Molecular Mechanisms in Tumorigenesis of Hepatocellular Carcinoma and in Target Treatments-An Overview.

Hepatocellular carcinoma is the most common primary malignancy of the liver, with hepatocellular differentiation. It is ranked sixth among the most common cancers worldwide and is the third leading cause of cancer-related deaths. The most important etiological factors discussed here are viral infection (HBV, HCV), exposure to aflatoxin B1, metabolic syndrome, and obesity (as an independent factor). Directly or indirectly, they induce chromosomal aberrations, mutations, and epigenetic changes in specific genes involved in intracellular signaling pathways, responsible for synthesis of growth factors, cell proliferation, differentiation, survival, the metastasis process (including the epithelial-mesenchymal transition and the expression of adhesion molecules), and angiogenesis. All these disrupted molecular mechanisms contribute to hepatocarcinogenesis. Furthermore, equally important is the interaction between tumor cells and the components of the tumor microenvironment: inflammatory cells and macrophages-predominantly with a pro-tumoral role-hepatic stellate cells, tumor-associated fibroblasts, cancer stem cells, extracellular vesicles, and the extracellular matrix. In this paper, we reviewed the molecular biology of hepatocellular carcinoma and the intricate mechanisms involved in hepatocarcinogenesis, and we highlighted how certain signaling pathways can be pharmacologically influenced at various levels with specific molecules. Additionally, we mentioned several examples of recent clinical trials and briefly described the current treatment protocol according to the NCCN guidelines.

Therapeutic targeting of PLK1 in TERT promoter-mutant hepatocellular carcinoma.

BACKGROUND: Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. METHODS: The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. RESULTS: PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. CONCLUSIONS: PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. KEY POINTS: TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.

Coexisting RET/PTC and TERT Promoter Mutation Predict Poor Prognosis but Effective RET and MEK Targets in Thyroid Cancer.

PURPOSE: To investigate the role of coexisting RET/PTC rearrangement and TERT promoter mutation in the prognosis and therapeutic targeting in papillary thyroid cancer (PTC). METHODS: A total of 669 PTC patients with complete clinical follow-up and genetic data were pooled from thyroid cancer datasets TCGA, MSK MetTropism, and MSK-IMPACT, from whom 163 patients (112 women and 47 men, 4 unknown) with wild-type BRAF/RAS were identified, with median age (IQR) of 46.00 (33.00, 61.00) years and median follow-up time (IQR) of 16.13 (8.09, 27.91) months for comparative genotype cohort analysis of mortality. RESULTS: There was a significant concurrence index between RET/PTC and TERT promoter mutations, being 2.040 (95% CI 1.110-3.747, P = 0.023). Mortality occurred in 5/100 (5%) patients harboring neither mutation, 2/18 (11.1%) patients harboring TERT promoter mutation alone, 0/31 (0%) patients harboring RET/PTC alone, and 7/14 (50%) patients harboring both genetic alterations, corresponding to HRs (95% CI) of 1 (Reference), 2.469 (0.405-14.02), 3.296e-09 (0-inf), and 9.019 (2.635-30.87), respectively, which remained essentially unchanged after adjustment for patient race, sex, and age. Similar results were observed with BRAF/RAS and TERT promoter mutations. Mechanistically, RET/PTC used the MAP kinase pathway to upregulate the mutated TERT, but not the wild-type TERT, and, correspondingly, targeting RET and MEK could suppress mutated TERT but not the wild-type TERT. CONCLUSION: Coexisting RET/PTC and TERT promoter mutation identify PTC as a unique clinical entity with high mortality, providing new implications for genetic-based prognostication and potential therapeutic targeting of RET and MEK guided by RET/PTC and TERT status.

TERT promoter mutations increase tumor aggressiveness by altering TERT mRNA splicing in papillary thyroid carcinoma.

CONTEXT: Telomerase reverse transcriptase promoter (TERT-p) mutations, which upregulate TERT expression, are strongly associated with tumor aggressiveness and worse prognosis in papillary thyroid carcinomas (PTCs). TERT expression is also observed in a proportion of PTCs without TERT-p mutations, but such tumors show less aggressiveness and better prognosis compared with TERT-p mutation-positive tumors. OBJECTIVE: TERT has multiple splicing variants whose relationships with the TERT-p status and clinicopathological characteristics remain poorly understood. We examined the relationship between the TERT-p mutational status, the TERT splicing pattern, and clinicopathological features. METHODS: We investigated the expression of two major variants, α deletion (dA) and ß deletion (dB), in a series of 207 PTCs operated between November 2001 and March 2020 in Nagasaki University Hospital and Kuma Hospital. RESULTS: The TERT-p mutations were found in 33 cases, and among 174 mutation-negative cases, 24 showed TERT expression. All cases were classified into three groups: the TERT-p mutation-negative/expression-negative group (mut-/exp-), the TERT-p mutation-negative/expression-positive group (mut-/exp+), and the TERT-p mutation-positive group (mut+/exp+). The +A + B/dB ratio in mut+/exp + was significantly higher than that in mut-/exp + PTCs. Analysis with clinicopathological data revealed that +A + B expression was associated with higher PTC aggressiveness, whereas dB expression counteracted this effect. Functional in vitro study demonstrated that dB strongly inhibited cell growth, migration, and clonogenicity, suggesting its tumor suppressive role. CONCLUSION: These results provide evidence that the TERT-p mutations alter the expression of different TERT splice variants, which, in turn, associates with different tumor aggressiveness.

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.

Predicting the prognosis of glioma patients with TERT promoter mutations and guiding the specific immune profile of immune checkpoint blockade therapy.

The telomerase reverse transcriptase promoter (TERTp) is frequently mutated in gliomas. This study sought to identify immune biomarkers of gliomas with TERTp mutations. Data from TCGA were used to identify and validate survival-associated gene signatures, and immune and stromal scores were calculated using the ESTIMATE algorithm. High stromal or immune scores in patients with TERTp-mutant gliomas correlated with shorter overall survival compared to cases with low stromal or immune scores. Among TERTp-mutant gliomas with both high immune and high stromal scores, 213 commonly shared DEGs were identified. Among 71 interacting DEGs representing candidate hub genes in a PPI network, HOXC6, WT1, CD70, and OTP showed significant ability in establishing subgroups of high- and low-risk patients. A risk model based on these 4 genes showed strong prognostic potential for gliomas with mutated TERTp, but was inapplicable for TERTp-wild-type gliomas. TERTp-mutant gliomas with high-risk scores displayed a greater percentage of naïve B cells, plasma cells, naïve CD4 T cells, and activated mast cells than low-risk score gliomas. TIDE analysis indicated that immune checkpoint blockade (ICB) therapy may benefit glioma patients with TERTp mutations. The present risk model can help predict prognosis of glioma patients with TERTp mutations and aid ICB treatment options.

Comparison of TERT and 5-Hydroxymethylcytocine immunohistochemistry in various thyroid carcinomas.

Telomerase reverse transcriptase (TERT) promoter mutation is associated with an aggressive clinical course in thyroid carcinomas. Therefore, detection of TERT promoter mutation is essential for proper patient management. 5-Hydroxymethylcytosine (5hmC) is an epigenetic marker involved in the DNA demethylation pathway, and its loss has been observed in various tumors. Loss of 5hmC has also been reported in thyroid carcinomas and is presented as a possible predictive biomarker for TERT promoter mutation and worse prognosis. This study evaluated the expression of TERT and 5hmC by immunohistochemistry (IHC) in 105 patients (44 in the TERT mutant group and 61 in the TERT wild group) with various thyroid carcinomas. H-scores were calculated using an image analyzer. The median H-scores of TERT IHC were significantly higher in the TERT mutant group than in the TERT wild group (47.15 vs. 9.80). The sensitivity and specificity of TERT IHC for predicting TERT promoter mutations were 65.9 and 65.7 %, respectively. Regardless of TERT promoter mutation status, the 5hmC H-scores were markedly lower in all subtypes of thyroid carcinomas compared to those in their normal counterparts. Significant differences in 5hmC H-scores were observed between N0 and N1 in total thyroid carcinomas, but not within the papillary thyroid carcinoma subgroup. In conclusion, TERT and 5hmC IHC have limitations in predicting the presence of TERT promoter mutations. The expression of 5hmC was downregulated in various thyroid carcinomas compared to that in normal and benign lesions, but comprehensive further studies are required to elucidate the role of 5hmC in thyroid carcinomas.

Complex phenotypic heterogeneity of combined hepatocellular-cholangiocarcinoma with a homogenous TERT promoter mutation.

To clarify the mechanism underlying the development and poor prognosis of combined hepatocellular-cholangiocarcinoma (cHCC-CCA), we characterized liver cancer driver mutations and poor prognostic markers in both the HCC and intrahepatic CCA (iCCA) components of a cHCC-CCA tumor. The telomerase reverse transcriptase (TERT) promoter mutation C228T was quantified by digital polymerase chain reaction using DNA from multiple microdissected cancer components of a single cHCC-CCA nodule. The protein expression of cancer-related markers, including TERT, was examined by serial thin-section immunohistochemistry and double-staining immunofluorescence. TERT promoter mutation and TERT protein expression were detected in all cancer components but not in noncancer regions. TERT promoter mutation frequencies were similar among components; those of TERT protein-positive cancer cells were higher in iCCA and mixed components than in HCC. The frequencies of Ki67- and p53-positive cells were similarly higher in iCCA and mixed components than in HCC. However, double-positive cells for the three proteins were unexpectedly rare; single-positive cells dominated, indicating phenotypic microheterogeneity in cancer cells within a component. Interestingly, HCC and CCA marker protein immunohistochemistry suggested dedifferentiation of HCC and transdifferentiation from HCC to iCCA in HCC and iCCA components, respectively. Such phenotypic intercomponent heterogeneity and intracomponent microheterogeneity were detected in a tumor nodule of cHCC-CCA uniformly carrying the early HCC driver mutation. Moreover, poor prognostic markers were randomly expressed without a regular pattern, consistent with the poor prognosis.

Risk stratification by combining common genetic mutations and TERT promoter methylation in papillary thyroid cancer.

PURPOSE: Risk stratification based on somatic mutations in TERT promoter and BRAF/RAS has been well established for papillary thyroid cancer (PTC), and there is emerging evidence showed that TERT promoter methylation was frequently observed in thyroid cancer patients with adverse features. This study was aimed to comprehensive explore the prognostic value of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation in PTC. METHODS: The relationships of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation with clinical characteristics and outcomes of PTC were analyzed in 382 patients with PTC. RESULTS: TERT promoter mutation and hypermethylation were collectively observed in 52 (13.6%) samples and associated with BRAF/RAS mutation, aggressive clinical characteristics, and poor clinical outcomes of PTC. Coexistence of BRAF/RAS and TERT alterations was found in 45 of 382 (11.8%) PTC patients and strongly associated with old patient age, extrathyroidal extension, advanced pathologic T stage and metastasis. Importantly, patients with both BRAF/RAS and TERT alterations had higher rates of tumor recurrence (13.6% vs 1.5%, P = 0.042) and disease progression (24.4% vs 3.3%, P < 0.001) than patients without any alterations, and cox regression analysis revealed that the coexistence of BRAF/RAS and TERT alterations, but not BRAF/RAS or TERT alterations alone, increased the risk of progression-free interval with an adjusted HR of 10.35 (95% CI: 1.79-59.81, P = 0.009). CONCLUSIONS: This study suggested that comprehensively analysis of BRAF/RAS mutations, TERT promoter mutation and methylation is an effective strategy to identify high-risk patients with PTC.

Prevalence of TERT Promoter Mutations in Orbital Solitary Fibrous Tumors.

The orbital manifestation of a solitary fibrous tumor (SFT) is exceptionally rare and poses specific challenges in diagnosis and treatment. Its rather exceptional behavior among all SFTs comprises a high tendency towards local recurrence, but it rarely culminates in metastatic disease. This raises the question of prognostic factors in orbital SFTs (oSFTs). Telomerase reverse transcriptase (TERT)-promoter mutations have previously been linked to an unfavorable prognosis in SFTs of other locations. We analyzed the prevalence of TERT promoter mutations of SFTs in the orbital compartment. We performed a retrospective, descriptive clinico-histopathological analysis of nine cases of oSFTs between the years of 2017 and 2021. A TERT promoter mutation was present in one case, which was classified with intermediate metastatic risk. Local recurrence or progress occurred in six cases after primary resection; no distant metastases were reported. Multimodal imaging repeatedly showed particular morphologic patterns, including tubular vascular structures and ADC reduction. The prevalence of the TERT promoter mutation in oSFT was 11%, which is similar to the prevalence of extra-meningeal SFTs of the head and neck and lower than that in other extra-meningeal compartments. In the present study, the TERT promoter mutation in oSFT manifested in a case with an unfavorable prognosis, comprising aggressive local tumor growth, local recurrence, and eye loss.

Association of Ultrasonography Features of Follicular Thyroid Carcinoma With Tumor Invasiveness and Prognosis Based on WHO Classification and TERT Promoter Mutation.

OBJECTIVE: To investigate the association of ultrasound (US) features of follicular thyroid carcinoma (FTC) with tumor invasiveness and prognosis based on the World Health Organization (WHO) classification and telomerase reverse transcriptase (TERT) promoter mutations. MATERIALS AND METHODS: This retrospective study included 54 surgically confirmed FTC patients with US images and TERT promoter mutations (41 females and 13 males; median age [interquartile range], 40 years [30-51 years]). The WHO classification consisted of minimally invasive (MI), encapsulated angioinvasive (EA), and widely invasive (WI) FTCs. Alternative classifications included Group 1 (MI-FTC and EA-FTC with wild type TERT), Group 2 (WI-FTC with wild type TERT), and Group 3 (EA-FTC and WI-FTC with mutant TERT). Each nodule was categorized according to the US patterns of the Korean Thyroid Imaging Reporting and Data System (K-TIRADS) and American College of Radiology-TIRADS (ACR-TIRADS). The Jonckheere-Terpstra and Cochran-Armitage tests were used for statistical analysis. RESULTS: Among 54 patients, 29 (53.7%) had MI-FTC, 16 (29.6%) had EA-FTC, and nine (16.7%) had WI-FTC. In both the classifications, lobulation, irregular margins, and final assessment categories showed significant differences (all Ps ≤ 0.04). Furthermore, the incidences of lobulation, irregular margin, and high suspicion category tended to increase with increasing tumor invasiveness and worse prognosis (all Ps for trend ≤ 0.006). In the WHO groups, hypoechogenicity differed significantly among the groups (P = 0.01) and tended to increase in proportion as tumor invasiveness increased (P for trend = 0.02). In the alternative group, punctate echogenic foci were associated with prognosis (P = 0.03, P for trend = 0.03). CONCLUSION: Increasing tumor invasiveness and worsening prognosis in FTC based on the WHO classification and TERT promoter mutation results were positively correlated with US features that indicate malignant probability according to both K-TIRADS and ACR-TIRADS.

TERT promoter mutations in atypical melanocytic lesions: A series of seven cases with adverse melanoma-specific outcome.

The majority of melanocytic proliferations can be readily categorized as benign or malignant based on histologic assessment under the microscope by a trained dermatopathologist. However, a subset of lesions, termed Atypical Melanocytic Proliferations (AMPs), are histologically ambiguous, leading to possible diagnostic error and suboptimal treatment. Mutations in the promoter region of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), are commonly found in melanomas but are rare in melanocytic nevi. In this study, we aimed to determine the prevalence of hot spot TERT promoter (TERT-p) mutations in AMPs with adverse melanoma-specific outcome. Studies were approved by respective institutional review boards. Using a multi-center database, we identified seven cases of melanocytic proliferations with a clinical follow-up period of at least 4 years, which were initially diagnosed as AMPs, and which recurred either as melanoma at site of prior biopsy or as metastatic melanoma. Sequencing of the TERT-p region showed hotspot mutations in three cases (43 %), suggesting that TERT-p mutations are enriched and could aid in the identification of AMPs with adverse outcome. In comparison with existing ancillary techniques for prognostication of AMPs, TERT-p mutation analysis may have advantages in terms of cost effectiveness and turnaround time, and is a promising diagnostic parameter with potential widespread utility.

Artificial Intelligence Imaging for Predicting High-risk Molecular Markers of Gliomas.

Gliomas, the most prevalent primary malignant tumors of the central nervous system, present significant challenges in diagnosis and prognosis. The fifth edition of the World Health Organization Classification of Tumors of the Central Nervous System (WHO CNS5) published in 2021, has emphasized the role of high-risk molecular markers in gliomas. These markers are crucial for enhancing glioma grading and influencing survival and prognosis. Noninvasive prediction of these high-risk molecular markers is vital. Genetic testing after biopsy, the current standard for determining molecular type, is invasive and time-consuming. Magnetic resonance imaging (MRI) offers a non-invasive alternative, providing structural and functional insights into gliomas. Advanced MRI methods can potentially reflect the pathological characteristics associated with glioma molecular markers; however, they struggle to fully represent gliomas' high heterogeneity. Artificial intelligence (AI) imaging, capable of processing vast medical image datasets, can extract critical molecular information. AI imaging thus emerges as a noninvasive and efficient method for identifying high-risk molecular markers in gliomas, a recent focus of research. This review presents a comprehensive analysis of AI imaging's role in predicting glioma high-risk molecular markers, highlighting challenges and future directions.

Differential diagnosis of small hepatocellular nodules in cirrhosis: surrogate histological criteria of TERT promoter mutations.

AIMS: The differential diagnosis of small hepatocellular nodules in cirrhosis between dysplastic nodules and hepatocellular carcinoma (HCC) remains challenging on biopsy. As TERT promoter (pTERT) mutations may indicate the nodules already engaged in the malignant process, the aim of this study was to identify histological criteria associated with pTERT mutations by detecting these mutations by ddPCR in small formalin-fixed paraffin-embedded (FFPE) hepatocellular nodules arising in cirrhosis. METHODS AND RESULTS: We built a bicentric cohort data set of 339 hepatocellular nodules < 2 cm from cirrhotic samples, divided into a test cohort of 299 resected samples and a validation cohort of 40 biopsies. Pathological review, based on the evaluation of 14 histological criteria, classified all nodules. pTERT mutations were identified by ddPCR in FFPE samples. Among the 339 nodules, ddPCR revealed pTERT mutations in 105 cases (31%), including 90 and 15 cases in the test and validation cohorts, respectively. On multivariate analysis, three histological criteria were associated with pTERT mutations in the test cohort: increased cell density (P = 0.003), stromal invasion (P = 0.036) and plate-thickening anomalies (P < 0.001). With the combination of at least two of these major criteria, the AUC for predicting pTERT mutations was 0.84 in the test cohort (sensitivity: 86%, specificity: 83%) and 0.81 in the validation cohort (sensitivity: 87%, specificity: 76%). CONCLUSIONS: We identified three histological criteria as surrogate markers of pTERT mutations that may be used in routine biopsy to more clearly classify small hepatocellular nodules arising in cirrhosis.

Neuropil-like islands are a possible pathogenetic link between glioblastoma and gangliocytoma/ganglioglioma in a case of synchronous bilateral brain tumors.

Neuropil-like islands (NIs) are a histologic hallmark of glioneuronal tumors with neuropil-like islands (GTNIs), but GTNIs are presently not considered a homogeneous entity. The essence of GTNI is likely its glial component, and NIs are now considered aberrant neuronal differentiation or metaplasia. The case we report herein is a 41-year-old woman who was synchronously affected by two brain tumors: one was a glioblastoma (glioblastoma multiforme, GBM), of isocitrate dehydrogenase (IDH)-wild type, with NIs in the left parietal lobe, and the other was histologically a composite gangliocytoma (GC)/anaplastic ganglioglioma (GG) with NIs in the right medial temporal lobe. While both tumors were genetically wild type for IDH, histone H3, and v-raf murine sarcoma viral oncogene homolog B1 (BRAF), the former tumor, but not the latter, was mutated for telomerase reverse transcriptase promoter gene (TERT). A recent systematic study using DNA methylation profiling and next-generation sequencing showed that anaplastic GG separate into other WHO tumor types, including IDH-wild-type GBM. It suggested a diagnostic scheme where an anaplastic GG is likely an IDH-wild-type GBM if it is a BRAF wild type, IDH wild type, and TERT promoter mutant tumor. The likely scenario in this patient is that the GBM results from the progression of GC/anaplastic GG due to the superimposed TERT promoter mutation and the propagation of newly generated GBM cells in the contralateral hemisphere. A systematic analysis using DNA methylation profiling and next-generation sequencing was not available in this study, but the common presence of NIs histologically noted in the two tumors could support this scenario. Although a sufficient volume of molecular and genetic testing is sine qua non for the accurate understanding of brain tumors, the importance of histologic observation cannot be overemphasized.

Comparative Clinicopathologic and Genomic Analysis of Hepatocellular Neoplasm, Not Otherwise Specified, and Hepatoblastoma.

Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathologic and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathologic characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids DNA- and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoters, were employed. We found that patients with HCN-NOS were older (P < .001) and more frequently classified as high risk (P < .01), yet they showed no significant differences in alpha fetoprotein levels or survival outcomes compared with those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs 11/24; P < .001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs 6/24; P < .05) and chromosome 11 (7/17 vs 1/24; P < .01) when compared with HB. Furthermore, the recurrent loss/LOH of chromosomes 3, 4p, 9, 15q, and Y was only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the 2 groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathologic features and greater structural complexity compared with HB. However, patients with HCN-NOS exhibit comparable alpha fetoprotein levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared with those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.

Establishment of a Prediction Model Based on Preoperative MRI Radiomics for Diffuse Astrocytic Glioma, IDH-Wildtype, with Molecular Features of Glioblastoma.

PURPOSE: In 2021, the WHO central nervous system (CNS) tumor classification criteria added the diagnosis of diffuse astrocytic glioma, IDH wild-type, with molecular features of glioblastoma, WHO grade 4 (DAG-G). DAG-G may exhibit the aggressiveness and malignancy of glioblastoma (GBM) despite the lower histological grade, and thus a precise preoperative diagnosis can help neurosurgeons develop more refined individualized treatment plans. This study aimed to establish a predictive model for the non-invasive identification of DAG-G based on preoperative MRI radiomics. PATIENTS AND METHODS: Patients with pathologically confirmed glioma in Huashan Hospital, Fudan University, between September 2019 and July 2021 were retrospectively analyzed. Furthermore, two external validation datasets from Wuhan Union Hospital and Xuzhou Cancer Hospital were also utilized to verify the reliability and accuracy of the prediction model. Two regions of interest (ROI) were delineated on the preoperative MRI images of the patients using the semi-automatic tool ITK-SNAP (version 4.0.0), which were named the maximum anomaly region (ROI1) and the tumor region (ROI2), and Pyradiomics 3.0 was applied for feature extraction. Feature selection was performed using a least absolute shrinkage and selection operator (LASSO) filter and a Spearman correlation coefficient. Six classifiers, including Gauss naive Bayes (GNB), K-nearest neighbors (KNN), Random forest (RF), Adaptive boosting (AB), and Support vector machine (SVM) with linear kernel and multilayer perceptron (MLP), were used to build the prediction models, and the prediction performance of the six classifiers was evaluated by fivefold cross-validation. Moreover, the performance of prediction models was evaluated using area under the curve (AUC), precision (PRE), and other metrics. RESULTS: According to the inclusion and exclusion criteria, 172 patients with grade 2-3 astrocytoma were finally included in the study, and a total of 44 patients met the diagnosis of DAG-G. In the prediction task of DAG-G, the average AUC of GNB classifier was 0.74 ± 0.07, that of KNN classifier was 0.89 ± 0.04, that of RF classifier was 0.96 ± 0.03, that of AB classifier was 0.97 ± 0.02, that of SVM classifier was 0.88 ± 0.05, and that of MLP classifier was 0.91 ± 0.03, among which, AB classifier achieved the best prediction performance. In addition, the AB classifier achieved AUCs of 0.91 and 0.89 in two external validation datasets obtained from Wuhan Union Hospital and Xuzhou Cancer Hospital, respectively. CONCLUSIONS: The prediction model constructed based on preoperative MRI radiomics established in this study can basically realize the prospective, non-invasive, and accurate diagnosis of DAG-G, which is of great significance to help further optimize treatment plans for such patients, including expanding the extent of surgery and actively administering radiotherapy, targeted therapy, or other treatments after surgery, to fundamentally maximize the prognosis of patients.

HPV-positive clinically advanced squamous cell carcinoma of the urinary bladder (aBSCC): A comprehensive genomic profiling (CGP) study.

BACKGROUND: Advanced bladder squamous cell carcinoma (aBSCC) is an uncommon form of urinary bladder malignancy when compared with the much higher urothelial carcinoma incidence. We studied the genomic alteration (GA) landscape in a series of aBSCC based on the association with human papilloma virus (HPV) to determine if differences in GA would be observed between the positive and negative groups. METHODS: Using a hybrid capture-based FDA-approved CGP assay, a series of 171 aBSCC were sequenced to evaluate all classes of GA. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on up to 114 loci. Programmed cell death ligand -1 (PD-L1) expression was determined by IHC (Dako 22C3) with negative expression when PD-L1 was 0, lower expression of positivity set at 1 to 49%, and higher expression set at ≥50% expression. RESULTS: Overall, 11 (6.4%) of the aBSCC were found to harbor HPV sequences (10 HPV16 and 1 HPV 11). HPV+ status was identified slightly more often in women (NS) and in younger patients (P = 0.04); 2 female patients with aBSCC had a prior history of SCC including 1 anal SCC and 1 vaginal SCC. HPV+ aBSCC had fewer GA/tumor (P < 0.0001), more inactivating mutations in RB1 (P = 0.032), and fewer inactivating GA in CDKN2A (P < 0.0001), CDKN2B (P = 0.05), TERT promoter (P = 0.0004) and TP53 (P < 0.0001). GA in genes associated with urothelial carcinoma including FGFR2 and FGFR3 were similar in both HPV+ and HPV- aBSCC groups. MTAP loss (homozygous deletion) which has emerged as a biomarker for PRMT5 inhibitor-based clinical trials was not identified in any of the 11 HPV+ aBSCC cases, which was significantly lower than the 28% positive frequency of MTAP loss in the HPV- aBSCC group (P < 0.0001). MTOR and PIK3CA pathway GA were not significantly different in the 2 groups. Putative biomarkers associated with immunotherapy (IO) response, including MSI and TMB status, were also similar in the 2 groups. PD-L1 expression data was available for a subset of both HPV+ and HPV- cases and showed high frequencies of positive staining which was not different in the 2 groups. CONCLUSIONS: HPV+ aBSCC tends to occur more often in younger patients. As reported in other HPV-associated squamous cell carcinomas, HPV+ aBSCC demonstrates significantly reduced frequencies of inactivating mutations in cell cycle regulatory genes with similar GA in MTOR and PIK3CA pathways. The implication of HPV in the pathogenesis of bladder cancer remains unknown but warrants further exploration and clinical validation.

5hmC Immunohistochemistry: A Predictor of TERT Promoter Mutational Status in Follicular Thyroid Carcinoma?

Telomerase reverse transcriptase (TERT) gene aberrancies correlate to adverse prognosis in follicular thyroid carcinoma (FTC). As loss of 5-hydroxymethylcytosine (5hmC) has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a cohort of follicular thyroid tumors with available TERT data. A total of 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential, and 1 oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression were assessed for 5hmC immunoreactivity using two antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Of the 10 tumor cases with aberrant TERT, only 1 scored negative with both antibodies (1/10; 10%), whereas the remaining 9 cases (9/10; 90%) exhibited some positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case was scored negative using RM236, and 2 cases (2/19; 11%) using 4D9. The differences between TERT promoter mutated and wild-type groups were non-significant. The sensitivity and specificity for 5hmC immunohistochemistry (IHC) to detect mutated cases were 10% and 100% (RM236) and 20% and 89% (4D9). Therefore, 5hmC IHC is not a sensitive marker for detecting TERT promoter mutations in follicular thyroid tumors.

TERT Promoter Mutation in Benign and Malignant Salivary Gland Tumors; A Cross-Sectional Study.

Background & Objective: Telomere-related tumorigenesis mechanisms in the salivary gland, including mutation in the promoter region of TERT, have been rarely investigated. Therefore, the present study aimed to investigate the mutation in the promoter region of TERT in benign and malignant salivary gland tumors. Methods: This was a descriptive-analytical cross-sectional study. Tissue samples of 54 patients with primary salivary gland tumors sent to the pathology department of Rasool-e-Akram Hospital from September 2017 to September 2021 were examined. Fifteen samples including two groups of the most common benign tumors (n=5; 3 pleomorphic adenomas and 2 Warthin tumors) and four groups of the most common malignant tumors (n=10; 3 mucoepidermoid carcinomas, 3 adenoid cystic carcinomas, 2 acinic cell carcinoma, and 2 salivary duct carcinoma) were selected. The promoter region of TERT, including well-known hot spot regions, is sequenced using the Sanger sequencing method. Data were analyzed using statistical software R version 4.1.2. Results: Of 15 salivary gland tumor specimens, consisting of 5 benign tumors and 10 malignant tumors after DNA sequencing, TERT promoter region mutation was only seen in one of the adenoid cystic carcinoma samples, located at -146 bp upstream from ATG (chr5: 1,295,250 C>T). Conclusion: TERT promoter mutation was not different in malignant and benign salivary tumors. Nonetheless, there are a few studies that report TERT promoter mutation in adenoid cystic carcinoma of the salivary gland, necessitating the need for further investigations.