TERT Promoter Mutations Research Articles

Molecular profile of adult primary leptomeningeal gliomatosis aligns with glioblastoma, IDH-wildtype.

Adult primary leptomeningeal gliomatosis (PLG) is a rare, rapidly progressive and fatal disease characterized by prominent leptomeningeal infiltration by a glial tumor without an identifiable parenchymal mass. The molecular profile of adult PLG has not been well-characterized. We report the clinical, pathological, and molecular findings of six adult PLG patients (five males and one female), median age 58 years. All cases exhibited pathological leptomeningeal enhancement at presentation. Leptomeningeal biopsy was diagnostic in five (of six) cases, revealing infiltration by an astrocytic glioma with mitotic activity, lacking microvascular proliferation or necrosis. One case was diagnosed at autopsy. All tumors were IDH-wildtype, with five harboring TERT promoter mutations. Additional mutations identified were PTEN in one case, TP53 in two cases, and NF1 in two cases. A chromosome profile with +7/-10 was found in four cases, whereas the remaining two showed either chromosome 7 or 7p gain only. Four cases showed chromosome 9p loss with CDKN2A/B homozygous deletion, one case showed hemizygous CDKN2A/B loss, and one case showed intact chromosome 9 and CDK4/GLI1 amplification. DNA methylation profiling was performed in four cases and revealed a match to glioblastoma (GBM) family and mesenchymal typical class with high confidence scores in two cases; the other two cases showed only suggestive combined scores for GBM family and mesenchymal atypical class. The molecular profile of all cases closely aligned with that of adult-type GBM, IDH-wildtype, CNS WHO grade 4. All patients succumbed to the disease. In five cases with extensive leptomeningeal disease at diagnosis, the course was rapid, with median survival of 24 days following palliative care. Only one case, with relatively localized disease at diagnosis, received chemoradiation therapy and survived 535 days, raising the possibility that early diagnosis and timely treatment could improve outcome. A detailed list of previously reported cases is provided in a supplementary table.

TERT promoter mutations in gliomas: Molecular roles in tumorigenesis, metastasis, diagnosis, prognosis, therapeutic targeting, and drug resistance.

Telomerase reverse transcriptase (TERT), a critical player in cellular immortalization, has emerged as a focal point of investigation due to its frequent promoter mutations in various human malignancies. TERT promoter mutations exhibit a significant role in tumorigenesis, fostering unbridled cellular proliferation and survival. This comprehensive review delves into the landscape of TERT promoter mutations and their profound implications in cancer, particularly within the context of gliomas. This article meticulously examines the intricate interplay between TERT promoter mutations and the metastatic cascade, shedding light on their capacity to orchestrate invasive behavior in gliomas. Moreover, this review describes the recent trends in therapeutic targeting of the TERT and dissects the evolving landscape of drug resistance associated with TERT mutations, providing insights into potential therapeutic challenges. In addition, the diagnostic and prognostic implications of TERT promoter mutations in gliomas are scrutinized, unraveling their potential as robust biomarkers. It also discusses the recent advancements in molecular diagnostics, illustrating the promise of TERT mutations as diagnostic tools and prognostic indicators. This review collectively aims to contribute to a deeper understanding of TERT promoter mutations in gliomas, offering a foundation for future research endeavors and paving the way for innovative strategies in glioma management.

BRAFV600E/pTERT double mutated papillary thyroid cancers exhibit immune gene suppression

IntroductionBRAFV600E mutation (BRAFmut) is common in papillary thyroid cancer (PTC), and most patients have an excellent outcome. However, a TERT-promoter mutation (pTERTmut) in the presence of BRAFmut (BRAFmutpTERTmut) has been demonstrated to confer a more aggressive behavior to PTC. Lymphocytic infiltration is often present in PTC. In this study, we sought to decipher the relationship between the BRAF and pTERT mutations and immune gene dysregulation in tumor samples from a cohort of 147 samples of PTC.MethodsThe abundance of 770 immune gene transcripts was determined by multiprex capture/detection and digital counting of mRNA transcripts using the NanoString nCounter® PanCancer Immune Profiling Panel.ResultsWe identified 40 immune transcripts differentially expressed in BRAFmutpTERTmutvs BRAFmutpTERT wildtype (pTERTwt) (P<0.05). Transcripts induced by BRAFmut alone were significantly repressed in BRAFmutpTERTmut samples, such as genes expressed by lymphoid cells, antigen-presenting cells, and cytotoxic cells, including chemokines, cytokines, checkpoint control proteins, interferon downstream markers, TNF superfamily proteins and BMP markers. A validation analysis using 444 samples from The Cancer Genome Atlas (TCGA) PTC dataset yielded similar results. Deconvolution analysis confirmed differences in the immune cell populations such as increased presence of M2 macrophages in the BRAFmutpTERTmut Mayo cohort and a lower abundance of M1 macrophages in the BRAFmutpTERTmut TCGA cohort compared to BRAFmutpTERTwt. Most of the immune gene pathways were enriched in the BRAFmutpTERTwt tumors in both Mayo and TCGA cohorts but not in BRAFmutpTERTmut. BRAFmutpTERTwt had higher stromal lymphocytes infiltration as compared to BRAFwtpTERTwt tumors, corroborating the transcriptomic findings.DiscussionTo our knowledge this is the first report of a potential link between TERT and the immune microenvironment, offering an explanation for the aggressive nature of BRAFmutpTERTmut PTC.

Molecular and immune landscape of hepatocellular carcinoma for therapeutic development.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, with an estimated 750,000 deaths in 2022. Recent emergence of molecular targeted agents (MTAs) and immune checkpoint inhibitors (ICIs) and their combination therapies have been transforming HCC care, but their prognostic impact in advanced-stage disease remains unsatisfactory. In addition, their application to early-stage disease is still an unmet need. Omics profiling studies have elucidated recurrent and heterogeneously present molecular aberrations involved in pro-cancer tumor (immune) microenvironment that may guide therapeutic strategies. Recurrent aberrations such somatic mutations in TERT promoter and TP53 have been regarded undruggable, but recent studies have suggested that these may serve as new classes of therapeutic targets. HCC markers such as alpha-fetoprotein (AFP), glypican-3 (GPC3), and epithelial cell adhesion molecule (EpCAM) have also been explored as therapeutic targets. These molecular features may be utilized as biomarkers to guide the application of new approaches as companion biomarkers to maximize therapeutic benefits in patients who are likely to benefit from the therapies, while minimizing unnecessary harm in patients who will not respond. The explosive number of new agents in the pipelines have posed challenges in their clinical testing. Novel clinical trial designs guided by predictive biomarkers have been proposed to enable their efficient and cost-effective evaluation. These new developments collectively facilitate clinical translation of personalized molecular-targeted therapies in HCC and substantially improve prognosis of HCC patients.

The molecular landscape of 227 adult granulosa cell tumors of the ovary: Insights into the progression from primary to recurrence

Adult granulosa cell tumors (AGCTs) of the ovary are characterized by their propensity for late recurrences and are primarily managed surgically due to the limited efficacy of systemic treatment. The FOXL2 p.C134W somatic mutation has been identified in ∼95% of AGCT cases, and TERT promoter alterations have been linked to worse overall survival. This study highlights the potential prognostic significance of FOXO1 mutations, suggesting that they may be associated with poorer overall survival and shorter time to recurrence. A total of 183 primary AGCTs and 44 recurrences without corresponding primary tumors were analyzed. The primary AGCTs were categorized into three groups: 77 non-recurrent tumors, 18 tumors which later recurred (including 9 cases with matched primary-recurrence pairs), and 88 tumors with unknown recurrence status. Targeted next-generation sequencing was conducted on 786 cancer-related genes to investigate their genetic profile. The study aimed to identify the molecular alterations associated with AGCT pathogenesis and recurrence rate, comparing primary vs. recurrent tumors, and primary-recurrent vs. primary non-recurrent cases. Our findings confirmed the high prevalence (99%) of the FOXL2 p.C134W mutation in AGCTs. Secondary truncating FOXL2 mutations were observed in 5% of cases. Two cases with typical AGCT morphology were FOXL2 wild-type, harboring mutations in KRAS or KMT2D instead, suggesting alternative genetic pathways. TERT promoter mutations were found in 43% of cases, more frequently in recurrences. Other recurrent mutations detected in the cohort included KMT2D (10%), FOXO1 (7%), CHEK2 (5%), TP53 (3.5%), PIK3CA (3.5%), and AKT1 (3%). Two recurrent, FOXL2-mutated cases also carried DICER1 mutations. One tumor exhibited MSI-High status and a TMB of 19 mut/Mb. Our results indicate the need for further investigation into the role of FOXO1 as a potential prognostic marker in AGCTs.

A molecular and immunohistochemical study of 37 cases of ovarian Sertoli-Leydig cell tumor.

This study provides an analysis of 37 ovarian Sertoli-Leydig cell tumors (SLCT), focusing on their morphological, immunohistochemical, and molecular features. The cohort was comprised of 9 well-differentiated, 25 moderately differentiated, and 3 poorly differentiated tumors. The immunohistochemical analysis was performed with 28 markers, including diagnostic markers and markers with possible predictive significance. The results showed high expression of sex cord markers (FOXL2, SF1, inhibin A, CD99, calretinin, ER, PR, AR), and variable expression of other markers such as CKAE1/3 (83%), CAIX (14%), and MUC4 (1%). Loss of PTEN expression was present in 14% of cases, and CTLA4 expression was seen in 43% of cases. All tumors were MMR proficient and HER2 and PD-L1 negative. The molecular analysis showed DICER1 mutations in 54.5% of cases, and a FOXL2 mutation in 6% of tumors. In addition, we detected 2 cases with TERT promoter mutation. RNA NGS sequencing identified significant differences in mRNA expression between DICER1MUT and DICER1WT tumors. The DICER1WT tumors showed increased expression of PRKCA, HNF1A, LDLR, and MAP2K5. On the contrary, the DICER1MUT cases showed increased expression of CDK6, NOTCH2, and FGFR2. The results of our study show that SLCTs exhibit distinct molecular features based on their degree of differentiation. We have confirmed that DICER1 mutations are characteristic of moderately and poorly differentiated SLCTs, while well-differentiated SLCTs may represent a distinct entity. DICER1MUT and DICER1WT tumors showed different mRNA expression profiles. The FOXL2 mutation is less common in these tumors and is mutually exclusive with the DICER1 mutation.

Longitudinal multimodal profiling of IDH-wildtype glioblastoma reveals the molecular evolution and cellular phenotypes underlying prognostically different treatment responses.

Despite recent advances in the biology of IDH-wildtype glioblastoma, it remains a devastating disease with median survival of less than 2years. However, the molecular underpinnings of the heterogeneous response to the current standard-of-care treatment regimen consisting of maximal safe resection, adjuvant radiation, and chemotherapy with temozolomide remain unknown. Comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent glioblastoma specimens from 106 patients was performed to investigate the molecular evolution and cellular phenotypes underlying differential treatment responses. While TERT promoter mutation and CDKN2A homozygous deletion were early events during gliomagenesis shared by initial and recurrent tumors, most other recurrent genetic alterations (eg, EGFR, PTEN, and NF1) were commonly private to initial or recurrent tumors indicating acquisition later during clonal evolution. Furthermore, glioblastomas exhibited heterogeneous epigenomic evolution with subsets becoming more globally hypermethylated, hypomethylated, or remaining stable. Glioblastoma that underwent sarcomatous transformation had shorter interval to recurrence and were significantly enriched in NF1, TP53, and RB1 alterations and the mesenchymal epigenetic class. Patients who developed somatic hypermutation following temozolomide treatment had significantly longer interval to disease recurrence and prolonged overall survival, and increased methylation at 4 specific CpG sites in the promoter region of MGMT was significantly associated with this development of hypermutation. Finally, an epigenomic evolution signature incorporating change in DNA methylation levels across 347 critical CpG sites was developed that significantly correlated with clinical outcomes. Glioblastoma undergoes heterogeneous genetic, epigenetic, and cellular evolution that underlies prognostically different treatment responses.

Mechanistic basis of atypical TERT promoter mutations

Non-coding mutations in the TERT promoter (TERTp), typically at one of two bases −124 and −146 bp upstream of the start codon, are among the most prevalent driver mutations in human cancer. Several additional recurrent TERTp mutations have been reported but their functions and origins remain largely unexplained. Here, we show that atypical TERTp mutations arise secondary to canonical TERTp mutations in a two-step process. Canonical TERTp mutations create de novo binding sites for ETS family transcription factors that induce favourable conditions for DNA damage formation by UV light, thus creating a hotspot effect but only after a first mutational hit. In agreement, atypical TERTp mutations co-occur with canonical driver mutations in large cancer cohorts and arise subclonally specifically on the TERTp driver mutant chromosome homolog of melanoma cells treated with UV light in vitro. Our study gives an in-depth view of TERTp mutations in cancer and provides a mechanistic explanation for atypical TERTp mutations.

TERT mutations and aggressive histopathologic characteristics of radioiodine-refractory papillary thyroid cancer.

Radioiodine (RI) ablation following thyroid-stimulating hormone suppression is an effective treatment for papillary thyroid cancer (PTC), typically leading to favorable outcomes. However, RI-refractory tumors exhibit aggressive behavior and poor prognoses. Recent studies highlight the role of genetic abnormalities in PTC signaling pathways, including the activation of telomerase reverse transcriptase (TERT), and the correlation of mutations with adverse outcomes. This study analyzed mutations in BRAF V600E and the TERT-promoter genes, comparing clinicopathological features between RI-refractory and RI-responsive PTCs. Among 82 RI-refractory patients, formalin-fixed, paraffin-embedded tissues from initial surgeries were available for 26. Another 89 without distant metastasis over 5 years formed a matched RI-responsive control group. Histopathologically, RI-refractory PTCs showed increased frequencies of small tumor clusters without fibrovascular cores, hobnail features, and a high height-to-width ratio of tumor cells. These tumors were more likely to exhibit necrosis, mitosis, lymph node metastasis, extrathyroidal extension, and involvement of resection margins. TERT-promoter mutations were statistically significantly associated with these aggressive clinicopathologic features. Immunohistochemically, decreased expression of sodium iodide symporter and thyroglobulin stimulating hormone receptor proteins was common in RI-refractory PTCs, along with lower levels of oncogenic proteins such as vascular endothelial cell growth factor, vascular endothelial cell growth factor receptor 2, and nuclear factor kappa-light-chain-enhancer of activated B cells. Total loss of PTEN expression was occasionally observed. In contrast, all cases tested positive for cytoplasmic β-catenin. RI-refractory PTCs are linked to TERT mutations and exhibit specific aggressive histopathologic features, particularly in tumor centers.

The value of NGS-based multi-gene testing for differentiation of benign from malignant and risk stratification of thyroid nodules.

Fine-needle aspiration (FNA) biopsy is typically used in conjunction with cytopathologic evaluation to differentiate between benign and malignant thyroid nodules. Even so, the cytology results for 20-30% of thyroid nodules are indeterminate. This study sought to evaluate the usefulness of next-generation sequencing (NGS)-based multi-gene panel testing for risk stratification and the differentiation of benign from malignant thyroid nodules. Thyroid nodule samples were obtained from a cohort of 359 patients who underwent FNA. An NGS-based multi-gene panel testing was conducted for these samples, in which single-nucleotide variants (SNVs) and small insertion/deletions (InDels) can be detected in 11 genes and fusion events can be identified in 5 genes. Surgical resection was conducted for 113 patients (113/359), and then histopathology results were obtained. In comparison to cytology alone, the diagnostic sensitivity of NGS combination cytology increased from 0.7245 (95% CI: 0.6289-0.8032) to 0.898 (95% CI: 0.8223-0.9437); the associated AUC was 0.8303 (vs. Cytology AUC: 0.7622, P < 0.001). BRAF V600E was identified in 136 patients, of whom 79 underwent surgery and were diagnosed with papillary thyroid carcinoma (PTC) pathologically. TERT promoter mutations or BRAF/RAS co-mutations with other genes were identified in 5 patients, while 4 patients were diagnosed with malignant thyroid cancer using the pathological method. RAS mutations were identified in 27 patients, while 10 patients underwent surgery, which showed that 3 patients were classified as PTC and 7 cases were benign. In addition, 4 RET fusions, 1 RET activation mutation, and 3 TP53 inactivation mutations were identified in the remaining 8 patients who have not undergone surgery. Negative genetic test results or variants with uncertain significance were identified in 183 patients. Among these patients, 12 malignant thyroid tumors, including 11 PTC and 1 MTC, were diagnosed in 20 patients who received surgery. Thyroid nodules coupled with BRAF V600E, TERT promoter variants, BRAF/RAS co-mutations with other genes, RET fusions, and RET activating mutations were classified as high-risk. Nodules with RAS mutations (NRAS, KRAS, HRAS) and TP53 inactivating mutations were considered to be in the intermediate-risk group, while those with non-pathogenic mutations (negative and variants of uncertain significance) were placed in the low-risk group. When combined with cytopathology, NGS increases the sensitivity of diagnosing benign and malignant thyroid nodules, and the reference is useful for patient risk stratification.

PATH-15. DIGITAL PCR ANALYSIS FOR DETECTINGTERT PROMOTOR MUTATION IN MENINGIOMAS

Abstract The WHO Classification of Brain Tumors 5th edition requires the evaluation of TERT promoter mutations and CDKN2A/B homozygous deletions for the diagnosis of malignant meningiomas. Digital PCR (dPCR) is an emerging method for highly sensitive quantitative analysis. In this study, we performed TERT promoter mutation analysis using Pyrosequencing and dPCR in specimens with atypical and anaplastic meningiomas to investigate its utility and clinical significance. Adult patients diagnosed with atypical or anaplastic meningiomas after tumor resection between January 2009 and June 2023 were included. Genomic DNA was extracted from the excised specimens. TERT promoter mutations was analyzed by using the Pyrosequence and dPCR methods and compared with the clinical course. A total of 37 meningioma cases (13 males and 24 females) with 64 specimens were included. Atypical meningioma, grade II (WHO 2016) at diagnosis was 53 specimens (83%) and anaplastic meningioma, grade III (WHO 2016) was 11 specimens (17%). Of these, 7 (10%) were diagnosed with TERT promoter mutations by pyrosequencing, and one case with a low frequency (15%) of mutations by dPCR that tested negative by pyrosequencing had repeated recurrences in a short period of time. The dPCR method is highly sensitive in detecting low frequency TERT promoter mutations in tumor cell subpopulations and may contribute to the determination of treatment by predicting the malignant course of the disease. The results suggest that dPCR could contribute to therapeutic decision-making by detecting low-frequency TERT promoter mutations in tumor cell subpopulations.

NCMP-10. EFFECT OF EXTENT OF RESECTION ON GLIOMA SEIZURE OUTCOMES BASED ON MOLECULAR GROUPS

Abstract INTRODUCTION Increased extent of resection (EOR) is a known predictor of seizure freedom in glioma patients. However, it is unknown how tumor genomic alterations affect EOR-induced seizure control. OBJECTIVE To evaluate the genotype-specific impact of EOR on post-operative seizure control METHODS Records of 553 glioma patients who underwent tumor resection from 2012-2023 were reviewed. All patient tumors were analyzed with next generation sequencing and FISH analysis. The unsupervised machine learning algorithm non-negative matrix factorization (NMF) was used to cluster the genomic data. Clinical variables including extent of resection of contrast enhancing tumor and non-enhancing tumor, temozolomide therapy, and radiation therapy were analyzed using Cox Proportional Hazards models. RESULTS NMF clustering of tumor sequencing data revealed four molecular groups: Group 1 (n=56): CDKN2a loss, EGFR copy number gain or mutation, TERT promoter (TERTp) mutation, and MGMT methylated; Group 2 (n=27): IDH mutation, TP53 mutation, and MGMT methylated; Group 3 (n=60): TP53 mutation, TERTp mutation and MGMT methylated; Group 4 (n=244): MGMT unmethylated. Thresholds in the post-operative residual non-enhancing tumor volume (NETV) were identified which marked the most difference in seizure control. For patients in Group 1, the NETV threshold that led to improved epilepsy outcomes was &amp;lt;94.7 cc (p=0.047), whereas the threshold for Group 4 was &amp;lt;89.1 cc (p=0.016). When analysis was conducted for Group 2, the threshold of 10.2 cc failed to reach significance (p=0.1). Interestingly, the threshold analysis for Group 3 found that having a NETV of &amp;gt; 10.1 cc showed improved seizure outcomes (p=0.016). A multivariable cox proportional hazard analysis found that twelve-month seizure freedom was predictive of better OS in group 1 (HR 0.02, p=0.05) and group 4 (HR 0.21, p=1.63e-9). CONCLUSION Our study reveals clinically distinct molecular groups of glioma that display different seizure responses. This data suggests a tumor-specific approach to aggressiveness of extent of resection in order to achieve optimal seizure control.

EPID-19. INTERACTION BETWEEN GERMLINE AND SOMATIC GENETICS TO ELUCIDATE RISK OF ADULT DIFFUSE GLIOMA

Abstract Acquired tumor alterations are part of the WHO diagnosis of glioma. However, germline predisposition to these alterations is unknown. GWAS in European and Asian populations identified germline variants in 30 regions, many in or near genes that also acquire alterations, suggesting that interactions between inherited and acquired genetics are involved in glioma development. To evaluate such interactions, we used 3686 glioma cases and 1889 controls. A case-control design and multinomial logistic regression were used to identify germline variants associated with development of TERT-promoter mutant gliomas, generating two interesting observations. First, TERT germline variants were associated with predisposition to IDHwt glioma that have a TERT promoter mutation, but not with IDH-mutant codeleted gliomas that have a TERT promoter mutation. Second, two novel regions were observed in this more homogeneous subgroup of TERT mutated tumors: variants in TXNDC11 (OR=2.66, p=5.7x10-8) and RUVBL2 (OR=3.1, p=5.8x10-8). High tumor expression of TXNDC11 is associated with poor prognosis in glioma and RUVBL2 interacts with TERT to affect telomerase activity. Similar analyses are being performed on additional clinically relevant acquired alterations. In parallel, case-case GWAS analyses were performed to evaluate germline variants associated with other tumor alterations. Comparing IDH-mutant versus IDHwt stratified by rs55705857 genotype, the most significant variants were PHLDB1 (p=1.2x10-13) and D2HGDH (p=2.1x10-10), highlighting the importance of these two variants independent of CCDC26 for the development of IDH-mutant glioma. As a parallel approach, we utilized recursive partitioning and regression trees (rpart) to classify IDH mutation status using the 30 known variants. The top four branches of the regression tree included CCDC26, D2HGDH, PHLDB1, and MAML2, as well as two Asian variants (RAB27A, CYP4F12). This highlights the importance of utilizing variants discovered in non-European populations in analyses. Overall, understanding these interactions are necessary to develop therapeutics, stratify clinical trials, and to develop relevant tumor models.

PATH-31. COMPLETE TUMOR DISAPPEARANCE ON BRAIN MRI AFTER STANDARD CHEMORADIATION THERAPY FOR A MOLECULAR GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is the most aggressive brain tumor. The WHO CNS5 classification has newly incorporated three molecular criteria in diagnosing molecular GBM, including a concurrent gain of chromosome 7 and loss of chromosome 10 (+7/-10), TERT promoter mutation, or EGFR amplification. Despite new classification, the Stupp protocol remains the standard of care for all GBMs. However, it is unclear if the treatment response of the molecular GBM with low grade features is the same as the limited response of the traditional histopathological GBM. CASE REPORT A 74-year-old male presented in August of 2023 with new onset left-sided facial numbness. The numbness resolved spontaneously that day. Brain MRI was notable for a diffuse left frontal T2/Flair hyperintense lesion extending across the corpus callosum into the right frontal lobe without definite enhancement. Brain biopsy from 8 sites of the tumor demonstrated a subset of areas with mild hyper-cellularity, suggesting a low-density infiltrating glioma. However, TERT promoter mutation (-124C&amp;gt;T), was detected along with wild-type IDH1, supporting molecular GBM. Studies also found unmethylated MGMT gene promotor. Brain MRIs after 3 and 6 months showed no tumor progression. The patient has remained asymptomatic apart from baseline mild forgetfulness. He was started on the 6-week standard concomitant temozolomide and radiation therapy. Follow-up brain MRI after the above treatment showed complete imaging resolution of the tumor. DISCUSSION It is very rare for complete imaging resolution of GBM on brain MRI after standard 6-week chemoradiation therapy. Our case suggests that molecular GBM with low-grade features on imagine and pathology might have a better response to the standard treatment than traditional histopathological GBM even though the molecular GBM patient has unfavorable prognostic factors such as unmethylated MGMT and older age (&amp;gt;70 years old). Our experience emphasizes the heterogenous response of molecular GBM to the current standard treatment.

PATH-33. THE IMPACT OF THE MOLECULAR CLASSIFICATION IN HISTOLOGICALLY DIAGNOSED GLIOBLASTOMAS

Abstract OBJECTIVE Glioblastomas have been diagnosed based on traditional histological findings (necrosis, microvascular proliferation) and molecular genetic findings (IDH-wildtype, H3-wildtype, TERT promoter mutation, EGFR amplification, +7/−10 chromosome copy-number changes). Thus, IDH-wildtype and H3-wildtype glioblastomas can be classified into those with molecular genetic findings (defined as molecular glioblastoma) and those with only histological findings (defined as non-molecular glioblastoma). We re-evaluated the molecular genetics of patients diagnosed histologically with glioblastoma at our institution and examined their characteristics. METHODS A total of 275 cases diagnosed histologically as glioblastoma between December 2000 and March 2022 were included. Sanger sequencing (IDH1/2, Histone H3, TERTp) and MLPA for copy number analysis (PDGFRA, EGFR, CDKN2A, PTEN, MDM2, CDK4, TP53) were performed. The genetic analysis results and survival periods were analyzed. RESULTS Among the 275 cases, 151 initial glioblastomas met the criteria of IDH wild-type and H3 wild-type. Genetic analysis results that could be evaluated were obtained in 89 of these cases. TERT promoter mutation was present in 43 cases (48%), with 37% (16 cases) showing EGFR amplification and 19% (8 cases) showing EGFR gain/PTEN loss (+7/-10) simultaneously. On the other hand, TERT promoter wild-type was found in 45 cases (51%), with EGFR amplification in only 1 case (2%) and EGFR ain/PTEN loss in 4 cases (9%), and these 5 cases were judged to correspond to molecular glioblastoma. Adding one case with TERT promoter evaluation failure but EGFR amplification, the integrated molecular/non-molecular glioblastoma cases were 49 (55%)/40 (45%), revealing that both tumor types exist in nearly equal proportions. Survival curve analysis showed that median overall survivals of molecular and non-molecular glioblastoma were 17 and 29 months, respectively, showing a two-fold difference, and non-molecular glioblastoma had significantly better prognosis (P=0.0136). CONCLUSION It was revealed that tumors diagnosed as glioblastoma based on conventional histological findings can be broadly classified into molecular and non-molecular glioblastoma, with significant differences in prognosis.

BIOM-24. GLIOBLASTOMA DNA ALTERATIONS AND RAPID EARLY PROGRESSION AFTER SURGERY

Abstract Glioblastoma is the most common primary brain cancer with overall poor prognosis. In the weeks following initial resection, approximately 50% of glioblastoma patients will demonstrate rapid early progression (REP) on MRI prior to the start of radiation therapy (RT). REP is associated with worse overall survival (OS), however associations between REP and biomarkers in glioblastoma have yet to be evaluated. A single institution sample of 100 patients (age 61.2 years median, 57 males) with a primary diagnosis of glioblastoma were included in this study. Patients underwent gross or subtotal resection followed by RT within 60 days of their surgery. Patients who received biopsy only were excluded. For this analysis, REP was defined as increased nodular enhancement at the border of the resection cavity between the post-operative MRI and RT planning MRI. DNA mutations were collected from commercial next generation sequencing (NGS) results. Biomarkers with a mutational incidence of &amp;gt;10% were used in the analysis, which included TERT promoter (N=80), EGFR (N=38), PTEN (N=40), TP53 (N=36), CDKN2A/2B (N=41), NF1 (N=16), MTAP (N=24), PIK3CA (N=14), and MGMT methylation status (N=32). Statistical analysis was performed using SPSS Statistics 28.0. Of the patients sampled, 45 met the classification of REP. The presence of EGFR alterations (p=0.001) and MGMT methylation (p=0.016) strongly correlated with REP. After multivariate linear regression, only EGFR alterations significantly correlated with REP (p=0.006). There was no significant correlation between REP or overall survival and mutations in TERT promoter, PTEN, TP53, CDKN2A/2B, NF1, MTAP, and PIK3CA. This is the first study to analyze molecular contributors to REP. We identified that EGFR alterations are most strongly correlated with REP. This suggests that patients with EGFR alterations identified on pathology should move rapidly to adjuvant therapies given their propensity to quickly re-grow after resection.

PATH-59. HISTOLOGIC CORRELATES FOR MOLECULAR FEATURES IN MENINGIOMAS

Abstract Histologic findings are the primary determinant of meningioma prognosis and post-operative management. However, the association of specific pathologic features with molecular alterations has not yet been comprehensively investigated. Here we utilized a large dataset of well-characterized meningiomas to identify correlates between clinically relevant histological and molecular features. Detailed histologic information was collected for 809 resected meningiomas, including cases with DNA methylation (n=380), copy number array (n=252), next-generation sequencing (n=52), RNA risk score (n=394), and/or TERT promoter profiling (n=238). Analyses were conducted on prognostic pathologic features, including WHO Grade, Ki-67, mitotic index (MI), brain invasion, and atypical histologic findings (necrosis, hypercellularity, prominent nucleoli, sheeting, and small cells). Correlation with molecular variables was performed using Fisher’s Exact Tests, including DNA methylation subgroup, RNA risk score, CNV profiles, and TERT promoter mutations. All statistics were corrected for multiple hypothesis testing. Nearly 65% of meningiomas exhibited at least one atypical histologic feature. Among said features, Ki-67/MI, WHO Grade, sheeting, and necrosis were associated with recurrent lesions and known clinical features of aggressive meningiomas. Several histologic features, including hypercellularity, elevated Ki-67/mitotic index, and WHO grade, also correlated with DNA methylation and RNA risk groups. Several chromosomal arm events were related to specific pathologic features (prominent nucleoli, Ki-67, MI, and grade), including chromosomes 1, 4, 10, 14, and 18. Our analysis highlights the continued value in screening for histologic markers of aggressive behavior in meningiomas, as they correlate with the presence of high-risk molecular features and may serve as proxy markers when advanced molecular testing is not available.

NIMG-21. EARLY-STAGE GLIOBLASTOMA AND MOLECULAR ANALYSIS REVEAL THE RADIOLOGICAL INITIATION OF IDH WILDTYPE GLIOBLASTOMA

Abstract Glioblastoma (GB), IDH wildtype, is an aggressive and malignant tumor that necessitate early surgical intervention upon radiological suspicion. Consequently, the natural growth dynamics of GB remain not fully understood. Previous studies have examined GB growth using multiple MRIs between initial radiological diagnosis of GB and preoperative MRI, but these studies have short-term intervals between MRIs (defined as shortGB-cohort). Unless GB growth is a constant phenomenon, shortGB cannot estimate radiological initiation. In this study, we focused on early-stage GB (eGB), characterized by small, nonspecific lesions on MRI that eventually progress to classic GB findings. We compared three cohorts: 49 cases of eGB. 68 cases of shortGBt, and 23 cases of eGB from previous publications (other-eGB). Molecular analyses revealed that TERT promoter mutations, EGFR amplification/gain, CDKN2A hemi/homozygous deletion, and PTEN hemi/homozygous deletion were consistent between eGB and shortGB. Our analysis indicated that the radiological initiation of GB in eGB was -0.84 years (95% confidence interval (CI): -1.09 to -0.68), compared to -0.35 years (95%CI: -0.46 to -0.28) in shortGB. In eGB, factors such as age &amp;lt; 65, MIB1 &amp;gt; 30%, TERTp mutation, and the presence of copy-number alterations (CNA) in EGFR/PTEN/CDKN2A, with or without TERTp mutation, correlated with faster tumor progression. In cases without these alterations, the estimated radiological initiation was -2.31 years (95%CI: -100 to -0.80), indicating slower progression. The ShortGB-cohort did not show any significant changes based on these factors, suggesting it does not actually reflect tumor origin. The other-eGB cohort presented radiological initiation between -0.92 and -0.15 year, consistent with our eGB-cohort. This study investigates the radiological origin of GB using extremely rare cases of eGB, providing insights into the radiological pathophysiology and its association with molecular alterations in IDH-wildtype GB.

TMOD-03.BRAFV600E MUTATION INDUCES TWIST1 UPREGULATION ASSOCIATED WITH MIGRATION AND CEREBRAL FLUID DISSEMINATION IN GLIOBLASTOMA

Abstract INTRODUCTION BRAFV600E-mutant glioblastoma (GBM) with epithelioid features frequently show cerebrospinal fluid dissemination (CSFD), which results in poor prognoses. BRAF mutation is worth investigating since BRAF or downstream MEK inhibitors show dramatic response for BRAFV600E-mutant GBM including CSFD. This suggests BRAF mutation could play a role for inducing the clinical manifestation of CSFD found in newly diagnosed and recurrent BRAFV600E-mutant GBM. Because, BRAFV600E-mutant GBM often recur with CSFD, elucidating the mechanism by which BRAFV600E-mutant GBM exhibits CSFD is important to improve prognosis. (Objective) To elucidate the molecular mechanism of CSFD involved in BRAFV600E-mutant GBM. METHODS We introduced the heterozygous BRAFV600E mutation in human induced pluripotent stem cells (iPSC) in the context of GBM genetic alterations, CDKN2A/2B, and PTEN deletion, TERT promoter mutation and with EGFRvIII overexpression. We implanted these iPSC-derived neural progenitor cells into immunodeficient mouse brains to establish a BRAFV600E-mutant GBM model. At the same time, we created a model with the same genetic background without BRAFV600E. We compared their gene expression, pathology, and migration patterns to identify BRAFV600E specific characteristics. RESULTS RNA sequencing revealed BRAFV600E-mutant tumors upregulated migration, and downregulated cell-to-cell adhesion genes. TWIST1 was one of the most upregulated genes, and was validated by qPCR. Concomitant with TWIST1 expression we detected downregulation of E-cadherin. The BRAFV600E GBM model histologically showed a cluster-like invasion phenotype, while non-BRAF tumors showed homogenous invasion with typical GBM pathological findings. Immunohistochemical staining showed relatively high TWIST1 expression in cluster-like tumor cells of BRAFV600E tumors. Upon re-engraftment of primary tumor-derived cells, secondary BRAFV600E tumors presented leptomeningeal dissemination. TWIST1 knockdown resulted in a decrease in the migratory ability of our BRAFV600E model in vitro, and is under investigation in vivo. CONCLUSION BRAV600E mutation induces TWIST1 upregulation, and might facilitate migration and CSFD. This work nominates TWIST1 and its regulated genes as targets for BRAFV600E-mutant GBM with CSFD.

STEM-05. SHARED ONCOGENIC MUTATIONS IN THE SUBVENTRICULAR ZONE AND GLIOBLASTOMA: GENETIC AND CLINICAL IMPLICATIONS

Abstract The subventricular zone (SVZ), a neurogenic niche in the adult brain, is suspected to harbor neural stem cells (NSCs) that can acquire cancer-driving mutations and give rise to glioblastoma (GBM). Nevertheless, the corroborating genetic data are restricted to a small patient cohort. This study aimed to validate the presence of low-level oncogenic mutations in the SVZ of GBM patients and assess their clinical implications.. We performed deep hybrid capture sequencing of 15 GBM-associated genes and TERT promoter amplicon sequencing on triple-matched samples from the SVZ, primary tumor, and blood of 60 GBM patients. Approximately 62% of patients (40 of 61 patients) showed shared oncogenic mutations between the tumor and tumor-free SVZ, including TERT promoter, PTEN, and TP53. While shared mutations did not affect overall clinical profiles, TERT promoter mutations were significantly enriched in the shared group (94.9% of patients, p ≤ 0.001). RNA sequencing revealed upregulation of proliferation-related genes in tumors with shared SVZ mutations, and these patients had worse progression-free and overall survival (p = 0.038 and 0.031, respectively). This study provides the largest-scale genetic validation of the SVZ as a potential cellular origin of GBM, highlighting the distinct genetic and clinical features associated with shared oncogenic mutations. These findings warrant further investigation into the role of SVZ mutations in GBM genesis and their potential as therapeutic targets.