Abstract BACKGROUND Diffuse midline glioma (DMG) is a devastating pediatric high-grade glioma (pHGG) located along the midline structures of the brain and spine. Palliative radiotherapy is the only approved treatment outside of clinical trials, with patients given 9–11-months survival post-diagnosis. Over the last 10-years, it has become common for patients to be prescribed precision therapies targeting genetic alterations, however largely, DMG harbor genetic alterations that are untargetable. METHODS To improve DMG patient outcomes, we are performing multi-omic analysis on 210 DMG, normal controls and other pHGG samples to functionally characterize the genomic landscape of these tumors and understand the protein-controlled functional consequences of recurring somatic alterations. RESULTS Genomic sequencing has been performed on 40/210 pHGG samples, identifying 290 unique mutations and several recurrent chromosomal alterations. Additionally, proteomic/phosphoproteomic profiling identified both unique and similar features of post-translational landscapes across genotypes. Examining H3.3K27M DMG cell-lines, SU-DIPG-XIII and UON-VIBE5, genomic sequencing identified 11 and 6 mutations respectively, however, only UON-VIBE5 possessed a genomically-predicted target, a PDGFRA-D842V mutation, potentially targetable by Avapritinib. Conversely, investigation of proteomes/phosphoproteomes revealed several potential therapeutic vulnerabilities. SU-DIPG-XIII possessed high enrichment of HDAC/BRD and CDK pathways and UON-VIBE5, ATM-centric pathways. In vitro proliferation assays of Avapritinib treated UON-VIBE5 failed to elicit a response at clinically relevant doses, while all proteomically-predicted therapies (HDAC – Fimepinostat, BRD – JQ1, CDK – Ribociclib and ATM – WSD-0628) successfully influenced tumor cell growth whilst leaving microglia controls cells unaffected. Avapritinib and WSD-0628 (CNS-active ATM inhibitor) drug predictions were further validated in the RA055 DMG model, which possesses similar genomic and proteomic features to UON-VIBE5. Avapritinib alone failed to influence tumor growth, while WSD-0628 notably reduced cell proliferation. The combination of these drugs, however, synergistically inhibited RA055 proliferation. CONCLUSIONS These findings thus provide preliminary evidence supporting the need to take a multi-omic approach in treating DMGs.

Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death in children and young adults. Current treatment strategies are centered on maximal safe resection, followed by radiotherapy, and interrogation of the tumor genome to identify targetable mutations. Unfortunately, we are yet to see an improvement in patient outcomes with a median overall survival remaining 15-months. To improve patient outcomes, we have begun to characterize the genome, proteome, and phosphoproteome of 168 pHGGs to better understand the functional consequences of their somatic alterations as well as their influence of the tumor microenvironment. Employing a novel ‘pharmaco-phospho-proteo-genomics’ pipeline, we have analyzed pHGG cell lines and tumor tissue specimens at diagnosis, relapse (partial resection congenital glioblastoma), and autopsy. Genomic profiling was conducted utilizing the 523-gene TruSight Oncology 500 (TSO500) next-generation sequencing panel. Simultaneously, tumor proteomes and phosphoproteomes were characterized using our high-throughput global phospho-proteomic profiling technique termed pHASED (phospho Heavy-labelled-spiketide FAIMS StEpped-CV DDA). High-fidelity tumor proteomic and phospho-proteomic data were identified and compared to normal control brain samples. Across 40 pHGG samples, we identified 290 unique somatic alterations with a high predicted impact severity and quantified 7,345 unique proteins and 3,327 phosphoproteins. Gene panel sequencing of a critical pediatric glioblastoma patient sample identified 18 somatic alterations, eight of which had a high predicted impact severity, however, none were targetable. Conversely, phosphoproteomic profiling identified enriched MAPK and PRKCB signaling, relative to normal brain tissues, thereby encouraging the use of the TGA/FDA approved therapies trametinib (MAPKs) and enzastaurin (PRKCB). In vitro investigations confirmed the utility of these treatment approaches and in vivo patient derives xenograft mouse models for this sample are under investigation. This pilot study provides critical data to support the benefit of interrogating the genome, proteome, and phospho-proteome of these devastating tumours to aid in the selection/development of effective treatment strategies.

Instrumented fixation of the C1-C2 motion segment is a standard surgical technique to stabilise that spinal segment. Instability at C1-C2 can arise from a number of conditions. Fixation of the C1 lateral mass usually involves dissection and exposure of the C2 nerve root and the posterior wall of the C2 lateral mass which can result in significant bleeding from the venous plexus. Whilst image guidance is increasing in accessibility, there are few public hospitals in Australia that have access to this technology. The authors describe their technique for insertion of a C1 lateral mass screw over a threaded K-wire to avoid extensive dissection of the C2 nerve root, reducing the risk of significant haemorrhage from the epidural venous plexus during the procedure. A retrospective analysis was undertaken on 18 consecutive patients who underwent C1-C2 instrumented fixation using this technique. Indications for C1-C2 instrumented fixation included traumatic injury (10 patients), failure of non-operative management of odontoid fractures (5 patients), pathological fractures of C2 (2 patients) and inflammatory conditions (1 patient). All patients underwent successful C1-C2 stabilisation using this technique. Blood loss did not exceed 400mls in any patient. There were no vertebral artery injuries and no patient experienced a neurological deterioration. The authors propose that their technique for insertion of a C1 lateral mass screw over a threaded K-wire is safe and effective with a low risk of neurological or vertebral artery injury. The technique may be considered as a slight modification of the Harm's procedure to reduce disturbance of the adjacent venous plexus and thereby reduction in intraoperative bleeding and operative time.

A 54-year-old man presented with two episodes of dysarthria and left facial droop. Both episodes resolved by the time of examination. MRI of the brain revealed a right frontotemporal, heterogeneously enhancing mass with surrounding vasogenic oedema, suggestive of a high-grade primary brain neoplasm. The patient was administered preoperative 5-aminolevulinic acid hydrochloride (Gliolan), and fluorescence-guided resection of the lesion was undertaken. Cryptococcus gattii infection was diagnosed from the specimen and the patient was given appropriate antifungal treatment. This is the first reported case of Gliolan-mediated fluorescence in a fungal abscess and highlights one of the potential pitfalls in fluorescence-guided surgery.