Non-neoplastic Tissue Samples Research Articles

The multispecies microbial cluster of Fusobacterium, Parvimonas, Bacteroides and Faecalibacterium as a precision biomarker for colorectal cancer diagnosis.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

TRIM25 promotes glioblastoma cell growth and invasion via regulation of the PRMT1/c-MYC pathway by targeting the splicing factor NONO

BackgroundUbiquitination plays an important role in proliferating and invasive characteristic of glioblastoma (GBM), similar to many other cancers. Tripartite motif 25 (TRIM25) is a member of the TRIM family of proteins, which are involved in tumorigenesis through substrate ubiquitination.MethodsDifference in TRIM25 expression levels between nonneoplastic brain tissue samples and primary glioma samples was demonstrated using publicly available glioblastoma database, immunohistochemistry, and western blotting. TRIM25 knockdown GBM cell lines (LN229 and U251) and patient derived GBM stem-like cells (GSCs) GBM#021 were used to investigate the function of TRIM25 in vivo and in vitro. Co-immunoprecipitation (Co-IP) and mass spectrometry analysis were performed to identify NONO as a protein that interacts with TRIM25. The molecular mechanisms underlying the promotion of GBM development by TRIM25 through NONO were investigated by RNA-seq and validated by qRT-PCR and western blotting.ResultsWe observed upregulation of TRIM25 in GBM, correlating with enhanced glioblastoma cell growth and invasion, both in vitro and in vivo. Subsequently, we screened a panel of proteins interacting with TRIM25; mass spectrometry and co-immunoprecipitation revealed that NONO was a potential substrate of TRIM25. TRIM25 knockdown reduced the K63-linked ubiquitination of NONO, thereby suppressing the splicing function of NONO. Dysfunctional NONO resulted in the retention of the second intron in the pre-mRNA of PRMT1, inhibiting the activation of the PRMT1/c-MYC pathway.ConclusionsOur study demonstrates that TRIM25 promotes glioblastoma cell growth and invasion by regulating the PRMT1/c-MYC pathway through mediation of the splicing factor NONO. Targeting the E3 ligase activity of TRIM25 or the complex interactions between TRIM25 and NONO may prove beneficial in the treatment of GBM.

Scoring System and Diagnosis of Papillary Thyroid Carcinoma Using Human Bone Marrow Endothelium Marker-1, Cytokeratin 19, and Galectin-3.

The increasing incidence of papillary thyroid carcinoma (PTC) and the inadequacy of routine histologic examination in its diagnosis necessitate the application of ancillary studies like immunohistochemistry. This research aimed to investigate the scoring system and diagnosis of PTC with cytokeratin 19 (CK19), human bone marrow endothelium marker-1 (HBME-1), and galectin-3. This experimental laboratory study was performed at Babol University of Medical Sciences, Mazandaran, Iran from April 2017 to March 2019. Neoplastic and nonneoplastic tissue samples of 100 cases with a diagnosis of PTC were selected by convenience sampling. CK19, HBME-1, and galectin-3 immunohistochemistry markers were used on tissue samples. Analysis was performed using the t test and the chi-square test, as well as the receiver operator characteristic (ROC) curve (significance level P < 0.05). The CK19 staining was observed in all 100 (100%) non-neoplastic tissues, but HBME-1 and galectin-3 were positive in 36 (36%) and 14 (14%) of non-neoplastic tissues, respectively. The intensity scores of all the markers and their total had significantly different means in PTC and non-neoplastic tissues (P < 0.001). A significant difference was observed between the total score of each marker and the total score of their combination (P < 0.001). The combination of all 3 markers with an 11.5 0 cut-off for the total score showed the most sensitive (0.99) and specific (1.00) results. Interpreting CK19, HBME-1, and galectin-3 with the aid of the proposed scoring system was fruitful. HBME-1 and galectin-3 can be used individually or in combination for the diagnosis of PTC.

CD10 expression in urinary bladder urothelial carcinoma is associated with high-tumor grade and stage.

Primary urinary bladder carcinoma is a common cancer worldwide. There is limited published data about CD10 immunoexpression pattern in urothelial bladder carcinoma (UBC). This study aims to examine CD10 immunoexpression in UBC and evaluate its relationship with clinicopathological parameters. The retrospective study examined 130 samples of UBC tissue and 30 samples of non-neoplastic urothelial bladder tissue, which were obtained from the Anatomic Pathology Department, King Abdulaziz University, Jeddah, Saudi Arabia. The project started in June 2019 and completed in February 2021. Tissue microarrays (TMA) were prepared from paraffin blocks and tissue sections prepared from the recipient blocks were used for immunohistochemistry studies utilizing CD10 antibody. The immunostaining results were recorded and analyzed. Positive staining of CD10 was observed in 64 (49%) cases of UBC and was not detected in any non-neoplastic urothelium samples. CD10-positive staining was identified in 36.7% and 66.7% of low and high-grade tumors, respectively. There was an association between positive CD10 immunostaining and high tumor grade (p=0.006) and muscularis propria invasion (p=0.007). There was no association between CD10 immunoexpression and age, gender, nodal and distant metastasis, lymphovascular invasion, and tumor recurrence. CD10 immunoexpression was not associated with the probabilities of overall survival (log rank 1.663, p=0.197) or disease-free survival (log rank 1.637, p=0.201). In UBC, CD10 immunoexpression is associated with higher tumor grade and muscle invasion, but it is not associated with patient survival or other clinicopathological parameters. CD10 immunoexpression cannot be used as a biomarker for poor prognosis in UBC.

Suppressive effect of YY1-mediated RGS22 regulation on the proliferation, migration and invasion of pancreatic ductal adenocarcinoma.

Regulator of G-protein signaling 22 (RGS22) is specifically expressed in the testis and in tumors of epithelial origin, but the expression and role of RGS22 in pancreatic cancer are unclear. In this study, 52 pairs of pancreatic ductal adenocarcinoma (PDAC) and adjacent non-neoplastic tissue samples with the corresponding clinical data were used to examine the expression of RGS22 and its relationship with PDAC prognosis. The findings showed that the expression of RGS22 was higher in the PDAC tissues than in the adjacent non-tumorous tissues and its expression was associated with the degree of blood vessel invasion. The in vitro experiments with PDAC cell lines and a normal control cell line showed that the proliferation, invasion, and metastasis of PDAC cells were suppressed by RGS22 overexpression and enhanced by RGS22 knockdown. The in vivo effect of RGS22 on PDAC xenografts was studied using subcutaneous implantation of tumor cells in BALB/cA-nu mice, and the results corroborated the in vitro findings. Analysis of the regulators of RGS22 showed that it was positively regulated by the transcription factor Yin Yang-1 (YY1). Thus, YY1-mediated RGS22 regulation suppressed the proliferation, migration, and invasion of PDAC.

Enrichment of Prevotella intermedia in human colorectal cancer and its additive effects with Fusobacterium nucleatum on the malignant transformation of colorectal adenomas

BackgroundOwing to the heterogeneity of microbiota among individuals and populations, only Fusobacterium nucleatum and Bacteroides fragilis have been reported to be enriched in colorectal cancer (CRC) in multiple studies. Thus, the discovery of additional bacteria contributing to CRC development in various populations can be expected. We aimed to identify bacteria associated with the progression of colorectal adenoma to carcinoma and determine the contribution of these bacteria to malignant transformation in patients of Han Chinese origin.MethodsMicrobiota composition was determined through 16S rRNA V3–V4 amplicon sequencing of autologous adenocarcinomas, adenomatous polyps, and non-neoplastic colon tissue samples (referred to as “tri-part samples”) in patients with CRC. Enriched taxa in adenocarcinoma tissues were identified through pairwise comparison. The abundance of candidate bacteria was quantified through genomic quantitative polymerase chain reaction (qPCR) in tissue samples from 116 patients. Associations of candidate bacteria with clinicopathological features and genomic and genetic alterations were evaluated through odds ratio tests. Additionally, the effects of candidate bacteria on CRC cell proliferation, migration, and invasion were evaluated through the co-culture of CRC cells with bacterial cells or with conditioned media from bacteria.ResultsPrevotella intermedia was overrepresented in adenocarcinomas compared with paired adenomatous polyps. Furthermore, co-abundance of P. intermedia and F. nucleatum was observed in tumor tissues. More notably, the coexistence of these two bacteria in adenocarcinomas was associated with lymph node involvement and distant metastasis. These two bacteria also exerted additive effects on the enhancement of the migration and invasion abilities of CRC cells. Finally, conditioned media from P. intermedia promoted the migration and invasion of CRC cells.ConclusionThis report is the first to demonstrate that P. intermedia is enriched in colorectal adenocarcinoma tissues and enhances the migration and invasion abilities of CRC cells. Moreover, P. intermedia and F. nucleatum exert additive effects on the malignant transformation of colorectal adenomas into carcinomas. These findings can be used to identify patients at a high risk of malignant transformation of colorectal adenomas or metastasis of CRC, and they can accordingly be provided optimal clinical management.

Abstract 1184: Early-stage lung cancer detection via circulating microbial DNA biomarkers and machine learning classification

Abstract Introduction: Human tissues, including tumors, are extensively colonized by taxonomically diverse microbes. Intra-tumoral microbial activity and events of cellular turnover and trafficking contribute to shedding of microbial nucleic acids into the blood stream. Here we characterized microbial signatures (mbDNA) present in primary-tumor tissue and in the blood of patients affected with different cancer types, with particular focus on lung cancer, and we demonstrated the discriminatory power of such microbial signatures for the identification and classification of lung cancer versus other cancer types. We further validated our findings using plasma-derived cell-free microbial DNA (cf-mbDNA) to discriminate between lung cancer and cancer-free control samples. Methods: We examined The Cancer Genome Atlas (TCGA) compendium of treatment-naïve, whole genome and transcriptomic sequencing datasets to extrapolate genetic signatures of microbial origin associated with 33 different tumor types collected from 10,481 patients, which included non-neoplastic tumor-adjacent tissue and blood samples. 7.2% of TCGA sequencing reads were classified as non-human, of which 35.2% could be taxonomically classified using a reference database containing 59,974 total microbial genomes. These taxonomically assigned data sets were then used to train machine learning models (using a 70/30 train/test split for all cancers) to discriminate between and within types and stages of cancer. Results: We demonstrated that mbDNA signatures from whole blood can be used to accurately classify the tissue of origin of 20 unique cancer types, including lung adenocarcinoma and lung squamous cell carcinoma. For lung adenocarcinoma we reported high discrimination between paired tumor tissue and normal-adjacent tissue (Avg. {AUROC,AUPR}={0.85,0.95}) and between primary tumor tissue and all-other cancer types (Avg. {AUROC,AUPR}={0.96,0.69}, n=32 cancer-types). We also demonstrated the high performance of blood-derived mbDNA when discriminating among TCGA cancer types: Avg. {AUROC,AUPR}={0.97,0.80}. Subsequent liquid biopsy results using plasma-derived cf-mbDNA offer compelling evidence that cf-mbDNA signatures can robustly discriminate adenocarcinoma lung-cancer samples from non-cancer controls. Conclusion: mbDNA holds considerable promise as a truly orthogonal means of detecting and classifying lung cancer independently from host genomic alternations. Using only mbDNA signatures we have demonstrated robust discrimination between cancer-free controls and lung cancer samples and have provided early evidence of the applicability of this approach to liquid biopsy. Our present efforts analyzing plasma cf-mbDNA with an expanded sample cohort will serve to fully validate this new class of liquid biopsy biomarkers for lung cancer detection. Citation Format: Gregory D. Sepich-Poore, Serena Fraraccio, Stephen Wandro, Rob Knight, Sandrine Miller-Montgomery, Eddie Adams. Early-stage lung cancer detection via circulating microbial DNA biomarkers and machine learning classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1184.

Knockdown of NUSAP1 inhibits cell proliferation and invasion through downregulation of TOP2A in human glioblastoma

ABSTRACT Nucleolar and spindle associated protein 1 (NUSAP1), an indispensable mitotic regulator, has been reported to be involved in the development, progression, and metastasis of several types of cancer. Here, we investigated the expression and biological function of NUSAP1 in human glioblastoma (GBM), an aggressive brain tumor type with largely ineffective treatment options. Analysis of the molecular data in CGGA, TCGA and Rembrandt datasets demonstrated that NUSAP1 was significantly upregulated in GBM relative to low grade gliomas and non-neoplastic brain tissue samples. Kaplan-Meier analysis indicated that patients with tumors showing high NUSAP1 expression exhibited significantly poorer survival in both CGGA (P = 0.002) and Rembrandt cohorts (P = 0.017). Analysis of RNA sequencing data from P3-cells with stable knockdown of NUSAP1 revealed topoisomerase 2A (TOP2A) as a possible molecule downregulated by the loss of NUSAP1. Molecular analysis of the CGGA data revealed a strong correlation between NUSAP1 and TOP2A expression in primary gliomas and recurrent gliomas samples. SiRNA knockdown of either NUSAP1 or TOP2A in U251, T98 and GBM derived patient P3 cells inhibited GBM cell proliferation and invasion, and induced cell apoptosis. Finally, stable knockdown of NUSAP1 with shRNA led to decreased tumor growth in an orthotopic xenograft model of GBM in mice. Taken together, NUSAP1 gene silencing induced apoptosis possibly through the downregulation of the candidate downstream molecule TOP2A. Interference with the expression of NUSAP1 might therefore inhibit malignant progression in GBM, and NUSAP1 might thus serve as a promising molecular target for GBM treatment.

Discriminating Neoplastic from Nonneoplastic Tissues Using an miRNA-Based Deep Cancer Classifier

Next-generation sequencing has enabled the collection of large biological data sets, allowing novel molecular-based classification methods to be developed for increased understanding of disease. miRNAs are small regulatory RNA molecules that can be quantified using next-generation sequencing and are excellent classificatory markers. Herein, a deep cancer classifier (DCC) was adapted to differentiate neoplastic from nonneoplastic samples using comprehensive miRNA expression profiles from 1031 human breast and skin tissue samples. The classifier was fine-tuned and evaluated using 750 neoplastic and 281 nonneoplastic breast and skin tissue samples. Performance of the DCC was compared with two machine-learning classifiers: support vector machine and random forests. In addition, performance of feature extraction through the DCC was also compared with a developed feature selection algorithm, cancer specificity. The DCC had the highest performance of area under the receiver operating curve and high performance in both sensitivity and specificity, unlike machine-learning and feature selection models, which often performed well in one metric compared with the other. In particular, deep learning had noticeable advantages with highly heterogeneous data sets. In addition, our cancer specificity algorithm identified candidate biomarkers for differentiating neoplastic and nonneoplastic tissue samples (eg, miR-144 and miR-375 in breast cancer and miR-375 and miR-451 in skin cancer).

Immunohistochemical identification of complement peptide C5a receptor 1 (C5aR1) in non-neoplastic and neoplastic human tissues.

The complement component C5a and its receptor C5aR1 are involved in the development of numerous inflammatory diseases. In addition to immune cells, C5aR1 is expressed in neoplastic cells of multiple tumour entities, where C5aR1 is associated with a higher proliferation rate, advanced tumour stage, and poor patient outcomes. The aim of the present study was to obtain a broad expression profile of C5aR1 in human non-neoplastic and neoplastic tissues, especially in tumour entities not investigated in this respect so far. For this purpose, we generated a novel polyclonal rabbit antibody, {5227}, against the carboxy-terminal tail of C5aR1. The antibody was initially characterised in Western blot analyses and immunocytochemistry using transfected human embryonic kidney (HEK) 293 cells. It was then applied to a large series of formalin-fixed, paraffin-embedded non-neoplastic and neoplastic human tissue samples. C5aR1 was strongly expressed by different types of immune cells in the majority of tissue samples investigated. C5aR1 was also present in alveolar macrophages, bronchial, gut, and bile duct epithelia, Kupffer cells, occasionally in hepatocytes, proximal renal tubule cells, placental syncytiotrophoblasts, and distinct stem cell populations of bone marrow. C5aR1 was also highly expressed in the vast majority of the 32 tumour entities investigated, where a hitherto unappreciated high prevalence of the receptor was detected in thyroid carcinomas, small-cell lung cancer, gastrointestinal stromal tumours, and endometrial carcinomas. In addition to confirming published findings, we found noticeable C5aR1 expression in many tumour entities for the first time. Here, it may serve as an interesting target for future therapies.

Long non-coding RNA (lncRNA) DSCAM-AS1 is upregulated in breast cancer.

Accumulating evidence highlights that long noncoding RNA (lncRNA) DSCAM-AS1 play a key regulatory role in different stages of cancer development and progression. This study aimed to investigate whether the expression of DSCAM-AS1 is deregulated in breast cancer. The relative expression of DSCAM-AS1 was measured in fifty breast cancerous and matched adjacent non-neoplastic tissue samples using quantitative real-time polymerase chain reaction (qPCR) technique. The association between DSCAM-AS1 expression and patients' clinicopathological features was evaluated. Sensitivity and specificity of the DSCAM-AS1 expression for diagnosing breast cancer was obtained by the receiver operating characteristic (ROC) curve analysis. Our results showed that the expression of DSCAM-AS1 was significantly up-regulated in breast cancerous tissues compared with the matched adjacent non-neoplastic tissues (P <0.05). Furthermore, we observed a significant association between the DSCAM-AS1 expression and lymph node metastasis (P =0.011) but no other clinicopathological characteristics (P >0.05). ROC curve analysis resulted in an area under the curve (AUC) of 0.67 and showed that the DSCAM-AS1 expression level may discriminate cancerous and non-cancerous tissues with 68% sensitivity and 76% specificity. This study provides further evidence that the expression of DSCAM-AS1 is deregulated in breast cancer and highlights its potential in breast cancer development.

Loss of COPZ1 induces NCOA4 mediated autophagy and ferroptosis in glioblastoma cell lines

Dysregulated iron metabolism is a hallmark of many cancers, including glioblastoma (GBM). However, its role in tumor progression remains unclear. Herein, we identified coatomer protein complex subunit zeta 1 (COPZ1) as a therapeutic target candidate which significantly dysregulated iron metabolism in GBM cells. Overexpression of COPZ1 was associated with increasing tumor grade and poor prognosis in glioma patients based on analysis of expression data from the publicly available database The Cancer Genome Atlas (P < 0.001). Protein levels of COPZ1 were significantly increased in GBM compared to non-neoplastic brain tissue samples in immunohistochemistry and western blot analysis. SiRNA knockdown of COPZ1 suppressed proliferation of U87MG, U251 and P3#GBM in vitro. Stable expression of a COPZ1 shRNA construct in U87MG inhibited tumor growth in vivo by ~60% relative to controls at day 21 after implantation (P < 0.001). Kaplan–Meier analysis of the survival data demonstrated that the overall survival of tumor bearing animals increased from 20.8 days (control) to 27.8 days (knockdown, P < 0.05). COPZ1 knockdown also led to the increase in nuclear receptor coactivator 4 (NCOA4), resulting in the degradation of ferritin, and a subsequent increase in the intracellular levels of ferrous iron and ultimately ferroptosis. These data demonstrate that COPZ1 is a critical mediator in iron metabolism. The COPZ1/NCOA4/FTH1 axis is therefore a novel therapeutic target for the treatment of human GBM.

KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer.

MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell-type-specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell-free DNA (cfDNA) in non-small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real-time methylation-specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh-frozen NSCLC tissues, their corresponding adjacent non-neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non-neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease-free survival (ΗR=0.239; P=0.001) and worse overall survival (OS; HR=0.342, P=0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression-free survival (PFS; HR=0.431; P=0.005) and worse OS (HR=0.306; P<0.001). Our data strongly suggest that the detection of KMT2C promoter methylation in plasma cfDNA predicts poor prognosis in patients with both operable and metastatic NSCLCs. KMT2C promoter methylation in plasma cfDNA therefore merits further evaluation and validation as a noninvasive circulating epigenetic biomarker.

Abstract PO-34: Subcutaneous panniculitis-like T-cell lymphoma with HAVCR2 mutation shows unique clinicopathologic features and gene expression profile

Abstract Recent studies identified germline HAVCR2 (TIM-3) mutations as the specific genetic predisposition to subcutaneous panniculitis-like T-cell lymphoma (SPTCL). However, distinction between HAVCR2-mutated and HAVCR2-wildtype SPTCLs remains elusive. We studied the prevalence of HAVCR2 mutation in a nation-wide cohort of SPTCL and investigated clinical and molecular distinction between HAVCR2-mutated and HAVCR2-wild-type SPTCLs. A multicenter nationwide cohort of 53 SPTCL cases was established; patients were diagnosed at seven Korean institutions between 2003 and 2020. Histologic features were reviewed by experienced hematopathologists and clinical features were retrospectively reviewed. Whole-exome sequencing (WES) and RNA-seq were performed using formalin-fixed, paraffin-embedded (FFPE) samples of 8 patients diagnosed in Seoul National University Hospital (SNUH), with matched non-neoplastic tissue samples from two patients. Direct sequencing of HAVCR2 exon 2 was successfully carried out using FFPE samples from 33 of the remaining 45 patients. Among 41 patients with available HAVCR2 mutation status, 28 (68.3%) were women, and the median age at diagnosis was 30 years (range 11–74). Ten patients (10/40; 25.0%) suffered hemophagocytic syndrome (HPS) or HPS-like systemic illness during the clinical course, and 14 patients (14/40; 35.0%) progressed during the follow-up. Six patients (6/41; 14.6%) died of disease progression or HPS. We found 18 patients (18/41; 43.9%) with HAVCR2 mutation; 15 patients harbored biallelic HAVCR2 Y82C mutation and 3 patients were noted for heterozygous HAVCR2 Y82C mutation. HAVCR2-mutated SPTCLs occurred in younger patients (median age 26.5 versus 37; Mann-Whitney p-value = 0.003), and were more often complicated by HPS or HPS-like systemic illness (10/18 versus 0/22; Fisher's exact p-value &amp;lt; 0.001), compared to HAVCR2-wild-type SPTCLs. Survival analysis using log-rank test revealed that HAVCR2-mutated SPTCLs had shorter progression-free survival, though statistical significance was not achieved (p-value = 0.081) WES results did not show recurrent genetic alteration other than HAVCR2 Y82C in 4 out of 8 patients. Mutations in genes involved in T/NK cell-associated inflammation (PVRL1, PVRL2, TICAM1, GZMA), epigenetic modification (BAZ2A, KMT2C, KMT2D, SETD1A), JAK-STAT signaling (IFNL2, PIAS3), and NF-kB pathway (PDCD11) were observed in individual cases. Gene set enrichment analyses (GSEA) using RNA-seq results showed significant enrichment of pathways involving TNF-alpha signaling via NF-kB (FDR q-value = 0.008), hypoxia (FDR q-value = 0.009), IL6-JAK-STAT3 signaling (FDR q-value = 0.026), apoptosis (FDR q-value = 0.121), and MTORC1 signaling (FDR q-value = 0.188) in HAVCR2-mutated SPTCLs. HAVCR2 Y82C hotspot mutation frequently occurs in Korean patients with SPTCL, which was characterized by unique clinicopathologic features. SPTCL with HAVCR2 Y82C was enriched with distinct cellular pathways, which remains to be further validated. Citation Format: Jiwon Koh, Insoon Jang, Seungchan Mun, Cheol Lee, Hee-Jung Cha, Young Ha Oh, Jin Man Kim, Young Hyeh Ko, Jae Ho Han, Heounjeong Go, Jooryung Huh, Kwangsoo Kim, Yoon Kyung Jeon. Subcutaneous panniculitis-like T-cell lymphoma with HAVCR2 mutation shows unique clinicopathologic features and gene expression profile [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-34.

The impact of DNA demethylation on the upregulation of the NRN1 and TNFAIP3 genes associated with advanced gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Very few therapeutic options are currently available in this neoplasia. The use of 5-Aza-2'-deoxycytidine (5-AZAdC) was approved for the treatment of myelodysplastic syndromes, and this drug can treat solid tumours at low doses. Epigenetic manipulation of GC cell lines is a useful tool to better understand gene expression regulatory mechanisms for clinical applications. Therefore, we compared the gene expression profile of 5-AZAdC-treated and untreated GC cell lines by a microarray assay. Among the genes identified in this analysis, we selected NRN1 and TNFAIP3 to be evaluated for gene expression by RT-qPCR and DNA methylation by bisulfite DNA next-generation sequencing in 43 and 52 pairs of GC and adjacent non-neoplastic tissue samples, respectively. We identified 83 candidate genes modulated by DNA methylation in GC cell lines. Increased expression of NRN1 and TNFAIP3 was associated with advanced tumours (P< 0.05). We showed that increased NRN1 and TNFAIP3 expression seems to be regulated by DNA demethylation in GC samples: inverse correlations between the mRNA and DNA methylation levels in the promoter of NRN1 (P< 0.05) and the intron of TNFAIP3 (P< 0.05) were detected. Reduced NRN1 promoter methylation was associated with III/IV TNM stage tumours (P= 0.03) and the presence of Helicobacter pylori infection (P= 0.02). The identification of demethylated activated genes in GC may be useful in clinical practice, stratifying patients who are less likely to benefit from 5-AZAdC-based therapies. KEY MESSAGES: Higher expression of NRN1 and TNFAIP3 is associated with advanced gastric cancer (GC). NRN1 promoter hypomethylation contributes to gene upregulation in advanced GC. TNFAIP3 intronic-specific CpG site demethylation contributes to gene upregulation in GC. These findings may be useful to stratify GC patients who are less likely to benefit from DNA demethylating-based therapies.

LncRNAs Predicted to Interfere With the Gene Regulation Activity of miR-637 and miR-196a-5p in GBM.

Rigorous molecular characterization of biological systems has uncovered a variety of gene variations underlying normal and disease states and a remarkable complexity in the forms of RNA transcripts that exist. A recent concept, competitive endogenous RNA, suggests that some non-coding RNAs can bind to miRNAs to modulate their role in gene expression. Here, we used several platforms, integrating mRNA, non-coding RNAs and protein data to generate an RNA-protein network that may be dysregulated in human glioblastoma multiforme (GBM). Publicly available microarray data for mRNA and miRNA were used to identify differentially expressed miRNAs and mRNAs in GBM relative to non-neoplastic tissue samples. Target miRNAs were further selected based on their prognostic significance, and the intersection of their target gene set with the differentially expressed gene set in Venn diagrams. Two miRNAs, miR-637 and miR-196a-5p, were associated with poor and better prognosis, respectively, in GBM patients. Non-coding RNAs, ENSG00000203739/ENSG00000271646 and TPTEP1, were predicted to be miRNA target genes for miR-637 and miR-196a-5p and positively correlated with the selected mRNA, CYBRD1 and RUFY2. A local protein interaction network was constructed using these two mRNAs. Predictions based on the ENSG00000203739/ENSG00000271646-miR-637-CYBRD1 and TPTEP1-miR-196a-5p-RUFY2 regulation axes indicated that the two proteins may act as an oncogene and tumor suppressor, respectively, in the development of GBM. These results highlight competitive endogenous RNA networks as alternative molecular therapeutic targets in the treatment of the disease.

Methylation silencing of TGF-\u03b2 receptor type II is involved in malignant transformation of esophageal squamous cell carcinoma

BackgroundAlthough massive studies have been conducted to investigate the mechanisms of esophageal squamous cell carcinoma (ESCC) carcinogenesis, the understanding of molecular alterations during the malignant transformation of epithelial dysplasia is still lacking, especially regarding epigenetic changes.ResultsTo better characterize the methylation changes during the malignant transformation of epithelial dysplasia, a whole-genome bisulfite sequencing analysis was performed on a series of tumor, dysplastic, and non-neoplastic epithelial tissue samples from esophageal squamous cell carcinoma (ESCC) patients. Promoter hypermethylation in TGF-β receptor type II (TGFBR2), an important mediator of TGF-β signaling, was identified. Further, we evaluated the methylation and expression of TGFBR2 in tumor samples through The Cancer Genome Atlas multiplatform data as well as immunohistochemistry. Moreover, treatment of ESCC cell lines with5-Aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, reactivated the expression of TGFBR2. The lentiviral mediating the overexpression of TGFBR2 inhibited the proliferation of ESCC cell line by inducing cell cycle G2/M arrest. Furthermore, the overexpression of TGFBR2 inhibited the tumor growth obviously in vivo.ConclusionsThe characterization of methylation silencing of TGFBR2 in ESCC will enable us to further explore whether this epigenetic change could be considered as a predictor of malignant transformation in esophageal epithelial dysplasia and whether use of a TGFBR2 agonist may lead to a new therapeutic strategy in patients with ESCC.

Relationship between polycomb-group protein BMI-1 and phosphatases regulating AKT phosphorylation level in endometrial cancer.

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.

Increased Expression of DNA Repair Gene RAD51 Associated with Shorter Overall Survival in Glioblastoma

The diagnosis of Glioblastoma (GBM) carries a poor prognosis. Despite surgical resection and high-dose adjuvant radiation, most treatment failures occur locally. We conducted a retrospective clinical-genetic study to identify factors which may mediate resistance of GBM to radiation, such as increased repair of DNA damage. We quantified the expression levels of key DNA repair enzymes in patient-derived tissue samples and examined their correlation with overall survival (OS). We retrospectively identified adult patients diagnosed with GBM, with tissue available in our institution’s archive, who were treated at our institution with maximal safe resection followed by adjuvant temozolomide and radiation (60 Gy). Total mRNA was extracted from formalin-fixed paraffin embedded (FFPE) tissue. mRNA gene expression profiles were obtained utilizing a digital gene expression panel. We compared the mRNA expression profiles of the GBM samples with a cohort of non-neoplastic temporal lobe FFPE tissue obtained from surgical resections for epilepsy. Normalized mRNA counts and differential expression (DE) comparisons were generated using analysis software. Kaplan-Meier (KM) and multivariate Cox proportional hazard (CPH) models were generated to examine factors associated with OS. Twenty-four GBM and 12 non-neoplastic brain tissue samples with sufficient quantity and quality of RNA were identified, processed, and analyzed. DE comparison identified 7 DNA repair genes that were significantly up-regulated in the GBM samples relative to the non-neoplastic brain tissue (more than 8-fold difference with adjusted p values all < 0.01). A multivariate CPH model that included age, tumor size, KPS, extent of resection, and the 7 differentially expressed DNA repair genes identified increased levels of RAD51 as being independently associated with an increased risk of death (HR = 10.11; p = 0.026), and RAD51 expression levels were over 9-fold higher in GBM samples versus non-neoplastic brain tissue. KM OS models demonstrated that GBM patients with high levels of RAD51 (above the cohort’s median) had significantly shorter OS compared to patients with low levels of RAD51 (10.6 months vs 18.2 months, KM log-rank p = 0.03). Within this small population of GBM patients that were uniformly treated with the current standard of care, we found that expression levels of RAD51, a key component of the homologous recombination DNA repair pathway, was significantly correlated with OS. These findings suggest that RAD51 mRNA levels could be used as a novel prognostic biomarker to improve outcome predictions in patients with GBM, and more importantly, inhibition of RAD51 protein activity during radiation could offer a new therapeutic target to improve the treatment of this deadly disease.

DNA Methylation-Based Method to Differentiate Malignant from Benign Thyroid Lesions.

Background: The differential diagnosis of thyroid nodules using fine-needle aspiration biopsy (FNAB) is challenging due to the inherent limitation of the cytology tests. The use of molecular markers has potential to complement the FNAB-based diagnosis and avoid unnecessary surgeries. In this study, we aimed to identify DNA methylation biomarkers and to develop a diagnostic tool useful for thyroid lesions. Methods: Genome-wide DNA methylation profiles (Illumina 450K) of papillary thyroid carcinoma (PTC = 60) and follicular thyroid carcinoma (FTC = 10) were compared with non-neoplastic thyroid tissue samples (NT = 50) and benign thyroid lesions (BTL = 17). The results were confirmed in publicly available databases from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) using the same DNA methylation platform. Two classifiers were trained to discriminate FTC and PTC from BTL. To increase the applicability of the method, six differentially methylated CpGs were selected and evaluated in 161 thyroid tumors and 69 BTL postsurgical specimens and 55 prospectively collected FNAB using bisulfite-pyrosequencing. Results: DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and FTC, respectively. The CpGs confirmed by GEO and TCGA databases showing high areas under the receiver operating characteristic curve in all sample sets were used to train our diagnostic classifier. The model based on six CpGs was able to differentiate benign from malignant thyroid lesions with 94.3% sensitivity and 82.4% specificity. A similar performance was found applying the algorithm to TCGA and GEO external data sets (91.3-97.4% sensitivity and 87.5% specificity). We successfully evaluated the classifiers using a bisulfite-pyrosequencing technique, achieving 90.7% sensitivity and 75.4% specificity in surgical specimens (five of six CpGs). The study comprising FNAB cytology materials corroborated the applicability and performance of the methodology, demonstrating 86.7% sensitivity and 89.5% specificity in confirmed malignant tumors, and 100% sensitivity and 89% specificity in cases with indeterminate cytology. Conclusions: A novel diagnostic tool with potential application in preoperative screening of thyroid nodules is reported here. The proposed protocol has the potential to avoid unnecessary thyroidectomies.