C228T Mutation Research Articles

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.

BIOM-71. EXPANDING THE SPECTRUM OFTERT PROMOTER NON-CANONICAL SEQUENCE VARIANTS IN CNS TUMORS: A CASE SERIES

Abstract Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A>C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A>C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (>0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (>0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A>C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A>C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.

8078 TERT Positive Thyroid Nodule in the Setting of Metastatic Melanoma

Abstract Disclosure: E. Langnas: None. S. Chao: None. K.M. Gomes: None. A. Bhan: None. Metastatic disease to the thyroid gland is uncommon with a reported incidence of up to 4% in post-surgical specimens. Lung cancer is the most common primary tumor site in autopsy series. Clinically, renal cell carcinoma is the most common primary tumor site. Metastatic melanoma to the thyroid gland is clinically rare, however, in autopsy series, it has been reported with incidence of up to 39%. A 61-year-old man presented to the endocrinology clinic for an incidentally detected thyroid nodule. He had recently presented with a chronic cough, prompting a chest x-ray that revealed pulmonary nodules. Subsequent computed tomography (CT) showed numerous bilateral lung nodules measuring up to 2.5 cm and an incidental thyroid nodule. Ultrasound of the thyroid identified a 2.7 cm mixed cystic-solid right thyroid nodule. No risk factors for thyroid cancer were identified. His TSH was 3.51uIU (0.45 - 5.33 uIU/mL). The thyroid nodule was biopsied, and pathology revealed atypical spindle cells with irregular nuclear membranes and scattered histiocytes. A further review with cytopathology suggested non-thyroidal origin of the cells. Molecular testing with AFIRMA reported a telomerase reverse transcriptase (TERT) promoter mutation at C228T. The sequencing was negative for TERT C250T, BRAF, RET, PETC1, PTC3 and MTC. Due to concerns about metastatic disease, a positron-emission tomography (PET)-CT was conducted, revealing hypermetabolic lesions in numerous bilateral lung nodules, right thyroid nodule, porta hepatis lymph node, and a soft tissue nodule within the greater curvature of the gastric body. Subsequent biopsies of the right lung and gastric body confirmed the diagnosis of metastatic melanoma. On further questioning, it was found that the patient had a history of a skin lesion on his back that was excised several years ago with pathology reporting tumoral melanosis. Malignant melanoma has a poor prognosis due to high recurrence and metastatic rates. In melanoma, TERT promoter mutations are the most common mutations in noncoding regulatory regions. TERT C228T mutations and C250T mutations are seen in malignant melanoma and studies have shown that TERT promoter mutations are associated with aggressive clinical behavior. These TERT promoter mutations can also be seen in various other cancers including primary thyroid cancers with a similar association with more aggressive thyroid tumor characteristics. Our case describes a patient with a thyroid nodule and molecular testing noting a TERT promoter mutation. Both primary thyroid cancer and metastatic disease to the thyroid should be considered in these cases. This case also highlights the importance of obtaining a thorough history of prior malignancy in patients with thyroid nodules to assess the risk of metastatic disease. Presentation: 6/1/2024

TERT C228T Mutation in Circulating Tumor DNA as a Clinical Marker in Hepatocellular Carcinoma Patients Attending a Tertiary Hospital in Egypt: A Case-Control Study

TERT C228T Mutation in Circulating Tumor DNA as a Clinical Marker in Hepatocellular Carcinoma Patients Attending a Tertiary Hospital in Egypt: A Case-Control Study

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.

Distinct transcriptional and prognostic impacts of TERT promoter mutations C228T and C250T in papillary thyroid carcinoma.

TERT promoter mutations C228T and C250T are associated with disease aggressiveness and poor clinical outcomes in patients with papillary thyroid carcinomas. However, very little is known about the transcriptional consequences of these mutations and whether they both carry similar oncogenic potential. Here we characterized the transcriptional disturbances and clinical outcomes associated with the presence of each of these two mutations using data derived from The Cancer Genome Atlas. We observed that tumors harboring the C228T mutation (n = 27) exhibited a 16-fold increase in TERT mRNA levels (P = 5.3 × 10-42), whereas C250T tumors (n = 8) showed only a two-fold increase in expression (P = 0.034). The C228T mutation was associated with the activation of signaling pathways controlling the cell cycle, cellular division, and extracellular matrix degradation. Univariate analysis demonstrated that the C228T mutation was associated with older age at diagnosis, large tumor size, lymph node invasion, and distant metastases at diagnosis. The C228T mutation was also associated with worse progression-free interval (PFI) in comparison to WT tumors (HR = 5,04; P < 0.001). This association remained significant in a multivariate analysis (HR = 3.74, P = 0.003) adjusting for BRAF-V600E status and ATA risk group. Our data indicate that TERT promoter mutations C228T and C250T have distinct transcriptional consequences in papillary thyroid carcinoma (PTC), suggesting a greater oncogenic potential for the C228T mutation. TERT promoter mutation C228T may be a useful prognostic marker to identify patients at high risk of distant recurrence. Clinical data for the C250T mutation is still limited, with no evidence up to date to confirm its prognostic significance.

Abstract 331: Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology

Abstract Introduction: Recurrent mutations C228T and C250T in the TERT gene promoter are prevalent in cancer and serve as important biomarkers for aggressive disease. Ion Torrent™ NGS sequencing, combined with Ion AmpliSeqTM HD technology, enables comprehensive genomic profiling with ultra-high sensitivity for low-frequency variants in cell-free DNA (cf-DNA) and highly heterogeneous solid tumor samples. However, the high GC content (&amp;gt;80%) and the presence of G repeats that are known to adopt a compact stacked three-G-quadruplex conformation in the TERT gene promoter region pose sequencing challenges such as low read depth and strand-bias, i.e. prematurely truncated reads on one strand. Methods: We designed nine AmpliSeqTM HD panels with amplicon sizes of 70 to 104 bp, ideal for cf-DNA and FFPE samples, to target the recurrent TERT promoter hotspots. The designs were tested alone and combined with a larger Ion AmpliSeqTM HD DNA research panel resulting in highly multiplexed libraries. The libraries were used for Ion 540TM chip template preparation on the Ion ChefTM instrument and subsequently sequenced using the Ion GeneStudioTM S5 System. Sequencing data was used to evaluate coverage of the target hotspots by each design for each condition, and additional panels were designed to test single amplicons covering both TERT promoter hotspots. Results: Through performance evaluation, we selected two overlapping amplicons, each effectively amplifying one of the two hotspots. Individually tested, these amplicons yielded approximately 3000 families each from a 10ng gDNA input and when combined into the larger Ion AmpliSeqTM HD DNA research panel, they produced several thousand reads across various libraries, providing molecular coverage of around 200 functional molecules. While capture efficiency was lower compared to TERT amplicon-only libraries, these improved amplicons allow downstream analysis of variants. A single-pool solution (single amplicon/s covering both hotspots) will be explored further using the second batch of amplicons/primers. Conclusions: The optimized amplicons in two pools developed in this study enable the detection of challenging yet clinically significant TERT promoter hotspot mutations using the ultra-sensitive Ion AmpliSeqTM HD technology. For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Citation Format: Anupma Sharma, Guobin Luo, Na Li, Giorgio Pea, Fiona Hyland. Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 331.

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.

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.

Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing

BackgroundActivation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.ResultsSequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.ConclusionsWe applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

PATH-50. GENETIC AND EPIGENETIC PROFILING OF MENINGIOMAS TO GAIN INSIGHTS INTO MOLECULAR HETEROGENEITY AND RISK STRATIFICATION.

Abstract BACKGROUND Meningiomas are slow-growing CNS tumors, with most being benign (WHO grade 1) and having indolent behavior. Limited data on molecular profiling of meningiomas and its clinical implications have hindered the implementation of effective therapies and new treatments. Aims &amp; Objectives: Perform DNA methylation profiling, copy number analysis, and targeted sequencing in meningiomas. MATERIALS &amp; METHODS 208 meningiomas, grade 1 (n=109), grade 2 (n=83), and grade 3 (n=16)were assessed for pTERT mutation, CDKN2A homozygous deletion, and H3K27me3 expression. Additionally, 35 diagnostically challenging meningiomas underwent DNA methylation profiling, copy number analysis, and targeted sequencing. RESULTS there were 199 adult and 9 pediatric cases, (mean age 44.5 years). The tumors were located in basal (53%) ,convexity (42%),and 5% in other locations.pTERT C228T mutation was detected in 4.8% and CDKN2A deletion in 2.4% cases. Loss of H3K27me3 expression was found in a significant proportion of tumors across all three grades. Methylation profiling identified four distinct molecular subgroups: Malignant, Intermediate, Benign A, and Benign B. Intermediate, Benign A, and Benign B subgroups included both WHO grade 1 and grade 2 cases. Mutations showed a strong association with specific methylation subgroups (NF2, AKT1, SMO, TRAF7, and pTERT). Loss of chromosome 22q was observed in all subgroups. 1p/14q co-deletion was predominantly seen in the malignant and intermediate subgroups (50%). CDKN2A loss was mainly observed in the malignant subgroup (50%). The DKFZ classifier successfully classified 71% of cases with an acceptable score. Methylation profiling significantly influenced PFS in WHO grade 1 and 2 meningiomas (p=0.0051). CONCLUSIONS Genome-wide DNA methylation profiling reveals clinically distinct molecular subgroups and heterogeneity within the same grade of meningiomas and is particularly valuable for identifying high-risk cases and predicting prognosis in WHO grade 1 and 2 meningiomas. The pTERT mutation serves as a significant and cost-effective marker for risk stratification in meningiomas.

BIOM-32. EVS CARRYING TENASCIN-C ENABLE TUMOR-SPECIFIC MUTATION ANALYSIS IN GLIOBLASTOMA PATIENTS

Abstract Extracellular vesicles (EVs) transport biological and specific information from tumors into the bloodstream, enabling non-invasive detection of tumor material and disease monitoring. Based on a proteomics screen, we performed immunophenotyping of eight glioma-related antigens (tenascin-C (TNC), integrin-beta 1 (ITGB1), profilin-1 (PFN1), CD44, CD133, GPNMB, HLA-II, SPARC) and tetraspanins (CD9, CD63 and CD81) in plasma EVs from GBM patients (before and after surgery (n = 38)), from matched GBM relapse patients (n = 11), and from healthy donors (HD, n = 12) using imaging flow cytometry. Double-positive TNC+/CD9+ EVs showed the strongest differences per mL of plasma in primary (FC = 7.6, p&amp;lt; .0001, ROC analysis AUC = 81%) and relapsed GBM (FC = 16.5, p&amp;lt; .0001; AUC = 90%) compared to HD subjects. High TNC signals were also observed in GBM-EVs by immunogold electron microscopy compared to HD-EVs. In paired analysis, TNC+/CD9+ EVs showed a 3.9-fold decrease after tumor removal (p&amp;lt; .001) and re-increased at GBM recurrence in these patients (FC = 8.4, p&amp;lt; .05; AUC = 84%). In tissue samples, TNC levels were 5.4-fold higher in GBM patients than in non-neoplastic cortex controls (p&amp;lt; .01) measured by immunohistochemistry and correlated positively with plasma TNC+/CD9+ EV levels in RTK-I/II GBM patients (r = 0.42, p&amp;lt; .05). Accordingly, spatial transcriptomics of GBM tissue sections revealed that TNC is specifically overexpressed in GBM cells. Furthermore, magnetic sorting of TNC in plasma EVs allowed detection of GBM-specific TERT*C228T mutations by digital droplet PCR. Mutated TERT DNA was enriched in TNC+ EVs (n = 13) compared to TNC- EVs (FC = 110, p&amp;lt; 0.0001), total EV DNA (FC = 36.7, p&amp;lt; 0.01), and cfDNA (FC = 7.2, p &amp;lt; 0.05). In conclusion, we identified TNC as a biomarker in circulating EVs from GBM patients that can be isolated and used for tumor-specific mutation analysis.

PATH-27. DROPLET DIGITAL PCR ENHANCES DETECTION OF HOTSPOT MUTATIONS IN CIRCULATING TUMOR DNA FROM CEREBROSPINAL FLUID OF PATIENTS WITH HIGH-GRADE GLIOMA

Abstract Molecular profiling of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) has the potential to enhance diagnosis, prognosis, and longitudinal monitoring for glioma patients through a minimally invasive approach. We previously detected somatic mutations from gliomas in CSF samples using MSK-IMPACT™, a 505-gene FDA-authorized next-generation DNA sequencing panel, and detected tumor-derived mutations in 50% of samples of patients with high-grade gliomas (HGG). To investigate whether we could enhance sensitivity of CSF ctDNA profiling and biomarker detection, we tested MSK-IMPACT™ pre-capture NGS residual DNA libraries from 43 CSF ctDNA samples by droplet digital PCR (ddPCR) for detection of key recurrent alterations in IDH1, BRAF, TERT, H3F3A, and TP53 using previously validated probes. Residual libraries contained a median of 18.0ng of DNA library (range, 0.0153-18.0ng). Of the 7 samples positive for IDH1 R132H, TERT C228T, or TERT C250T mutations in MSK-IMPACT™, we found that ddPCR accurately detected all mutations (7/7, 100%), confirming sensitivity of ddPCR in CSF ctDNA. We also tested 36 mutation-negative samples by MSK-IMPACT™ for the corresponding undetected mutations, of which 7 were tested for two mutations. Overall, ddPCR detected 41.9% (18/43) of previously undetected mutations. Specifically, we found that ddPCR rescued hotspot mutations such as BRAF V600E (2/3, 66.7%), IDH1 R132G/H (3/8, 37.5%), TERT C228T (11/26, 42.3%), and TERT C250T (2/4, 50.0%). Collectively, our results suggest that a combined-modality approach involving MSK-IMPACT™ and ddPCR enables comprehensive assessment of limited CSF ctDNA samples with high sensitivity and provides a minimally invasive methodology to molecularly profiling glioma.

Screening for a practical method to monitor the status of patients with metastatic bladder cancer at the circulating cell-gene level

Identifying a novel method to monitor metastatic bladder cancer status at the cell-gene level could lead to earlier appropriate therapeutic intervention and better outcomes. In this study, we evaluated a practical method to monitor the cancer status at the circulating cell-gene level before and after treatment in fourteen patients with metastatic bladder cancer who were indicated for systemic drug therapy. Patients were assessed via imaging before and after drug treatment, and cell-free DNA (cfDNA) analysis was performed to detect three parameters: cfDNA level, ERRB2 gene copy numbers, and telomerase reverse transcriptase (TERT) gene mutations. We hypothesized that decreased cfDNA levels, a normal copy number of ERB-B2 receptor tyrosine kinase 2 (ERBB2), and the absence of the TERT C228T mutation indicate cancer suppression. We found that a > 1.8-fold increase in cfDNA levels, increased copy number of ERBB2, or the existence of the TERT C228T mutation indicated disease progression. Stable cfDNA levels, normal ERBB2 copy number, and the absence of TERT C228T mutations indicate a stable cancer status. Collectively, our results show that the combination of cfDNA concentration, TERT mutation, and ERBB2 copy number may be useful for determining the efficacy of drug therapy in patients with metastatic bladder cancer.

Concurrent TERT promoter C228T and C250T mutations in diffuse gliomas: Rare occurrence of intratumoral heterogeneity.

Concurrent TERT promoter C228T and C250T mutations in diffuse gliomas: Rare occurrence of intratumoral heterogeneity.

P11.28.ATERT C228T: A NOVEL MOLECULAR INDEPENDENT PROGNOSTIC FACTOR IN PATIENTS WITH GLIOBLASTOMA IDH WILD TYPE

Abstract BACKGROUND TERT promoter (TERTp) variants regulate TERT expression and are the most frequent mutations in glioblastoma (GB), specifically c. -124C&amp;gt;T and c.-146C&amp;gt;T, known as C228T and C250T respectively. The role on prognosis of TERTp mutation is unclear and its clinical impact remains unknown. We assess the association of the status of TERTp considering the mutation subtype (C228T vs C250T vs TERTp wild type (WT)) with outcomes in patients (pts) with GB IDH-WT. MATERIAL AND METHODS We expanded a retrospective multicenter study to include pts with GB treated between 2010-2022 with standard treatment (radiotherapy with concomitant and adjuvant temozolomide). Clinical, molecular, and histological data were collected from medical reports. C228T and C250T TERTp mutations were assesed by PCR/Sanger sequencing (93%) or NGS (7%). MGMTp methylation and IDH mutation were tested by MS-PCR and immunohistochemistry/sequencing, respectively. Median overall survival (mOS) was estimated using the Kaplan-Meier method and compared between strata through the log-rank test. Multivariate Cox regression models were performed to adjust the effect of C228T mutation by MGMTp methylation, age, Karnofsky (KPS) and type of surgery and to assess the potential role of the interaction of both molecular factors RESULTS A total of 255 pts have been enrolled with a median follow up of 14.2 months; 63% were male, 35% older than 65 years old, 70% had a Karnofsky Index &amp;gt;80%, 40% had gross total resection and 51% had MGMTp methylation. A total of 86 pts (34%) were treated with bevacizumab in latter lines.The presence of TERTp mutation C228T was observed in 54.7% (N=140), C250T in 22.7% (N=58) and the rest (N=57) were TERTp WT. The median OS was 16.4 months (95% CI 14.7 - 18). The C250T mutation was significantly associated with better OS 21.7 months (95% CI 16.1 - 32; p=0.041). Multivariate analysis confirmed that C228T TERTp mutation confers worse prognosis independently of methylation status, age, KPS and type of surgery (HR 1.55; 1.07-2.24 p=0.02). In a further multivariate analysis to assess the potential role of the interaction between both molecular factors, pts with GB unmethylated MGMTp/C228T TERTp mutation had the worse prognoses (HR = 2.83, 95% CI: 1.59-5.03, p &amp;lt; 0.001), followed by the unmethylated MGMTp/C250T (HR = 2.06, 95% CI: 1.08 - 3.94, p = 0.028), and methylated MGMTp/C228T TERTp (HR = 1.80, 95% CI: 1.01 - 3.17, p = 0.041). CONCLUSION We report that TERTp C228T mutation is an independent prognostic factor of worse survival regardless of MGMT methylation status, age, KPS and type of surgery, in pts with GB IDH-WT treated homogeneously with standard treatment. We purpose to consider it as an important new prognostic molecular factor in the design of new clinical trials.

Concurrent PIK3CA and TERT Mutation Promote the Proliferation and Invasion of Thyroid Carcinoma Cells and may be Caused by Up-regulating the Expression of GABPA/GABPB1

Our previous research demonstrated that TERT and concurrent PIK3CA mutations predict worse overall survival in patients with poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma. However, the molecular mechanism underlying the synergistic oncogenic operations of the two oncogenes is unclear. This study aimed to explore further the effect of TERT and PIK3CA co-mutation on the malignant biological phenotype of thyroid carcinoma and its possible mechanism. PIK3CA E545K mutation plasmid was transfected into thyroid anaplastic cancer cell line (C643) with TERT promoter mutation, then CCK-8 and transwell invasion assays were used to investigate the ability of cell proliferation and invasion, respectively. RT-qPCR and western blot were performed to detect the expression of PIK3CA, TERT, GABPA and GABPB1. GABPA/GABPB1 siRNA plasmid was transfected with C643 cells, then the ability of cell proliferation and invasion were identified. We also detected the expression of PIK3CA and TERT. C643 cells carry TERT promoter mutation C228T. Concurrent PIK3CA E545K and TERT mutation markedly enhanced the proliferation and invasion of C643 cell in vitro, with significantly increased mRNA/protein expression of PIK3CA, TERT, GABPA and GABPB1. Knocking down GABPA markedly inhibited cell proliferation. Knocking down of GABPB1 significantly decreased the proliferation and invasion of C643 cells, with much lower expression of PIK3CA and TERT. TERT and PIK3CA co-mutations promote the proliferation and invasion of thyroid anaplastic carcinoma cells and may be caused by up-regulating the expression of GABPA and GABPB1.

Collision tumor of a papillary and follicular thyroid carcinoma: a case report

BackgroundPapillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are common differentiated thyroid cancers, but the detection of a collision tumor is an extremely rare event. Case presentationThe patient was a 69-year-old Japanese female with multiple cervical lymph node swellings and a thyroid tumor. Preoperative fine needle aspiration cytology of the enlarged lymph node revealed a cytological diagnosis of papillary thyroid carcinoma (PTC). A total thyroidectomy, right cervical dissection and paratracheal dissection were performed. Histopathological and immunohistochemical analyses of resected specimens revealed a collision tumor of PTC and FTC. Multiple metastases of papillary carcinoma were found in the dissected lymph nodes. In the PTC lesion, IHC for BRAF (V600E) was positive but negative for the FTC lesion. Genetic analyses further revealed a TERT promoter C228T mutation in PTC and a NRAS codon 61 mutation in FTC. The patient died of recurrent cancer 8 months after surgery.ConclusionsA case of a collision tumor of PTC and FTC is very rare, and even fewer cases have been subjected to genetic scrutiny. The present case was successfully diagnosed by pathological examination using immunohistochemical and genetic analyses. The TERT promoter mutation in the PTC lesion was consistent with the aggressive behavior of the cancer.

PTERT C250T mutation: A potential biomarker of poor prognosis in metastatic melanoma

Melanoma is the most aggressive form of skin cancer and the leading cause of death from cutaneous tumors. Several studies have associated alterations in the TERT promoter region (pTERT) with gene overexpression, aggressiveness and poor prognosis of the disease. The aim of this study was to clarify the role of pTERT molecular status in paired samples of primary melanoma and metastasis using tissue and plasma to establish a correlation with disease progression and survival.A total of 88 FFPE tissue samples from 53 patients with advanced melanoma were analyzed. Of these, 35 had paired samples. We also examined cfDNA samples from plasma of 25 patients. We detected a good correlation between primary tumors and metastases in pTERT mutation and methylation status. We were also able to identify pTERT mutations in plasma samples that correlated with mutational status in tissue samples. Interestingly, the C250T mutation was associated with worse survival and higher TERT mRNA expression, compared to the other most common mutation: C228T. In addition, hyper-methylation of the promoter region seems to be related to the progression of pTERT wild type (WT) patients. These results suggest that TERT gene alterations plays an important role during tumor progression, with the detection of the C250T mutation in tissue and plasma as a potential biomarker of poor prognosis in patients with advanced melanoma.