Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy.

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. Patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations (“non-R132H IDH1/2 mutations”). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1R132H have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1R132H mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.

Background The randomized phase II INTELLANCE-2/EORTC_1410 trial on EGFR-amplified recurrent glioblastomas showed a trend towards improved overall survival when patients were treated with depatux-m plus temozolomide compared with the control arm of alkylating chemotherapy only. We here performed translational research on material derived from this clinical trial to identify patients that benefit from this treatment. Methods Targeted DNA-sequencing and whole transcriptome analysis was performed on clinical trial samples. High-throughput, high-content imaging analysis was done to understand the molecular mechanism underlying the survival benefit. Results We first define the tumor genomic landscape in this well-annotated patient population. We find that tumors harboring EGFR single-nucleotide variations (SNVs) have improved outcome in the depatux-m + TMZ combination arm. Such SNVs are common to the extracellular domain of the receptor and functionally result in a receptor that is hypersensitive to low-affinity EGFR ligands. These hypersensitizing SNVs and the ligand-independent EGFRvIII variant are inversely correlated, indicating two distinct modes of evolution to increase EGFR signaling in glioblastomas. Ligand hypersensitivity can explain the therapeutic efficacy of depatux-m as increased ligand-induced activation will result in increased exposure of the epitope to the antibody–drug conjugate. We also identified tumors harboring mutations sensitive to “classical” EGFR tyrosine-kinase inhibitors, providing a potential alternative treatment strategy. Conclusions These data can help guide treatment for recurrent glioblastoma patients and increase our understanding into the molecular mechanisms underlying EGFR signaling in these tumors.

The EGFR gene is one of the most frequently mutated and/or amplified gene both in lung adenocarcinomas (LUAD) and in glioblastomas (GBMs). Although both tumor types depend on the mutation for growth, clinical benefit of EGFR tyrosine kinase inhibitors (TKIs) has only been observed in LUAD patients and, thus-far, not in GBM patients. Also in LUAD patients however, responses are restricted to specific EGFR mutations only and these ‘TKI-sensitive’ mutations hardly occur in GBMs. This argues for mutation-specific (as opposed to tumor-type specific) responses to EGFR-TKIs. We here discuss potential reasons for the differences in mutation spectrum and highlight recent evidence for specific functions of different EGFR mutations. These mutation-specific effects likely underlie the differential treatment response between LUAD and GBMs and provide new insights into how to target EGFR in GBM patients.

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Background The fifth edition of the WHO Classification of Tumors of the Central Nervous System (WHO CNS 5), includes molecular parameters for both diagnosis and grading in addition to histological features. For IDH-mutant astrocytoma, homozygous deletion (HD) of CDKN2A/B now results in WHO grade 4, even in the absence of microvascular proliferation or necrosis. CDKN2A/B deletions can be determined by various techniques including shallow and targeted sequencing, and using genome wide DNA-methylation arrays. Various algorithms to call deletions also exist for each platform. Concordance between the various techniques and algorithms is however unknown. Because of the importance to properly call CDKN2A/B deletions, we compared two techniques to call HD in IDH-mutant astrocytoma patients. Methods Samples from 110 IDH-mutant astrocytoma patients enrolled in the GLASS-NL study, and therefore samples from at least two surgical resections per patient, were available. Overall survival (OS) was measured from date of first surgery. Both DNA-methylation data and shallow whole-genome sequencing (sWGS) was collected from 219 samples from 101 patients. For DNA-methylation analysis, HD of CDKN2A/B was defined by &lt;-0.6 log2 intensity combined with visual assessment on samples with log2 intensity between -0.6 and -0.2 (method 1), or by using a hard cutoff of &lt;-0.415 log2 intensity, as determined by Shirahata et al. (method 2). Absolute copy number profiles were estimated by ACE from sWGS data, HD of CDKN2A/B was defined by a loss of at least 1.2 CDKN2A alleles (method 3). Agreement score and Cohen’s kappa (κ) as an index for interrater agreement was calculated. Results Method 1 and method 2 called an HD of CDKN2A/B in 39 and 33 cases, respectively; method 1 called an HD of CDKN2A/B in 9 cases in which method 2 did not detect an alteration. The agreement was strong with 95% agreement and κ = 0.842. Method 3 called an HD of CDKN2A/B in 45 cases of which 12 and 19 were not detected by method 1 and method 2 respectively. Method 3 showed an almost perfect level of agreement with method 1, with 97% agreement and κ = 0.911, and a moderate agreement with method 2, with 89% agreement and κ = 0.671. Survival analysis showed that there was no significant difference in survival when patients were stratified by CDKN2A/B status of the initial sample of all methods. However, when stratified by CDKN2A/B status of the first recurrence, all methods showed a stronger association with OS (method 1: p = 0.001, HR = 2.61[1.44-4.72]; method 2: p = 0.0024, HR = 2.34[1.33-4.11]; method 3: p = 0.0099, HR = 2.34[1.2-4.55]). Conclusion Different methods and different cutoffs to determine the presence of a HD in CDKN2A/B may result in different test outcomes. Quality of the DNA, tumor cell percentage of the sample and ploidy are likely to influence the call of homozygous CDKN2A/B deletion. Ring tests are recommended to improve reliability.

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Tumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.