Novel gene fusions identified as new drug targets in paediatric glioma and their pre-clinical characterisation

Gliomas are the most common paediatric brain tumours, accounting for about 50% of all brain tumours in children. They are typically classified by the putative cell type they arise from based on morphology, the location they are found and malignancy grade. The most common glioma found in adults is glioblastoma, which is a WHO grade IV tumour with an average survival of less than 15 months. Extensive work has gone into characterising, modelling and treating this tumour in vivo and in vitro, but it is now clear that glioblastoma in children is biologically very different from that in adults. Paediatric high-grade gliomas do, however, share their aggressiveness with their adult counterparts, with few patients achieving long-term survival - dictating an urgency to find more precise therapies for these patients. Additionally, relatively little work has focused on low-grade gliomas to date. Nevertheless, these tumours also deserve a lot of attention, because low-grade gliomas make up 30-50% of all paediatric brain tumours and more than half of all paediatric gliomas. Although the tumour itself does not necessarily lead to a massively reduced life span and should rather be considered a chronic disease, the impact on the patients’ and families’ lives caused by the therapy load and possible recurrences remains a major clinical burden. Unlike high-grade gliomas, which are very heterogeneous with multiple oncogenic drivers, most low-grade gliomas are driven by alterations in the mitogen-activated protein kinase (MAPK) pathway through different mechanisms. Examples are BRAF alterations, FGFR1 mutations, and NTRK or MYB fusions. After analysing around 200 paediatric glioma samples by whole-genome/whole-exome and RNA sequencing within the ICGC PedBrain Tumour Project and the INFORM personalised medicine study, fusions involving an additional candidate gene (ALK) were found to be of interest in terms of their pattern of occurrence and availability of targeted inhibitors. Clinically these fusions arose in an interesting patient population, affecting infants (<2 years old) with histologically malignant tumours that however showed outcomes more similar to low-grade glioma than glioblastoma. PPP1CB:ALK was the most common gene fusion found in this context, which was then modelled in vivo using two different state-of-the-art methodologies: in utero electroporation and p0 injection using the RCAS-tva system. Through in utero electroporation, a novel mouse model was generated that nicely recapitulates the human tumours. Different ALK-specific inhibitors already used in clinical trials for other tumours, like non-small cell lung cancer or neuroblastoma, were tested on the tumour cells in an in vitro sphere culture setting and in addition also in vivo on allografted tumour cells. The results show a promising effect of the third-line, blood-brain-barrier penetrant ALK inhibitor lorlatinib, with IC50 values below 1.3nM and a significant increase in lifespan with a decrease in tumour signal, respectively. Thus, the project led from the genomic discovery of a novel driving event in paediatric glioma through to its modelling and identification of a promising new option for therapy. Further proof-of-concept application with other oncogenic combinations leads to the conclusion that the mouse model strategy and the methodology behind this can be further applied to test other candidate genes and specific inhibitor therapies. The overall aim is thus to accelerate the approval of targeted drugs by authorities after running a stratified clinical trial on a small infant patient population carrying the gene of interest, to enable patients to get the most potent therapy with the fewest side effects

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors

Surgical Debridement Is Superior to Sole Antibiotic Therapy in a Novel Murine Posttraumatic Osteomyelitis Model

<div><p>Introduction</p><p>Bone infections after trauma, <i>i</i>.<i>e</i>. posttraumatic osteomyelitis, pose one of the biggest problems of orthopedic surgery. Even after sufficient clinical therapy including vast debridement of infected bone and antibiotic treatment, regeneration of postinfectious bone seems to be restricted. One explanation includes the large sized defects resulting from sufficient debridement. Furthermore, it remains unclear if inflammatory processes after bone infection do affect bone regeneration. For continuing studies in this field, an animal model is needed where bone regeneration after sufficient treatment can be studied in detail.</p><p>Methods</p><p>For this purpose we created a stable infection in murine tibiae by <i>Staphylococcus aureus</i> inoculation. Thereafter, osteomyelitic bones were debrided thoroughly and animals were subsequently treated with antibiotics. Controls included debrided, non-infected, as well as infected animals exclusively treated with antibiotics. To verify sufficient treatment of infected bone, different assessments detecting <i>S</i>. <i>aureus</i> were utilized: agar plates, histology and RT-qPCR.</p><p>Results</p><p>All three detection methods revealed massive reduction or eradication of <i>S</i>. <i>aureus</i> within debrided bones 1 and 2 weeks postoperatively, whereas sole antibiotic therapy could not provide sufficient treatment of osteomyelitic bones. Debrided, previously infected bones showed significantly decreased bone formation, compared to debrided, non-infected controls.</p><p>Discussion</p><p>Thus, the animal model presented herein provides a reliable and fascinating tool to study posttraumatic osteomyelitis for clinical therapies.</p></div

Results of qRT-PCR detecting Staph aureus DNA.

<p>The amount of <i>S</i>. <i>aureus</i> within the infected bone of deb.W1 and 2 and nondeb.W1 and 2 in comparison to initial amount of bacteria before debridement (Day 0) is depicted. Significant differences between deb. and nondeb.W1 and 2 could be found.</p

GRAM staining of infected tibiae.

<p>Bacteria could only be observed within the defect site, whereas surrounding tissue showed complete absence of GRAM-positive bacteria. Numbers in overview above correspond with insets, which are enlarged below. Scale bar represents 10 μm.</p

GRAM staining of defects of deb.W1 and 2 and nondeb.W1 and 2.

<p>Whole defect was examined and characteristic photographs were taken in order to detect <i>S</i>. <i>aureus</i>. In deb.W1, <i>S</i>. <i>aureus</i> could be detected in two animals sporadically. No bacteria at all could be descried after 2 weeks of antibiotic treatment in deb.W2. Characteristic clusters of <i>S</i>. <i>aureus</i> could be detected in all animals of nondeb.W1 and 2. Scale bar represents 10 μm.</p

Bone smears on culture plates as infection control.

<p>Culture plates (CHROMagar Staph aureus, BD, Franklin (USA)) with smears of infected bone before debridement and smears taken from animals of deb.W1 and 2 and nondeb.W1 and 2.</p

Sequence of surgical procedures.

<p>Upper row from left to right: 1. Drilling a 1 mm hole into proximal mouse tibia; 2. Inoculation of bacteria with a hamilton syringe; 3. Sealing of the defect with bone wax. Lower row from left to right: 1. Exposition of infected bone; 2. Debriding infected bone tissue with injection needle; 3. Thorough flushing of debrided bone.</p

Surgical debridement Is superior to sole antibiotic therapy in a novel murine posttraumatic osteomyelitis model

$\bf{Introduction}$ Bone infections after trauma, $\textit{i.e.}$ posttraumatic osteomyelitis, pose one of the biggest problems of orthopedic surgery. Even after sufficient clinical therapy including vast debridement of infected bone and antibiotic treatment, regeneration of postinfectious bone seems to be restricted. One explanation includes the large sized defects resulting from sufficient debridement. Furthermore, it remains unclear if inflammatory processes after bone infection do affect bone regeneration. For continuing studies in this field, an animal model is needed where bone regeneration after sufficient treatment can be studied in detail. $\bf{Methods}$ For this purpose we created a stable infection in murine tibiae by $\textit{Staphylococcus aureus}$ inoculation. Thereafter, osteomyelitic bones were debrided thoroughly and animals were subsequently treated with antibiotics. Controls included debrided, non-infected, as well as infected animals exclusively treated with antibiotics. To verify sufficient treatment of infected bone, different assessments detecting $\textit{S. aureus}$ were utilized: agar plates, histology and RT-qPCR. $\bf{Results}$ All three detection methods revealed massive reduction or eradication of $\textit{S.aureus}$ within debrided bones 1 and 2 weeks postoperatively, whereas sole antibiotic therapy could not provide sufficient treatment of osteomyelitic bones. Debrided, previously infected bones showed significantly decreased bone formation, compared to debrided, non-infected controls. $\bf{Discussion}$ Thus, the animal model presented herein provides a reliable and fascinating tool to study posttraumatic osteomyelitis for clinical therapies