TERTp Mutations Research Articles

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 < 65, MIB1 > 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.

PATH-27. MOLECULAR, IMAGING, AND SURVIVAL MANIFESTATIONS IN MOLECULAR GLIOBLASTOMAS: INSIGHTS FROM A MULTI-NATIONAL STUDY

Abstract Apart from the traditional definition of IDH-wildtype GBM based on histological characteristics, IDH-wildtype diffuse gliomas previously assigned to histological grade 2 or 3 are now defined as IDH-wildtype GBM in the presence of qualifying molecular markers (TERTp mutation, EGFR amplification, or chromosome +7/-10) (“molecular GBM”) in the 2021 WHO classification. However, the clinical significance of molecular GBM is unrevealed. This collaboration aims to comprehensively evaluate the clinical, molecular, imaging characteristics, and prognosis of molecular GBMs in a multi-national dataset and stratify survival. Retrospective chart and imaging review was performed in 258 molecular GBM patients from eight centers across three continents. Clinical, molecular, imaging, and survival characteristics were analyzed. The median age of molecular GBM patients was 58 years (range: 23-83) with 139 males, with TERTp mutation (83 of 214 [38.8%]), EGFR amplification (160 of 204 [78.4%]), and chromosome +7/-10 (82 of 143 patients [57.3%]). The majority of tumors were located at the frontal and temporal lobes (48.4%) and exhibited non-contrast-enhancing (CE) tumors (73.7%). Gliomatosis cerebri pattern was shown with 27 patients (12.4%). The median OS was 28.1 months (95% confidence interval 22.1-34.1). There was significantly shorter survival in patients with high Ki-67 index (> 20) compared to those with low Ki-67 index (median OS 19.3 vs. 31.5 months, log-rank test P = 0.002), while no survival differences were found according to histological grade or molecular diagnostics. Survival was significantly shorter in patients with infiltrative imaging appearance compared to those without (median OS 24.7 vs 41.0 months, log-rank test P = 0.014). In conclusion, survival within molecular GBM patients may be heterogeneous and associated with Ki-67 index and infiltrative imaging appearance.

PATH-18. CURRENT STATUS OF CANCER GENOMIC PROFILING (CGP) TESTS IN BRAIN TUMOR TREATMENT - COMPLEMENTING BRAIN TUMOR PATHOLOGICAL DIAGNOSIS WITH CGP-

Abstract INTRODUCTION Cancer genomic profiling (CGP) tests are currently used in the search for therapeutic targets and have become an auxiliary tool in diagnosis according to the new WHO classification (WHO 2021), which requires the integration of pathomorphological and molecular diagnostics. Here, we report on brain tumor cases at our institution that underwent CGP testing. METHODS From May 2020 to the present, 27 brain tumor cases that underwent CGP testing at our institution were included in this study. There were 14 males and 13 females, ranging from 18 to 70 years (average 48.4 years). The tests conducted included Foundation One CDx, GeneMineTop, and NCC OncoPanel. RESULTS The cases that underwent CGP testing included 13 cases of glioblastoma IDH-wildtype, 3 cases of astrocytoma, IDH-mutant, grade 4, 4 cases of astrocytoma IDH-mutant, grade 3, 3 cases of oligodendroglioma, 1p/19q codeleted, grade 3, 1 case of astrocytoma, IDH-mutant, grade 2, 1 case of ependymoma, 1 case of pilocytic astrocytoma and 1 case of PitNEC. CGP testing enabled molecular diagnostics such as IDH mutation and TERTp mutation. Based on the results, therapeutic suggestions were possible for 5 out of 25 cases (20%) (BRAF V600E mutation 1 case, IDH1 R132H mutation 1 case, TMB high 3 cases). Checkpoint inhibitors were used in a case with high TMB. Genetic risk assessment by a genetic counselor was conducted for all cases, and one high TMB case was diagnosed as hereditary cancer. A germline pathogenic variant in MSH6 was detected, leading to a diagnosis of Lynch syndrome. CONCLUSION Molecular diagnostics through CGP testing served as an aid in diagnosis according to WHO2021. Cases where drug suggestions were feasible underwent treatment with targeted molecular therapies and checkpoint inhibitor. We experienced cases where hereditary cancer syndromes such as Lynch syndrome were diagnosed through the detection of germline pathogenic variants.

QLTI-06. NEXT GENERATION SEQUENCING SIGNIFICANTLY IMPROVES THE DIAGNOSTIC ACCURACY IN GLIOBLASTOMAS

Abstract The 2021 WHO Classification of CNS Tumours recognizes the presence of TERT promoter (pTERT) mutations, combined +7/-10 chromosome copy-number alterations, or EGFR amplification as a defining molecular feature(s) of glioblastoma, independent of histology. We evaluated these biomarkers in a cohort of Indian patients using a single-run next-generation sequencing (NGS) platform at a relatively low depth of 100. Seventy-four adult diffuse gliomas were selected from the Exsegen Genomics Research Pvt Ltd Biobank established during the trial CTRI/2021/09/036861 after reviewing their clinical and pathological data. Paired surgical tissue and blood samples underwent whole-exome sequencing on an Illumina NovaSeq-6000 (Illumina, San Diego, USA). Single-nucleotide variations (SNVs) were processed using the DRAGEN pipeline to generate Mutation Annotation Format (MAF) files. Copy number variations (CNVs) analysis was performed using GATK and GISTIC2.0. Thirty-nine glioma samples with IDH1R132H mutations were excluded. A total of 32/35 (91.4%) IDH-wildtype tumours met the 2021 WHO criteria for glioblastoma, IDH-wildtype and were included. Twenty-four glioblastomas also underwent whole-transcriptome analysis. Microvascular proliferation (n=24, 75%) and necrosis (n=30, 93.9%) were present in the majority. pTERT mutations were identified in 23 (71.9%) samples, with C228T mutations (n=15/23, 65.2%) being more common than C250T mutations (n=8/23, 34.8%). EGFR amplification, defined as ≥5 copies by NGS CNV, was seen in 15 (46.9%) samples, with a strong correlation between EGFR CNV and logEGFR expression (r=0.79; p=0.017). Whole-arm +7/-10 genotype was evident in 17 (53.1%) tumours, and another 5 (15.6%) showed partial copy-number alterations. Notably, two gliomas without necrosis and proliferation initially diagnosed as oligodendrogliomas due to misinterpretation of the IDH immunostain, were reclassified as glioblastoma, IDH-wildtype. The data was validated using Sanger Sequencing and targeted panel sequencing to a depth of 500. Even low depth NGS sequencing, that is cost effective and time efficient offers significant improvements in diagnostic molecular neuropathology.

Evaluation of a Cytology-Molecular Co-Test in Liquid-Based Cytology-Processed Urine for Defining Indeterminate Categories of the Paris System

Introduction: Urine cytology using the Paris system (TPS) classification is useful for the detection and monitoring of bladder urothelial carcinoma (UC). However, the categories “atypical urothelial cells” (AUCs) and “suspicious for high-grade urothelial carcinoma” (SHGUC) do not establish a clear diagnosis. This pilot study aimed to investigate whether the presence of mutations in fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT) genes, in urine processed with liquid-based cytology (LBC) could enhance the diagnostic performance of cytology, particularly in defining the indeterminate categories of AUC and SHGUC. Methods: Urine samples from 82 UC patients with primary tumors or under surveillance and 10 healthy individuals were examined. The ThinPrep method was used for cytology followed by DNA isolation from urine sediments. Targeted molecular analysis was achieved in 70 cases (63 patients and 7 controls) for exons 7 and 10 of the FGFR3 gene and the TERT gene promoter (pTERT), using PCR and Sanger sequencing. Molecular results were correlated with TPS cytology categories and validated by histopathological findings following cystoscopy. Results: In healthy subjects, cytology was negative for high-grade urothelial carcinoma (NHGUC) and no mutations were found. No mutations were found in patients with NHGUC cytology, except for one case with equivocal cystoscopy that carried a pTERT mutation. In high-grade urothelial carcinoma cytology (HGUC) (15/20, 75%) of the cases with histologically confirmed UC, molecular analysis revealed the presence of pTERT without FGFR3 mutations. In SHGUC and AUC cytology, FGFR3 and/or pTERT mutations were detected in 3/4 (75%) and 4/4 (100%) histologically confirmed UC cases, respectively. Cytology sensitivity was 85.7% increasing to 100% with the combined cytology-molecular test, whereas specificity remained unchanged at 86.3%. Conclusions: This pilot study suggests that the incorporation of FGFR3/pTERT molecular testing in urine LBC could enhance the diagnostic value of cytology by diagnosing bladder urothelial carcinoma in indeterminate cytology categories.

Brain tumoroids: treatment prediction and drug development for brain tumors with fast, reproducible and easy-to-use personalized models.

generation of patient avatar is critically needed in neuro-oncology for treatment prediction and preclinical therapeutic development. Our objective was to develop a fast, reproducible, low-cost and easy-to-use method of tumoroids generation and analysis, efficient for all types of brain tumors, primary and metastatic. tumoroids were generated from 89 patients: 81 primary tumors including 77 gliomas, and 8 brain metastases. Tumoroids morphology, cellular and molecular characteristics were compared with the ones of the parental tumor by using histology, methylome profiling, pTERT mutations and multiplexed spatial immunofluorescences. Their cellular stability overtime was validated by flow cytometry. Therapeutic sensitivity was evaluated and predictive factors of tumoroid generation were analyzed. All the tumoroids analyzed had similar histological (N=21) and molecular features (N=7) than the parental tumor. Median generation time was 5 days. Success rate was 65 %: it was higher for high grade gliomas and brain metastases versus IDH mutated low grade gliomas. For high-grade gliomas, neither other clinical, neuro-imaging, histological nor molecular factors were predictive of tumoroid generation success. The cellular organization inside tumoroids analyzed by MACSima revealed territories dedicated to specific cell subtypes. Finally, we showed the correlation between tumoroid and patient treatment responses to radio-chemotherapy and their ability to respond to immunotherapy thanks to a dedicated and reproducible 3D analysis workflow. patient-derived tumoroid model that we developed offers a robust, user-friendly, low-cost and reproducible preclinical model valuable for therapeutic development of all type of primary or metastatic brain tumors, allowing their integration into forthcoming early-phase clinical trials.

Comparative analysis of molecular and histological glioblastomas: insights into prognostic variance.

Whether molecular glioblastomas (GBMs) identify with a similar dismal prognosis as a "classical" histological GBM is controversial. This study aimed to compare the clinical, molecular, imaging, surgical factors, and prognosis between molecular GBMs and histological GBMs. Retrospective chart and imaging review was performed in 983 IDH-wildtype GBM patients (52 molecular GBMs and 931 histological GBMs) from a single institution between 2005 and 2023. Propensity score-matched analysis was additionally performed to adjust for differences in baseline variables between molecular GBMs and histological GBMs. Molecular GBM patients were substantially younger (58.1 vs. 62.4, P = 0.014) with higher rate of TERTp mutation (84.6% vs. 50.3%, P < 0.001) compared with histological GBM patients. Imaging showed higher incidence of gliomatosis cerebri pattern (32.7% vs. 9.2%, P < 0.001) in molecular GBM compared with histological GBM, which resulted in lesser extent of resection (P < 0.001) in these patients. The survival was significantly better in molecular GBM compared to histological GBM (median OS 30.2 vs. 18.4 months, P = 0.001). The superior outcome was confirmed in propensity score analyses by matching histological GBM to molecular GBM (P < 0.001). There are distinct clinical, molecular, and imaging differences between molecular GBMs and histological GBMs. Our results suggest that molecular GBMs have a more favorable prognosis than histological GBMs.

Diffusion-weighted MRI precisely predicts telomerase reverse transcriptase promoter mutation status in World Health Organization grade IV gliomas using a residual convolutional neural network.

Telomerase reverse transcriptase promoter (pTERT) mutation status plays a key role in making decisions and predicting prognoses for patients with World Health Organization (WHO) grade IV glioma. This study was conducted to assess the value of diffusion-weighted imaging (DWI) for predicting pTERT mutation status in WHO grade IV glioma. MRI data and molecular information were obtained for 266 patients with WHO grade IV glioma at the hospital and divided into training and validation sets. The ratio of training to validation set was approximately 10:3. We trained the same residual convolutional neural network (ResNet) for each MR modality, including structural MRIs (T1-weighted, T2-weighted, and contrast-enhanced T1-weighted) and DWI*, to compare the predictive capacities between DWI and conventional structural MRI. We also explored the effects of different regions of interest on pTERT mutation status prediction outcomes. Structural MRI modalities poorly predicted the pTERT mutation status (accuracy = 51%-54%; area under the curve [AUC]=0.545-0.571), whereas DWI combined with its apparent diffusive coefficient maps yielded the best predictive performance (accuracy = 85.2%, AUC = 0.934). Including the radiological and clinical characteristics did not further improve the performance for predicting pTERT mutation status. The entire tumour volume yielded the best prediction performance. DWI technology shows promising potential for predicting pTERT mutations in WHO grade IV glioma and should be included in the MRI protocol for WHO grade IV glioma in clinical practice. This is the first large-scale model study to validate the predictive value of DWI for pTERT in WHO grade IV glioma.

Using deep learning to decipher the impact of telomerase promoter mutations on the dynamic metastatic morpholome.

Melanoma showcases a complex interplay of genetic alterations and intra- and inter-cellular morphological changes during metastatic transformation. While pivotal, the role of specific mutations in dictating these changes still needs to be fully elucidated. Telomerase promoter mutations (TERTp mutations) significantly influence melanoma's progression, invasiveness, and resistance to various emerging treatments, including chemical inhibitors, telomerase inhibitors, targeted therapy, and immunotherapies. We aim to understand the morphological and phenotypic implications of the two dominant monoallelic TERTp mutations, C228T and C250T, enriched in melanoma metastasis. We developed isogenic clonal cell lines containing the TERTp mutations and utilized dual-color expression reporters steered by the endogenous Telomerase promoter, giving us allelic resolution. This approach allowed us to monitor morpholomic variations induced by these mutations. TERTp mutation-bearing cells exhibited significant morpholome differences from their wild-type counterparts, with increased allele expression patterns, augmented wound-healing rates, and unique spatiotemporal dynamics. Notably, the C250T mutation exerted more pronounced changes in the morpholome than C228T, suggesting a differential role in metastatic potential. Our findings underscore the distinct influence of TERTp mutations on melanoma's cellular architecture and behavior. The C250T mutation may offer a unique morpholomic and systems-driven advantage for metastasis. These insights provide a foundational understanding of how a non-coding mutation in melanoma metastasis affects the system, manifesting in cellular morpholome.

Nomogram of magnetic resonance imaging (MRI) histogram analysis to predict telomerase reverse transcriptase promoter mutation status in glioblastoma.

Telomerase reverse transcriptase promoter (pTERT) status is a strong biomarker to diagnose and predict the prognosis of glioblastoma (GBM). In this study, we explored the predictive value of preoperative magnetic resonance imaging (MRI) histogram analysis in the form of nomogram for evaluating pTERT mutation status in GBM. The clinical and imaging data of 181 patients with GBM at our hospital between November 2018 and April 2023 were retrospectively assessed. We used the molecular sequencing results to classify the datasets into pTERT mutations (C228T and C250T) and pTERT-wildtype groups. FireVoxel software was used to extract preoperative T1-weighted contrast-enhanced (T1C) histogram parameters of GBM patients. The T1C histogram parameters were compared between groups. Univariate and multivariate logistic regression analyses were used to construct the nomogram, and the predictive efficacy of model was evaluated using calibration and decision curves. Receiver operating characteristic curve was used to assess model performance. Patient age and percentage of unenhanced tumor area showed statistically significant differences between the pTERT mutation and pTERT-wildtype groups (P<0.001). Among the T1C histogram features, the maximum, standard deviation (SD), variance, coefficient of variation (CV), skewness, 5th, 10th, 25th, 95th and 99th percentiles were statistically significantly different between groups (P=0.000-0.040). Multivariate logistic regression analysis showed that age, percentage of unenhanced tumor area, SD and CV were independent risk factors for predicting pTERT mutation status in GBM patients. The logistic regression model based on these four features showed a better sample predictive performance, and the area under the curve (AUC) [95% confidence interval (CI)], accuracy, sensitivity, specificity were 0.842 (0.767-0.917), 0.796, 0.820, and 0.729, respectively. There were no significant differences in the T1C histogram parameters between the C228T and C250T groups (P=0.055-0.854). T1C histogram parameters can be used to evaluate pTERT mutations status in GBM. A nomogram based on conventional MRI features and T1C histogram parameters is a reliable tool for the pTERT mutation status, allowing for non-invasive radiological prediction before surgery.

Intraoperative rapid molecular diagnosis aids glioma subtyping and guides precise surgical resection.

The molecular era of glioma diagnosis and treatment has arrived, and a single rapid histopathology is no longer sufficient for surgery. This study sought to present an automatic integrated gene detection system (AIGS), which enables rapid intraoperative detection of IDH/TERTp mutations. A total of 78 patients with gliomas were included in this study. IDH/TERTp mutations were detected intraoperatively using AIGS in 41 of these patients, and they were guided to surgical resection (AIGS detection group). The remaining 37 underwent histopathology-guided conventional surgical resection (non-AIGS detection group). The clinical utility of this technique was evaluated by comparing the accuracy of glioma subtype diagnosis before and after TERTp mutation results were obtained by pathologists and the extent of resection (EOR) and patient prognosis for molecular pathology-guided glioma surgery. With NGS/Sanger sequencing and chromosome detection as the gold standard, the accuracy of AIGS results was 100%. And the timing was well matched to the intraoperative rapid pathology report. After obtaining the TERTp mutation detection results, the accuracy of the glioma subtype diagnosis made by the pathologists increased by 19.51%. Molecular pathology-guided surgical resection of gliomas significantly increased EOR (99.06% vs. 93.73%, p < 0.0001) and also improved median OS (26.77 vs. 13.47 months, p = 0.0289) and median PFS (15.90 vs. 10.57 months, p = 0.0181) in patients with glioblastoma. Using AIGS intraoperatively to detect IDH/TERTp mutations to accurately diagnose glioma subtypes can help achieve maximum safe resection of gliomas, which in turn improves the survival prognosis of patients.

Evaluation of prognostic biomarkers in meningiomas and their clinical implications in settings with limited resources.

The 5th edition of the World Health Organization (WHO) Central Nervous System (CNS) tumor classification for meningiomas acknowledges the clinical relevance of genomic profiling studies and emphasizes the importance of incorporating molecular information alongside histopathological features, leading to more accurate diagnoses and improved patient care. We analyzed 206 meningioma samples (108 histological grade 1, 89 grade 2, and 9 grade 3) to study pTERT mutations, CDKN2A/B homozygous deletion, loss of H3K27me3, and p16 expression. The association of these molecular markers with survival outcomes was also assessed. pTERT mutation was found in 4.85% of cases, predominantly occurring in histological grade 2 (11.24%), while none of the histological grade 1 or 3 meningiomas exhibited this mutation. CDKN2A/B gene deletion was absent in grade 1 and detected in 2.24% of grade2, and 33.3% of histological grade 3 cases. There was a significant increase in loss of H3K27me3 with higher tumor grades, while p16 loss was observed in over 50% of cases across all histological grades. The presence of pTERT mutation and CDKN2A/B homozygous deletion resulted in the reclassification of 5.33% (11/206) of meningiomas as integrated grade 3. pTERT mutation and CDKN2A/B deletion, emerged as prognostically relevant markers, showing significant differences in progression-free survival (PFS) between integrated grade 3 and histological grade 2 meningiomas (P = .0002). pTERT mutations are the most clinically relevant genetic alterations in meningiomas. Routine testing for pTERT mutations can identify high-risk cases of histologically grade 2 meningiomas, providing crucial prognostic information for treatment planning. CDKN2A/B alteration is rare and not cost-effective in assessing meningiomas. Immunohistochemical assessment of p16 and H3K27me3 expression lacks significant prognostic value. Assessment of pTERT mutations offers a cost-effective and valuable diagnostic tool for meningiomas.

Clinicopathologic and Molecular Characterization of High-Risk Human Papillomavirus-Positive Carcinomas of the Urinary Tract.

Human papillomavirus (HPV) is a well-known cause of squamous cell carcinomas of anogenital and oropharyngeal regions, where treatment strategies and prognosis depend on HPV status. The significance of HPV status in tumors arising along the urinary tract is not well established. To provide detailed clinical, morphologic, immunohistochemical, and molecular analysis of HPV+ urinary tract carcinomas (UTCs). We identified and retrospectively examined 12 HPV+ UTCs, confirmed by high-risk HPV in situ hybridization. The HPV+ UTCs originated from the urethra (9) and urinary bladder (3); 5 of 12 (42%) presented with nodal metastasis. On morphology, HPV+ UTCs were predominantly basaloid; well-differentiated squamous areas were focally seen. Available immunohistochemistry (IHC) showed strong staining for p16 (11 of 11), p63 (12 of 12), cytokeratin (CK) 903 (11 of 11), and CK5/6 (11 of 11); variable staining for GATA3 (8 of 12) and CK7 (4 of 11); and rare uroplakin II staining (1 of 12). Molecular analysis revealed the most frequently altered genes: KMT2C (42%), PIK3CA (42%), and KMT2D (25%). In contrast to published conventional urothelial and squamous cell carcinoma molecular data, TERTp mutation was rare (8%), and no TP53 or CDKN2A aberrations were identified. During available follow-up (11 of 12; median, 39 months), 6 patients required treatment for recurrence; ultimately, 1 died of disease, 2 were alive with disease, and 8 had no evidence of disease. Finally, we provide 11 HPV- squamous predominant UTCs for IHC and molecular comparisons; notably, a subset of HPV- UTCs was positive for p16 IHC (27%), making p16 IHC a less-specific surrogate marker for HPV status at this site. HPV+ UTCs show distinct clinical, morphologic, and molecular characteristics, suggesting important roles for HPV in UTC.

Telomerase Reverse Transcriptase-Promoter Mutation in Young Patients with Bladder Tumors.

The TERT (Telomerase Reverse Transcriptase) gene promoter mutation is one of the most prevalent mutations in urothelial bladder tumors and this mutation is related to bladder tumor progression. Our purpose was to evaluate the presence of this mutation in a population of patients who were first diagnosed at age ≤ 40 years and to examine its relationship with tumor characteristics and progression. A molecular study was performed to detect the two most prevalent mutations in the TERT promoter (C228T and C250T). The study included 29 patients, with a mean follow-up of 152 months. There were no statistically significant differences in the clinical or tumor characteristics according to the presence or absence of the mutation. Although the mutation group showed poorer recurrence-free survival (RFS), there was no statistically significant difference and there was no difference in progression-free survival by group (p > 0.05). The pTERT mutations in bladder tumor cells occurred less frequently in younger patients than in older patients, a finding that could indicate different mechanisms of carcinogenesis. The trend towards lower RFS in patients with mutated pTERT needs to be confirmed by further studies, given the small number of patients included in these studies due to the low incidence of bladder tumors in this age group.

The C250T Mutation of TERTp Might Grant a Better Prognosis to Glioblastoma by Exerting Less Biological Effect on Telomeres and Chromosomes Than the C228T Mutation.

TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients. Overall, there were no differences in outcomes between patients with mutated TERTp-wt or TERTp. However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp-wt (p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively (p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes. In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp-wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

MGMT unmethylation and high levels of CD47 and TIGIT indicate a poor prognosis in adult diffuse gliomas.

In 2021, the World Health Organization published a new classification system for central nervous system tumors. This study reclassified the adult diffuse glioma (ADG) into astrocytoma, oligodendroglioma, and glioblastoma (GBM) according to the new tumor classification. The association of TERT promoter (pTERT) mutation, MGMT methylation, and CD47/TIGIT expression with patient prognosis was investigated. Immunohistochemical analysis showed that the expression levels of CD47 and TIGIT in tumor tissues were significantly higher than those in normal brain tissues. CD47 levels were higher in GBM and grade 4 astrocytoma tissues. TIGIT expression was also higher in patients with GBM. The high expressions of CD47, TIGIT, and CD47/TIGIT were positively correlated with MGMT unmethylation but not pTERT mutation. Moreover, MGMT unmethylation was associated with poor overall survival in astrocytoma. High CD47, TIGIT, and CD47/TIGIT levels were associated with significantly reduced survival in ADG and GBM. GBM, MGMT unmethylation, and high CD47 expression were independent prognostic factors for overall survival in ADG. Collectively, these results showed that the MGMT unmethylation and high levels of CD47 and TIGIT are associated with a poor prognosis in ADG. Patients with high CD47 and TIGIT expression may benefit from anti-CD47 and TIGIT immunotherapy.

Comparative Cyto-Histological Genetic Profile in a Series of Differentiated Thyroid Carcinomas

Introduction: Molecular tests can contribute to improve the preoperative diagnosis of thyroid nodules. Tests available are expensive and not adapted to different populations. Aim: This study aimed to compare the cyto-histological genetic profile and to evaluate the reliability of molecular tests using ultrasound-guided fine needle aspiration cytology (US-FNAC) in accurately diagnosing differentiated thyroid carcinomas (DTCs) and predicting biologic behavior of papillary thyroid carcinomas (PTCs). Materials and Methods: The series included 259 patients with paired cyto-histological samples totaling 518 samples. The genetic alterations were analyzed via PCR/Sanger sequencing. The association with clinicopathologic features was evaluated in PTCs. Results/Discussion: From the 259 patients included, histologies were 50 (19.3%) benign controls and 209 (80.7%) DTC cases, from which 182 were PTCs; cytologies were 5.8% non-diagnostic, 18.2% benign, 39% indeterminate, and 37.1% malignant. In histology, indeterminate nodules (n = 101) were 22.8% benign and 77.2% malignant. Mutation frequencies in cytology and histology specimens were, respectively, TERTp: 3.7% vs. 7.9%; BRAF: 19.5% vs. 25.1%; and RAS: 11% vs. 17.5%. The overall cyto-histological agreement of the genetic mutations was 94.9%, with Cohen’s k = 0.67, and in indeterminate nodules agreement was 95.7%, k = 0.64. The identified mutations exhibited a discriminative ability in diagnosing DTC with a specificity of 100% for TERTp and BRAF, and of 94% for RAS, albeit with low sensitivity. TERTp and BRAF mutations were associated with aggressive clinicopathological features and tumor progression in PTCs (p &lt; 0.001). The obtained good cyto-histological agreement suggests that molecular analysis via US-FNAC may anticipate the genetic profile and the behavior of thyroid tumors, confirming malignancy and contributing to referring patients to surgery.

Plasma ctDNA liquid biopsy of IDH1, TERTp, and EGFRvIII mutations in glioma.

Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene mutations IDH1, TERTp, and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy. We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient). DNA was isolated from plasma and analyzed for IDH1, TERTp, and EGFRvIII mutations using ddPCR. Total cfDNA was significantly associated with tumor grade, tumor volume, and both overall and progression-free survival for all gliomas as well as the grade 4 glioblastoma subgroup, but was not reliably associated with changes in tumor volume/progression during the patients' postoperative time course. IDH1 mutation was detected with 84% overall sensitivity across all plasma samples and 77% in the preoperative samples alone; however, IDH1 mutation plasma levels were not associated with tumor progression or survival. IDH1m plasma levels were not associated with pre- or postsurgery progression or survival. The TERTp C228T mutation was detected in the plasma ctDNA in 88% but the C250T variant in only 49% of samples. The EGFRvIII mutation was detected in plasma in 5 out of 7 patients (71%) with tissue EGFRvIII mutations in tumor tissue. Plasma ctDNA mutations detected with ddPCR provide excellent diagnostic sensitivity for IDH1, TERTp-C228T, and EGFRvIII mutations in glioma patients. Total cfDNA may also assist with prognostic information. Further studies are needed to validate these findings and the clinical role of ctDNA in glioma.

Clinical, histological, and molecular differences in melanoma due to different TERT promoter mutations subtypes. A retrospective cross-sectional study in 684 melanoma patients.

Differences in survival according to the pTERT mutation subtypes (-124C > T, -146C > T, and tandem -138_139CC > TT) have been observed. The present study aimed to describe the clinical as the histopathological and molecular cutaneous melanoma features according to the presence of the three most prevalent pTERT mutation subtypes (-124C > T, -146C > T, and tandem -138_139CC > TT). A retrospective cross-sectional study including 684 patients was designed, and a Partial Least-Squares Discriminant Analysis (PLS-DA) was performed. After the PSL-DA, it was observed that the tandem -138_139CC > TT subtype differs from the other subtypes. The model demonstrated that the -124C > T and the -138_139 CC > TT subtypes were associated with fast-growing melanomas (OR 0.5, CI 0.29-0.86, p = .012) and with Breslow >2 mm (OR 0.6, CI 0.37-0.97, p = .037), compared to the -146C > T mutation. Finally, the -124C > T appeared to be more associated with the presence of TILs (non-brisk) than the -146C > T (OR 0.6, CI 0.40-1.01, p = .05). These findings confirmed that the -124C > T and the tandem -138_139 CC > TT subtypes are both highly associated with the presence of features of aggressiveness; however, only the -124C > T was highly associated with TILs. This difference could explain the worse survival rate associated with the tandem -138_139CC > TT mutations.

Multimodal MRI-based radiomic nomogram for predicting telomerase reverse transcriptase promoter mutation in IDH-wildtype histological lower-grade gliomas.

The presence of TERTp mutation in isocitrate dehydrogenase-wildtype (IDHwt) histologically lower-grade glioma (LGA) has been linked to a poor prognosis. In this study, we aimed to develop and validate a radiomic nomogram based on multimodal MRI for predicting TERTp mutations in IDHwt LGA. One hundred and nine IDH wildtype glioma patients (TERTp-mutant, 78; TERTp-wildtype, 31) with clinical, radiomic, and molecular information were collected and randomly divided into training and validation set. Clinical model, fusion radiomic model, and combined radiomic nomogram were constructed for the discrimination. Radiomic features were screened with 3 algorithms (Wilcoxon rank sum test, elastic net, and the recursive feature elimination) and the clinical characteristics of combined radiomic nomogram were screened by the Akaike information criterion. Finally, receiver operating characteristic curve, calibration curve, Hosmer-Lemeshow test, and decision curve analysis were utilized to assess these models. Fusion radiomic model with 4 radiomic features achieved an area under the curve value of 0.876 and 0.845 in the training and validation set. And, the combined radiomic nomogram achieved area under the curve value of 0.897 (training set) and 0.882 (validation set). Above that, calibration curve and Hosmer-Lemeshow test showed that the radiomic model and combined radiomic nomogram had good agreement between observations and predictions in the training set and the validation set. Finally, the decision curve analysis revealed that the 2 models had good clinical usefulness for the prediction of TERTp mutation status in IDHwt LGA. The combined radiomics nomogram performed great performance and high sensitivity in prediction of TERTp mutation status in IDHwt LGA, and has good clinical application.