole-exome sequencing (WES) from a patient who subsequently died of intrachemotherapy leptomeningeal progression. Results In combination, the detection of both somatic biallelic SMARCB1 mutations and the loss of INI-1 protein expression confirmed the diagnosis of AT/RT. Trisomy 8 was detected in a minority of AT/RT cells. The overall somatic mutation rate was 1.9 Mb. A total of 98 somatic mutations were detected of which 20/49 non-synonymous coding mutations were predicted to be potentially damaging to protein function. These 20 genes were stratified into 4 functionally discrete groups: Group 1). tubulin / actin dynamics; Group 2). cell adhesion; Group 3). metabolism; Group 4). cytotoxic resistance mechanisms. Molecular pathway analysis predicted that loss of NOTCH1 signalling could be an important cellular node that was perturbed by a combination of LNX1 and POFUT1 mutations. AT/RT cells did not express any endocrine differentiation markers. Furthermore, 90% of AT/RT cells demonstrated intense nuclear p53 expression despite no TP53 mutations or HDM2 amplification being detected. Conclusions This is the first adult AT/RT to have undergone next-generation sequencing using WES. We have shown that the genomic landscape can be relatively complex in comparison to pediatric primary AT/RT(Lee RS et al 2012. J Clin Invest; 122(8):2983-2988). First, we detected a somatic mutation rate which was four times higher (1.9 Mb versus <0.5 Mb) than pediatric primary AT/RT, but which was comparable to the mutation rate of pediatric metastatic AT/RT. Second, we detected a Chr8 CNV in a minority of cells. Given that Chr8 harbors the loci for a variety of pro-metastatic genes, including C-MYC, MT3-MMP and BNIP3, we hypothesize that the intra-chemotherapy tumor progression was possibly caused by a clonal population of trisomy 8 cells which over-expressed the respective proteins of these specific genes. Given the dedifferentiated phenotype of AT/RT cells and that NOTCH1 signalling is known to be essential for maintaining the pituitary stem cell pool, we hypothesize that the combined effects of LNX1 and POFUT1 mutations could have been important events in the generation of an AT/RT cancer-initiating cell, through loss of NOTCH1 signalling. In this regard, we are currently investigating the expression of NOTCH1 signalling proteins, as well as elucidating the cause of the high levels of nuclear p53 expression within this AT/RT, which we also hypothesize was related to perturbations in NOTCH1 signalling. Note: This abstract was not presented at the meeting. Citation Format: Swethajit Biswas, Madeleine Wood, Abhijit Joshi, Nick Bown, Lisa Strain, Philip Kane, Tommy Martinsson, Javier Garrido, James Campbell, Amanda Swain, Alan Ashworth. Whole-exome sequencing of an adult pituitary atypical teratoid rhabdoid tumor (AT/RT) - A not so simple genome as pediatric AT/RT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2014-5082