Abstract
BACKGROUND
Glioblastoma patients showing hypermethylation of the promoter of the O6-methylguanine-methyltransferase (MGMT) gene have significantly improved survival when treated with ...temozolomide compared to patients with hypomethylated MGMT promoters. However, the prognostic and predictive significance of partial MGMT promoter methylation is unclear.
METHODS
The National Cancer Database was queried for patients newly diagnosed in 2018 with histopathologically-confirmed IDH-wildtype glioblastoma. MGMT promoter methylation status was correlated with overall survival (OS) using multivariable Cox regression with Bonferroni correction for multiple testing (p< 0.008 was significant).
RESULTS
3,663 patients were identified with newly diagnosed, histopathologically-confirmed IDH-wildtype glioblastoma and known MGMT promoter methylation. Of the 2,807 patients who received single agent chemotherapy (i.e. likely temozolomide), the MGMT promoter was unmethylated, partially methylated, hypermethylated, and methylated not otherwise specified (NOS) in 57.9%, 5.0%, 3.1%, and 34.0% of cases, respectively, with the later group likely consisting predominantly of hypermethylated cases. Multivariable analysis demonstrated that among patients receiving temozolomide, those with unmethylated MGMT promoters had significantly worse OS than patients with partially methylated MGMT promoters (hazard ratio (HR) 1.94; 95% confidence interval (CI), 1.54-2.44; p< 0.001). However, the OS of hypermethylated patients (HR 1.02; 95% CI, 0.72-1.46; p=0.9) and methylated NOS patients (HR 0.99; 95% CI, 0.78-1.26; p=0.928) were not statistically different from the OS of partially methylated patients.
CONCLUSIONS
IDH-wildtype glioblastoma patients with partial methylation of the MGMT promoter treated with temozolomide have improved OS compared to their unmethylated counterparts, supporting the use of temozolomide therapy in these patients.
Abstract
Introduction
Following the results from the REGOMA study, regorafenib has become the first chemotherapeutic option for recurrent glioblastoma, IDH-wildtype, in many countries. However, ...predictive factors for response to regorafenib are scarcely recognized. The objective of this study was to identify molecular predictive factors for response to regorafenib using a clinically available platform.
METHODS
We analyzed a prospective cohort of 30 patients harboring recurrent glioblastoma, IDH-wildtype, and treated with regorafenib. Next-generation sequencing (NGS) analysis was performed on DNA extracted from paraffin-embedded tissues using a rapid, cheap, and clinically validated platform. MGMT methylation was assessed using methylation-specific PCR, and EGFRvIII expression was assessed using RT-PCR.
RESULTS
In our series, six-month progression-free survival (PFS) was 30% and median overall survival (OS) was 7.5 months: these data are consistent with current literature. Among clinical variables, gross-total resection was endowed with a positive prognostic value for PFS (p=0.0296, log-rank test). NGS analysis revealed a mutation in the EGFR pathway (EGFR and/or PIK3CA) in 18% of cases; a mutation in the mitogen-activated protein-kinase (MAPK) pathway (RAS and/or RET) in 18% of cases; no mutations in the remaining cases. Patients carrying MAPK pathway mutation had a poor response to regorafenib treatment, with a significantly shorter PFS and a nonsignificantly shorter OS compared to EGFR-mutated patients (for PFS, 2.5 vs 4.5 months, p=0.0061; for OS, 7 vs 9 months, p=0.1076). By combining NGS analysis with RT-PCR for EGFRvIII, we identified 14 patients with EGFR pathway activation, who had a significantly longer PFS and OS after regorafenib treatment. Multivariate analysis confirmed that MAPK pathway mutations predicted a scarce response to regorafenib treatment.
Conclusions
Through an easy-to-use and cheap platform, we identified a mesenchymal, MAPK-altered signature in IDH-wildtype glioblastoma, predictive of scarce response to regorafenib at recurrence. We thus provide a molecular selection criterion to implement in the clinical practice.
Abstract
BACKGROUND
In the pathological classification of brain tumors, newly revised in 2021, molecular genetic analysis of gliomas has become more important for diagnosis and understanding of the ...pathogenesis of the disease. Gliomas are prone to recurrence and malignant progression, and collecting tumor tissue during every genetic analysis is highly invasive. Therefore, techniques have been developed to analyze tumor-derived circulating cell-free DNA (ccfDNA) in the cerebrospinal fluid as a substitute for tissue collection. However, the amount of ccfDNA derived from the cerebrospinal fluid is extremely small and the number of cases for whom genetic analysis is possible is currently limited. In this study, we attempted to establish an efficient method for the integrated analysis of glioma-related genes by in vitro amplification of cerebrospinal fluid-derived ccfDNA from glioma patients and a highly sensitive assay.
METHODS
Tumor-derived ccfDNA was extracted and purified from 1 ml of cerebrospinal fluid obtained by lumbar puncture using the Maxwell RSC instrument in five glioma cases. Hot spots of mutations in IDH1 and H3F3A genes were analyzed by high resolution melting (HRM), TERT mutation by droplet digital PCR, MGMT promoter methylation by methylation-specific HRM, and chromosome 1p/19q loss by multiplex ligation-dependent probe amplification. For samples in which the amount of ccfDNA was too small to analyze, DNA was amplified approximately 500-fold by DNA polymerase using the GenomiPhi kit and analyzed by the same assay method.
RESULTS AND CONCLUSIONS
Although two of the five cases required amplification with the GenomiPhi kit, it was possible to analyze all cases for glioma-related genes as described above. Although the amount of ccfDNA obtained from low-grade gliomas and gliomas that are not in contact with the ventricles or subarachnoid space is small, this amplification method may increase the possibility of liquid biopsy using cerebrospinal fluid.
Abstract
The relative inaccessibility of a patient’s glioma throughout their disease course precludes individualized insights into tumor biology and responses thereof to therapeutic challenge. We ...propose that serial cerebrospinal fluid (CSF) samples can be used to perform glioma espionage in the form of biomarker discovery for analytes that are sensitive to tumor burden (for monitoring) and candidate therapies (for biological impact). To that end, we are routinely leveraging neurosurgical access to the CNS to collect CSF samples intraoperatively and from chronically implanted Ommaya reservoirs for longitudinal CSF access (NCT04692337, NCT04692324). Global proteomic analysis of CSF was performed on the Somalogic platform – an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Using each patient’s ranked protein list (glioma vs. 3 control patients), enrichment analysis was performed in a discovery cohort of five patients to identify conserved proteins of glioma. To reveal candidate glioma monitoring, further enrichment analysis was performed to identify proteins that decreased with resection and increased with recurrence, using two sets of paired patient samples. Forty-four proteins met these enrichment criteria; this candidate high-grade glioma proteomic signature was highly significantly enriched in an independent cohort of twelve glioma patients (FDR=0 for all patients). The HGG signature decreased in abundance following resection in serial samples from two other patients, confirming decreased tumor burden. Top proteins included LANC2, DPYL3, Sorcin, Integrin A1B1, and lactate dehydrogenase (FC of glioma vs. control: 145.3x, 113.9x, 78.0x, 71.6x, and 62.7x, respectively). Interestingly, despite having explicitly chosen samples with little-to-no hemoglobin in the discovery cohort, this HGG signature was highly enriched for plasma-like proteins (FDR=0), suggesting a potential biological impact of blood-brain barrier disruption on the glioma extracellular proteome. In conclusion, our data demonstrate that the glioma extracellular proteome can feasibly be accessed to perform biomarker discovery (“espionage”) via serially-acquired CSF samples.
Abstract
While tumors of the nervous system (NS) are rare, they contribute to significant morbidity and mortality. Despite advances in imaging technology, initial diagnosis and characterization of ...treatment response remains problematic for devastating tumors such as glioblastoma (GBM). A liquid biopsy can provide a minimally-invasive and serial source of biomarkers to supplement current diagnostic methods. Here, we utilize a unique characteristic of NS tumors including GBM and schwannoma/neurofibroma – the overexpression of the a2 variant of the IL-13 receptor which is not detectable in normal tissue. Previously, we have shown that IL13Ra2 is elevated in the plasma of some GBM patients above baseline levels. We hypothesize that the presence of circulating IL13Ra2 not only predicts the level of this receptor in the tumor tissue, but also has clinical prognostic value. Here, we used ELISA to measure the plasma levels of IL13Ra2 in the GBM (n=77) and schwannoma/neurofibroma (n=15) patients and correlated these levels to tumor levels, diagnosis, and clinical characteristics. We have demonstrated that elevated levels of plasma IL13Ra2 correlated with tumor presence compared to non-tumor controls for both GBM and schwannoma/neurofibroma patients, indicating this biomarker’s diagnostic utility. Further, for both GBM and schwannoma/neurofibroma patients the plasma concentration of IL13Ra2 is correlated to its tumor concentration, indicating that sampling patient plasma could provide insight into the tumor’s composition. Finally, elevated levels of IL13Ra2 protein in the tumor and plasma of GBM patients predicted longer patient survival. These exciting findings lay the groundwork for a plasma-based NS tumor liquid biopsy.
Abstract
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.
Abstract
BACKGROUND
Adverse events (AE) including seizures cause significant morbidity in patients with GBM. We propose a novel method for assessing genomic predictors of AEs using results from a ...clinical targeted sequencing platform with variant function analysis.
METHODS
We identified 1,011 consecutive adult patients with newly diagnosed, histologically confirmed IDH-wildtype GBM with targeted exome NGS (Oncopanel) at Dana-Farber Cancer Institute from 2013-2019. Seizure at presentation was retrospectively identified as an AE. Biologic function (high loss, low loss, neutral, low gain and high gain) was assigned to variants using a three-tiered approach leveraging a genetic variant database (OncoKB), followed by analysis using protein prediction tools (Sift, Polyphen2 and Provean). Univariate logistic regression was performed for each relevant altered gene against the outcome of interest with false-discovery rate correction. Genes associated with seizure at presentation were included iteratively in a multivariate logistic model including other predictors of the outcome.
RESULTS
Our analysis included 470 GBM patients with 107 genes and 12 whole chromosome or arm level candidate variants covered by all versions of Oncopanel and with >10% alteration. Seizure at presentation occurred in 143/463 patients (31%) and was associated with EGFR amplification (high gain) (OR: 2.76, 95% CI: 1.4-5.3, p = 0.04). In a multivariate analysis (including age, sex, and preoperative tumor volume), EGFR amplification remained statistically significant (OR: 1.5, 95% CI: 1.0-2.2, p = 0.03).
CONCLUSION
Genomic biomarkers based on functional variant analysis of a routine clinical panel may predict adverse events in GBM. Seizure at presentation was independently associated with EGFR amplification. Our ongoing analysis will look at predictors of myelosuppression, thromboembolism, pseudoprogression and early progression using a similar approach. Identifying molecular risk factors could improve the management of patients through supportive care and consideration of prophylactic therapies.
Abstract
Methylation classification is an essential component for integrative diagnosis in glioma, however, the DNA methylation classification is not always available for all the samples. We ...hypothesized that Raman spectroscopy might be suitable to predict the glioma methylome, based upon its ability to create a molecular fingerprint of the tumor and would provide biological insights into the discriminatory features. Raman Spectroscopy was used for molecular fingerprinting of the regions of interest using 1mm2 FFPE tissue spots from 45 patient samples with LGm1 to LGm6 methylation subtypes. Spectral information was then used to train a convolutional neural network (CNN), capable of detecting the glioma methylation subtypes. 70 % of the dataset was used for model training while the remaining 30% for validation. We demonstrate that Raman spectroscopy can accurately and rapidly classify gliomas according to their methylation subtype from achieved FFPE samples, as a novel way to obtain classification. For each sample we ran Ward linkage clustering with a variable number of clusters (from 2 to 7), with the majority cluster corresponding to tumor spots and the others corresponding to (various types of) non-tumor spots. The average accuracy over all samples was 90:3%, the average precision was 99:6% and the average recall was 90:2%. We show that Raman spectroscopy together with artificial intelligence can predict the methylome of glioma samples and augment the ability to classify these tumors retrospectively. The non-destructive nature of this method and the ability to be applied on FFPE samples directly, allows the histopathologist to reuse of the same slide for subsequent staining and downstream analyses.
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