Search

Glioblastoma inevitably recurs, but no standard regimen has been established for treating this recurrent disease. Several reports claim that reoperative surgery can improve survival, but the effects of reoperation timing on survival have rarely been investigated. We, therefore, evaluated the relationship between reoperation timing and survival in recurrent GBM. A consecutive cohort of unselected patients (real-world data) from three neuro-oncology cancer centers was analyzed (a total of 109 patients). All patients underwent initial maximal safe resection followed by treatment according to the Stupp protocol. Those meeting the following criteria during progression were indicated for reoperation and were further analyzed in this study: (1) The tumor volume increased by >20-30% or a tumor was rediscovered after radiological disappearance; (2) The patient's clinical status was satisfactory (KS = 70% and PS WHO = gr. 2); (3) The tumor was localized without multifocality; (4) The minimum expected tumor volume reduction was above 80%. A univariate Cox regression analysis of postsurgical survival (PSS) revealed a statistically significant effect of reoperation on PSS from a threshold of 16 months after the first surgery. Cox regression models that stratified the Karnofsky score with age adjustment confirmed a statistically significant improvement in PSS for time-to-progression (TTP) thresholds of 22 and 24 months. The patient groups exhibiting the first recurrence at 22 and 24 months had better survival rates than those exhibiting earlier recurrences. For the 22-month group, the HR was 0.5 with a 95% CI of (0.27, 0.96) and a p-value of 0.036. For the 24-month group, the HR was 0.5 with a 95% CI of (0.25, 0.96) and a p-value of 0.039. Patients with the longest survival were also the best candidates for repeated surgery. Later recurrence of glioblastoma was associated with higher survival rates after reoperation., 65269705; 00159816; 00209805; Ministerstvo Zdravotnictví Ceské Republiky, MZCR: 00098892, NU20-03-00148, NU21-03-00195, Ministry of Health of the Czech Republic [NU21-03-00195, NU20-03-00148, 00098892, 00209805, 65269705, 00159816]

Introduction: Glioblastoma (GBM) is the most frequent primary astrocytoma characterized by an aggressive and diffuse growth and a poor prognosis. The median survival of patients with GBM is only 12 to 16 months from diagnosis despite conventional therapy. It is, therefore, important to identify new therapeutic targets, which would allow a more effective treatment. In our present study, we focus on long non-coding RNAs (lncRNAs), the regulators of gene expression in both physiological conditions and GBM pathology, and their potential as targets for future therapy. Material and Methods: Our study included 219 GBM patients and 29 intractable epilepsy patients as controls who signed an informed consent prior to treatment. Previously, cDNA libraries were prepared from 77 rRNA-depleted RNA samples and sequenced, and the expression of 11 significantly dysregulated lncRNAs was validated in 188 specimens by RT-qPCR. Results were newly validated in an independent TCGA-GBM cohort. Moreover, LINC00634 expression was upregulated in U251 and T98G cells. Cells transfected with an empty vector served as negative controls (NCs). The effect of upregulated LINC00634 expression on viability, migration, and clonogenicity was studied in vitro, and, in the case of U251-derived cell line, in vivo in immunodeficient mice with the focus on GBM growth and diffuse character. Results: Out of the 538 significantly dysregulated lncRNAs (P < 0.001) identified by transcriptome sequencing, a panel of 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM was validated previously by qPCR in another cohort (P < 0.0001 for all lncRNAs) and newly in an independent TCGA-GBM dataset. The upregulated LINC00634 expression in U251 and T98G cells was confirmed by qPCR to be approximately 11,000-fold and 600-fold, respectively, compared to the expression in NCs. Although no significant effect on viability, migration and clonogenicity was observed in vivo, a less diffuse growth pattern was observed in U251-derived GBMs with upregulated expression of LINC00634 in vivo compared with NCs. Conclusion: A significant dysregulation of lncRNAs was previously observed in GBM compared to non-tumor controls using both transcriptome sequencing and RT-qPCR and was newly confirmed also by analyzing the TCGA-GBM dataset. We also showed possible effect of LINC00634 upregulation on the growth pattern of U251-derived GBM tumors in vivo. Our study shows that lncRNAs are dysregulated in GBM and could, therefore, serve as promising diagnostic biomarkers in GBM as well as potential therapeutic targets. The research was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102, funded by the European Union - Next Generation EU). Citation Format: Marek Vecera, Lenka Radova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Dagmar Al Tukmachi, Tana Machackova, Petra Pokorna, Jiri Sana, Ondrej Slaby. Long non-coding RNAs are dysregulated in glioblastoma and LINC00634 may affect the diffuse growth of U251-derived tumors in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3765.

Brain metastases (BMs) comprise a heterogeneous group of the most frequent intracranial tumors in adults, most originating in lung, breast, renal cell, and colorectal carcinomas and melanomas. Despite the recent improvements in imaging methodology resulting in earlier BM identification and advancements in treatment strategies, BMs are still a significant cause of patient morbidity. Furthermore, BMs frequency increases due to more prolonged survival of cancer patients and population aging. Since the most widely used prognostic scoring systems for BMs require prior knowledge of the primary origin and up to 14% of BMs are classified as BMs of unknown primary, there is an urgent unmet need for accurate biomarkers for identification of BM origin. MiRNAs are non-coding RNAs with an approximate length of 22 nucleotides, functioning as post-transcriptional regulators of gene expression. Dysregulated miRNA expression profile has been observed in many pathological processes, including the complex and not fully understood metastatic cascade. These molecules are very stable and present not only in tissues but also in human body fluids, including blood plasma and cerebrospinal fluid (CSF). Based on these facts, both tissue and circulating miRNAs are extensively studied as potential diagnostic biomarkers. Specific miRNA signatures of BMs were obtained using high-throughput miRNA profiling (Illumina small RNA sequencing) on 3 types of samples (metastatic tissue, blood plasma, CSF) from a cohort of 30 patients with BMs originating in the 5 tumor types – lung, breast, renal cell and colorectal carcinomas and melanomas (6 patients per group, 87 samples in total, only 3 CSF samples from RCC patients available). We identified significantly differentially expressed miRNAs in BM tissues with the ability to differentiate between primary origins. Tissue miRNAs could identify BMs originating from breast, colorectal and renal cell carcinomas and melanomas with high specificity and sensitivity. Interestingly, the heterogeneity of lung carcinomas was also characteristic for the corresponding BMs, making it challenging to distinguish accurately from other BMs. Even though the tissue-specific miRNA signature was the most precise, our results suggest a significant diagnostic potential of circulating miRNAs from CSF for BM patients. Therefore, these short and stable molecules could potentially help identify the origin of BMs of unknown primary. The research is supported by project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Dagmar Al Tukmachi, Michaela Ruckova, Marek Vecera, Tana Machackova, Petra Pokorna, Marketa Hermanova, Michal Hendrych, Leos Kren, Ivana Roskova, Vaclav Vybihal, Hana Valekova, Radim Jancalek, Jiri Sana, Martin Smrcka, Ondrej Slaby. Tumor tissue and cerebrospinal fluid microRNA profiles enable the classification of brain metastasis accordingly to their origin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3758.

BACKGROUND Brain metastases (BMs) are intracranial tumors that frequently occur in adult cancer patients. Because the prognosis of patients with BM individually varies, it would be useful to improve prognostic scoring tools by including new high-performance biomarkers. MicroRNAs (miRNAs) appear to be promising in this regard as they are highly stable and, thus, suitable for both next-generation sequencing (RNA-Seq) and retrospective analyses in formalin-fixed and paraffin-embedded (FFPE) tissues.Material and METHODS Total RNA enriched for miRNAs was isolated from 71 freshly frozen histopathologically confirmed BMs with origin in 5 tumor types (ca lung - 37%, melanoma - 23%, ca breast - 18%, RCC - 15%, CRC - 7%) using mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Sequencing libraries were prepared from RNA using the QIAseq miRNA Library Kit (Qiagen) and sequenced using the NextSeq 500 platform (Illumina). The miRNA molecules were subsequently transcribed from total RNA samples isolated from a retrospective set of 119 FFPE tissues using the TaqMan Advanced miRNA cDNA Synthesis Kit, and the expression of selected miRNAs was validated in a pilot experiment by qPCR using TaqMan Fast Advanced Master Mix and appropriate TaqMan MicroRNA Assays (all from Thermo Fisher Scientific). RESULTS The differential analysis identified 373 miRNAs with significantly different expression among the 5 BMs groups (p< 0.001). A molecular classifier based on the expression of 32 miRNAs was able to classify all samples correctly. Out of these, seven, including miR-122-5p, miR-141-3p, miR-146a-5p, miR-194-5p, miR-200c-3p, miR-211-3p, and miR-215-5p, were chosen for subsequent validation and their significantly different expression in 5 BMs groups was validated. CONCLUSIONS Presented results confirm the importance of studying dysregulated miRNA expression in BM and the diagnostic potential of validated miRNAs. The study was prepared with the grant support of the Ministry of Health of the Czech Republic - grant No. NV18-03-00398.

INTRODUCTION Modern comprehensive local treatment of BM provides the possibility of adequate local control while maintaining a satisfactory quality of life. METHODS Patients after metastasectomy and postoperative radiotherapy treated between 2007-2020 were evaluated. Local control was assessed separately at the site of the operated MM (localPFS) and separately in other parts of the brain (distalPFS). RESULTS A total of 118 patients were enrolled (70 with WBRT), 54% were women, the median age was 60 years, the median performance status Karnofsky 80%. The most common primary tumor was lung carcinoma (39/118, 33%). Targeted stereotactic radiotherapy was significantly more often indicated in operations performed after 2016 (p < 0.001). In the period 2013-2019, gross total resection was achieved significantly more often (83 vs 64%; p = 0.061). The median follow-up is 49 months. The median overall survival (OS) is 9 months (6.2 - 12), the median localPFS 22 months (14 - unattainable), median distPFS 11 months (6.8 - 27 months), median extracranialPFS 11 months (5.9 - 15). Stratification of patients according to the prognostic index brainmetgpa.com led to significant separation of patients for OS (p = 0.00017). In a multivariate analysis, stereotactic radiotherapy was statistically significant positive prognostic factor for OS compared to WBRT (median OS 17 vs. 5.6 months, HR 0.59, p = 0.018) with no effect on localPFS. CONCLUSION Our results of comprehensive local treatment of BM are comparable with the results presented by important foreign studies from prestigious oncology centers. We described targeted stereotactic postoperative radiotherapy as an independent positive prognostic factor after brain metastasectomy. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV18-03-00398, NV19-03-00501 and NV19-03-00559 and Supported by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705).

Meningiomas are the most common primary tumors of the central nervous system (CNS). Atypical meningioma (AM) recurs in 40% of patients despite total resection and radiotherapy (RT). No consensus on optimal adjuvant management was found, and it is difficult to identify patients insensitive to RT in real-life clinical practice. A promising group of biomarkers represent microRNAs (miRNAs), short non-coding RNAs that regulate most biological processes, including cell proliferation, differentiation, and apoptosis. The study aims to identify tissue miRNAs capable of predicting patients with AM who could benefit from the indicated adjuvant RT. The study includes 80 patients with AM in the exploratory phase and 400 patients with meningioma in the validation phase. Total RNA enriched with miRNAs was isolated from FFPE tissue using the mirVana miRNA Isolation Kit (TF Scientific). Subsequently, RNA quantity and quality controls were measured using NanoDrop 2000 (TF Scientific) and Qubit 2.0 (TF Scientific) instruments. A global miRNA expression profile was generated using the TaqMan Array Human MicroRNA Cards (TF Scientific), which allow the detection of up to 754 miRNAs simultaneously. Obtained data were processed and integrated through bioinformatics algorithms with clinicopathological data of patients with AM.The study identified significantly dysregulated miRNAs among AM patients with and without recurrence (p < 0.05). Results also suggest dysregulated miRNA expression profile in AM patients with indicated RT who did/did not develop a recurrence (p < 0.05). Lists of individual miRNAs and detailed graphical analyses will be included in the conference presentation.The results will help predict the prognosis of surgically intervened patients more accurately and can help determine which patients will benefit from the adjuvant RT. This research is supported by the AZV grant from the Ministry of Health of the Czech Republic (reg. No. NV19-03-00559) and Conceptual development of research organization (FNBr, 65269705). All rights reserved.

Brain metastases are the most frequent intracranial tumors in adults and the cause of death in almost one-fourth of cases. The incidence of brain metastases is steadily increasing. The main reason for this increase could be the introduction of new and more efficient therapeutic strategies that lead to longer survival but, at the same time, cause a higher risk of brain parenchyma infiltration. In addition, the advances in imaging methodology, which provide earlier identification of brain metastases, may also be a reason for the higher recorded number of patients with these tumors. Metastasis is a complex biological process that is still largely unexplored, influenced by many factors and involving many molecules. A deeper understanding of the process will allow the discovery of more effective diagnostic and therapeutic approaches that could improve the quality and length of patient survival. Recent studies have shown that microRNAs (miRNAs) are essential molecules that are involved in specific steps of the metastatic cascade. MiRNAs are endogenously expressed small non-coding RNAs that act as post-transcriptional regulators of gene expression and thus regulate most cellular processes. The dysregulation of these molecules has been implicated in many cancers, including brain metastases. Therefore, miRNAs represent promising diagnostic molecules and therapeutic targets in brain metastases. This review summarizes the current knowledge on the importance of miRNAs in brain metastasis, focusing on their involvement in the metastatic cascade and their potential clinical implications.

Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.

BACKGROUND One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may then play a key role in their biology, particularly in germ and stem cells. A number of piRNA molecules are also promising biomarkers useful for prognosis and prediction of disease progression.Material and METHODS Glioblastoma stem cells were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture differentiated cells. RNA was isolated from the selected paired primocultures and subsequently checked for quality by capillary electrophoresis. NEBNext Small RNA Library Prep Set was used for the preparation of cDNA libraries. Sequencing analysis was performed using the Next500/550 High Output v2 Kit for 75 cycles and the NextSeq500 sequencer. RESULTS Twenty paired primary cultures were prepared to verify the expression of CD133 and SOX2 in GSCs, the ability to establish tumors in immunodeficient mice, and the ability to differentiate. At this time, we finished sequencing of piRNA molecules and acquired data are bioinformatically analysed. Subsequently, we will perform analysis of selected piRNAs to be diagnostic and prognostic biomarkers on the GBM samples and non-tumor brain controls. All results will be presented at the conference. CONCLUSION Based on recent studies, it is evident that piRNA expression can have a significant impact on GBM biology. Their expression within GSCs may then provide a tool for identifying and targeting these cells and could be usable as a diagnostic and prognostic biomarker in GBM patients.The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501, NV19-03-00559, Conceptual development of research organization FNBr, 65269705

Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all p < 0.0001), and piR-9491 and piR-12488 reduced GBM cells’ ability to form colonies in vitro. In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen (p = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.

The aim of this retrospective study is to provide real-world evidence in glioblastoma treatment and to compare overall survival after Stupp's regimen treatment today and a decade ago. A current consecutive cohort of histologically confirmed glioblastoma irradiated from 1/2014 to 12/2017 in our cancer center was compared with an already published historical control of patients treated in 1/2003–12/2009. A total of new 155 patients was analyzed, median age 60.9 years, 61% men, 58 patients (37%) underwent gross total tumor resection. Stupp's regimen was indicated in 90 patients (58%), 65 patients (42%) underwent radiotherapy alone. Median progression-free survival in Stupp's regimen cohort was 6.7 months, median OS 16.0 months, and 2-year OS 30.7%. OS was longer if patients were able to finish at least three cycles of adjuvant chemotherapy (median 23.3 months and 43.9% of patients lived at 2 years after surgery). Rapid early progression prior to radiotherapy was a negative prognostic factor with HR 1.87 (p = 0.007). The interval between surgery and the start of radiotherapy (median 6.7 weeks) was not prognostically significant (p = 0.825). The median OS in the current cohort was about 2 months longer than in the historical control group treated 10 years ago (16 vs. 13.8 months) using the same Stupp's regimen. Taking into account differences in patient's characteristics between current and historical cohorts, age, extent of resection, and ECOG patient performance status adjusted HR (Stupp's regimen vs. RT alone) for OS was determined as 0.45 (p = 0.002).

Diffuse gliomas with K27M histone mutations (H3K27M glioma) are generally characterized by a fatal prognosis, particularly affecting the pediatric population. Based on the molecular heterogeneity observed in this tumor type, personalized treatment is considered to substantially improve therapeutic options. Therefore, clinical evidence for therapy, guided by comprehensive molecular profiling, is urgently required. In this study, we analyzed feasibility and clinical outcomes in a cohort of 12 H3K27M glioma cases treated at two centers. Patients were subjected to personalized treatment either at primary diagnosis or disease progression and received backbone therapy including focal irradiation. Molecular analyses included whole-exome sequencing of tumor and germline DNA, RNA-sequencing, and transcriptomic profiling. Patients were monitored with regular clinical as well as radiological follow-up. In one case, liquid biopsy of cerebrospinal fluid (CSF) was used. Analyses could be completed in 83% (10/12) and subsequent personalized treatment for one or more additional pharmacological therapies could be recommended in 90% (9/10). Personalized treatment included inhibition of the PI3K/AKT/mTOR pathway (3/9), MAPK signaling (2/9), immunotherapy (2/9), receptor tyrosine kinase inhibition (2/9), and retinoic receptor agonist (1/9). The overall response rate within the cohort was 78% (7/9) including one complete remission, three partial responses, and three stable diseases. Sustained responses lasting for 28 to 150 weeks were observed for cases with PIK3CA mutations treated with either miltefosine or everolimus and additional treatment with trametinib/dabrafenib in a case with BRAFV600E mutation. Immune checkpoint inhibitor treatment of a case with increased tumor mutational burden (TMB) resulted in complete remission lasting 40 weeks. Median time to progression was 29 weeks. Median overall survival (OS) in the personalized treatment cohort was 16.5 months. Last, we compared OS to a control cohort (n = 9) showing a median OS of 17.5 months. No significant difference between the cohorts could be detected, but long-term survivors (>2 years) were only present in the personalized treatment cohort. Taken together, we present the first evidence of clinical efficacy and an improved patient outcome through a personalized approach at least in selected cases of H3K27M glioma.