Patients with recurrent glioblastoma achieving response to bevacizumab combined with chemotherapy have clinical improvement and prolonged survival. High gene expression of angiotensinogen (AGT) is associated with a poor bevacizumab response. Because AGT expression is epigenetically regulated, we aimed to investigate whether AGT promoter methylation in tumor tissue predicts response to bevacizumab combination therapy in patients with recurrent glioblastoma. The study included 159 patients with recurrent glioblastoma, treated with bevacizumab combination treatment (training cohort, n = 77; validation cohort, n = 82). All patients could be evaluated for treatment response and biomarkers. DNA methylation of 4 CpG sites in the AGT promoter was measured using pyrosequencing. A model for nonresponse was established using logistic regression analysis. In the training cohort, lower methylation of each of the four CpG sites in the AGT promoter was significantly associated with nonresponse (all P < 0.05). Moreover, the mean methylation level of all four CpG sites was associated with an increased likelihood of not achieving response to bevacizumab combination therapy (twofold decrease: odds ratio = 3.01; 95% confidence interval: 1.41-6.44; P = 0.004). We developed a model for nonresponse in the training cohort, where a threshold of mean AGT promoter methylation levels was set to below 12%. The model could predict bevacizumab nonresponse with 96% specificity. Importantly, this predictor was also significantly associated with nonresponse in the validation cohort (P = 0.037). Taken together, our findings suggest that low AGT promoter methylation in tumor tissue predicts nonresponse to bevacizumab combination treatment in patients with recurrent glioblastoma. We have, thus, established and successfully validated a predictor for nonresponse that can be used to identify patients who will not benefit from bevacizumab combination therapy.Abbreviations 95% CI, 95% confidence interval; AGT, angiotensinogen; AUC, area under the receiving operating characteristic curve; CEBP, CCAAT/ enhancer-binding protein; CR, complete response; EIAED, enzyme-inducing anti-epileptic drug; OR, odds ratio; PD, progressive disease; PR, partial response; PS, ECOG performance status; SD, stable disease; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor A. 964Molecular Oncology 14 (2020) 964-973 ª

Thanks to clinically newly introduced inhibitors of the mesenchymal–epithelial transition (MET) receptor tyrosine-kinase, MET-gene copy number gain/amplification (MET-GCNG/GA) and increased expression of the MET protein are considered very promising therapeutic targets in lung cancer and other malignancies. However, to which extent these MET alterations occur in malignant mesothelioma (MM) remains unclear. Thus, we investigated by well-established immunohistochemistry and fluorescence in situ hybridization methods, the frequency of these alterations in specimens from 155 consecutive MMs of different subtypes obtained from pleural or peritoneal biopsies and pleurectomies. Thirty-three benign reactive mesothelial proliferations (RMPs) were used as controls. MET-protein upregulation was observed in 35% of all MM-cases, though restricted to predominantly epithelioid MMs. We detected low-/intermediate-level MET-GCNG/GA in 22.2% of MET-overexpressing MMs (7.8% of whole MM-cohort) and no MET-GCNG/GA in the other 77.8%, suggesting other upregulating mechanisms. In contrast, 100% of RMPs exhibited no MET-upregulation or MET-GCNG/-GA. Neither MET exon 14 skipping mutations nor MET-fusions were detected as mechanisms of MET overexpression in MM using RNA next-generation sequencing. Finally, in two cohorts of 30 MM patients with or without MET overexpression (MET-positive/-negative) that were matched for several variables and received the same standard chemotherapy, the MET-positive cases showed a significantly lower response rate, but no significant difference in progression-free or overall survival. Our results imply that MET overexpression occurs in a substantial fraction of predominantly epithelioid MMs, but correlates poorly with MET-amplification status, and may impact the likelihood of response to mesothelioma standard chemotherapy. The predictive significance of MET-IHC and -FISH for possible MET-targeted therapy of MM remains to be elucidated.

Background Glioblastoma is an aggressive brain tumour with a median survival of less than 15 months despite aggressive standard treatment consisting of neurosurgery, radiotherapy plus concomitant and adjuvant chemotherapy with temozolomide. There is no standard treatment at recurrence, and all have limited efficacy. In this study we will enroll glioblastoma patients from the Danish clinical trial “Protarget” to get a better understanding of resistance mechanisms and vulnerabilities in glioblastoma to give personalized medicine to glioblastoma patients at recurrent disease. We hypothesise that two connected factors are responsible for the failure to successfully target glioblastoma: i) intra-tumour heterogeneity and within this ii) cancer stem cells. Material and Methods We will use tumour tissue from glioblastoma patients included in the Danish phase 2, prospective, non-randomized clinical trial “Protarget” to establish short-term cultured neurospheres to preserve the heterogeneity of the tumour including cancer stem cells. We will then perform single cell RNA sequencing before and after drug screening. This setup will allow for a new approach to identify drug vulnerabilities at the single cell level. If successful in identifying drugs that leads to a clinical response, according to the "ProTarget" guidelines, we will consider scaling up the effort, to allow broader evaluation of efficacy. Our plan is to enroll 10 patients as a start. Patients will be selected based on ProTarget inclusion criteria and molecular profiling. We will further select patients with the following characteristics:1. IDH wildtype 2. High tumour purity (&gt;50%). 3. High degree of intra-tumour heterogeneity. Results Enrollment for "Protarget" began in 2020 and our cohort will be part of this study, which is why enrollment has already started, but the first patient has not been enrolled yet. Clinical trial registry number for "Protarget" is NCT04341181. Conclusion This new and flexible approach to identify drug vulnerabilities at the single cell level in glioblastoma in order to find targeted therapies is a promising tool for future treatment of glioblastoma patients.