Adjacent Normal Research Articles

Highly sensitive deep panel sequencing of 27 HPV genotypes in prostate cancer biopsies results in very low detection rates and indicates that HPV is not a major etiological driver of this malignancy.

Human papillomavirus (HPV) has been proposed to contribute to the carcinogenesis of prostate cancer. However, previous studies have yielded conflicting results. This study aims to add useful information to the ongoing discussion concerning the association between HPV infection and prostate cancer. We used two high-throughput next-generation sequencing (NGS) approaches to detect HPV RNA in malignant and adjacent normal (AN) prostate tissue (cohorts 1 and 2) and HPV DNA from carcinogenic and probably/possibly carcinogenic-classified HPV types (cohort 3) in malignant prostate, AN prostate, and benign prostatic hyperplasia (BPH) tissues. In total, 0% (cohort 1: 0/83, cohort 2: 0/16) of the malignant prostate tissue samples and 0% (cohort 1: 0/23, cohort 2: 0/8) of the AN prostate tissue samples were positive for HPV RNA. A total of 8.3% (1/12) of the BPH samples, 0% (0/28) of the AN samples, and 0.8% (1/132) of the malignant prostate samples were positive for HPV16 DNA. However, the normalized read count of the HPV16-positive malignant sample was close to the cut-off. In addition, no other carcinogenic-classified HPV types were detected in any of the BPH, AN, or malignant prostate tissue samples. Our study does not support HPV infection as a major contributor to the etiology of prostate cancer.

IL-17 expression and prognosis of laryngeal squamous cell carcinoma.

Objective: To investigate the roles of proinflammatory cytokine IL-17 in the prognosis of laryngeal squamous cell carcinoma (LSCC). Methods: In all, 67 LSCC patients were enrolled to evaluate the expression of IL-17, IL-23, CD8, CD31, and vascular endothelial growth factor by immunohistochemical staining. Cumulative survival time was determined by the Kaplan-Meier approach, and prognostic variables for overall survival (OS) were identified by Cox proportional-hazards models. Results: The expression of IL-17 and IL-23 was remarkably higher in LSCC tissue than in adjacent normal (AN) tissue (P < .001). The Kaplan-Meier analysis indicated that patients with cervical lymph node metastasis (CLNM), advanced tumor node metastasis (TNM) stage, poor-differentiated grade, high microvessel density (MVD) in LSCC tissues, and high IL-17 expression in AN tissues had poorer OS (all P < .05). Cox regression analysis revealed that CLNM (HR = 7.246; 95% CI: 2.598-20.208; P < .001), high MVD in tumor tissues (HR = 4.233; 95% CI: 1.482-12.092; P = .007), and low CD8 expression in AN tissues (HR = 3.996; 95% CI: 1.442-11.074; P = .008) were significantly associated with poorer OS in LSCC, while IL-17 expression in AN tissue was not. Conclusion: High IL-17 expression in AN tissues could indicate poor prognosis and may be a prognostic marker for LSCC. Low CD8 expression in AN tissues may represent poor frontline antitumor immunity and be associated with poor prognosis.

Abstract PO-100: Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway

Abstract Introduction The enrichment of Fusobacterium nucleatum has been reported in both oral rinses and lesion samples from oral squamous cell carcinoma (OSCC) patients based on several case-control sequencing analyses, but studies on the potential role of F. nucleatum in OSCC tumorigenesis are still limited. In this study, we sought to elucidate the relationship between F. nucleatum and host transcriptome dysregulation in potentially carcinogenic pathways. Method The association between microbiota dysbiosis and host gene transcriptome was profiled by bacterial 16S rRNA V3-V4 amplicon sequencing and host gene long non-coding RNA sequencing (lncRNA-seq) for tumor and adjacent normal (AN) tissues from OSCC patients. Cell migration and invasion assays were performed using the in vitro bacteria-cell co-culture model. EMT-related molecular mechanisms induced by F. nucleatum were further validated by IncRNA-seq, real-time PCR, and western blot for bacteria-infected cells, respectively, at the genetic and proteomic levels. Result Significant enrichment of Fusobacterium was observed in OSCC tumor samples when compared to the paired AN samples. Correlation analysis between mucosal oral bacteria and host transcriptome in the OSCC tumor microenvironment elucidated the positive relationship between Fusobacterium and EMT pathways including significantly upregulated genes INHBA, SNAI2, and LAMC2. Using in-vitro cell models, we further observed that F. nucleatum significantly promoted cell migration and invasion, and significantly dysregulated the expression of several EMT factors including the increased inducer inhibin beta A (INHBA), core regulators SNAI1 (snai1) and SLUG (snai2), effectors vimentin (VIM) and N-cadherin (CDH2), and decreased E-cadherin (CDH1). Following the INHBA/SMAD pathway induced by F. nucleatum infection, the expression of an essential extracellular matrix glycoprotein, Laminin 332, was dramatically enhanced, which may trigger the phosphorylation of phosphatidylinositol 3-kinase and AKT kinase in the downstream PI3K/AKT pathway for the promoted cell migration and invasion ability. Conclusion Our study confirms the enrichment of Fusobacterium in the OSCC tumor microenvironment and reveals that F. nucleatum infection induces the INHBA/SAMD pathway and subsequently increases Laminin 332 expression, which may further promote the phosphorylation of PI3K/AKT and enhance OSCC migration and invasion. Citation Format: Hengyan Zhu, Liuyang Cai, Zigui Chen, Jason Y. K. Chan. Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-100.

Abstract 3307: A novel targeted RNA sequencing approach for identification of aggressive prostate cancer

Abstract Introduction: Prostate cancer (PC) is a highly heterogeneous disease and can be either aggressive, requiring immediate treatment, or remain slow growing and even without treatment present no significant symptoms throughout the man’s life. Currently, there is an unmet need for new tools that can clearly distinguish indolent from aggressive PC in order to guide treatment decisions.PC is a highly heritable disease and so far, approximately 450 genomic regions associated with PC risk have been defined in genome-wide association studies. Most of these regions are however devoid of annotated genes and consequently the underlying mechanisms behind the associations are not easily elucidated. Accordingly, yet uncharacterized genes in PC-associated regions could be involved in PC development/progression. Methods: We quantified gene expression levels by Kallisto on total RNAseq data from fresh frozen prostate tissue samples from 134 localized PCs, 33 matched adjacent normal (AN) areas as well as 15 PC cell lines. Furthermore, we performed targeted RNAseq covering approximately seven million base pairs divided among 101 PC-risk associated regions (GWAS hits) for in-depth transcriptomic analyses in the same set of samples. HISAT and StringTie were used to identify novel transcripts (genes) with the criteria that a given gene had to have at least 10 reads in more than 50% of all samples to be included. Patient genotyping was performed on Illumina’s global screening array. Results: In totalRNA seq data we observed significant dysregulation of 10,256 genes (5,685 up- and 4,571 downregulated) between AN and localized PC, including upregulation of know PC-associated genes such as SIM2, PCAT7, TDRD1, and TFF3. Interestingly, we found a correlation between high transcription of TFF3 and high risk of biochemical recurrence (BCR) after radical prostatectomy and furthermore a trans-acting eQTL, the PC-risk associated SNP rs1938781. Here, heterozygote patients with the risk-allele had higher risk of BCR than patients homozygous for the reference allele, indicating a potential functional relationship between the PC risk-SNP and TFF3. Additionally, we detected 207 previously unannotated non-coding genes in the capture RNAseq data, where 148 (71.5%) were undetectable in the matched total RNAseq data indicating the existence of stably albeit lowly expressed genes in the genomic areas containing PC-risk SNPs. Citation Format: Jacob Fredsøe, Christa Haldrup, Iver Nordentoft, Benedicte Ulhøi, Michael Borre, Karina D. Sørensen. A novel targeted RNA sequencing approach for identification of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3307.

Abstract 3778: Genome-Scale CRISPRa and CRISPRi screening for lncRNA drivers of prostate cancer progression

Abstract Background: Overtreatment of indolent prostate cancer (PC) and delayed treatment of aggressive PC is common due to suboptimal risk stratification tools, thus warranting identification of novel prognostic biomarkers. Although a few long non-coding RNAs (lncRNAs) with biomarker potential in PC are known, the majority of lncRNAs remain uncharacterized. Here, we aimed to identify novel lncRNA biomarker candidates. We hypothesized that strong candidates would have a functional role in driving PC progression in addition to their expression being linked to PC prognosis, and we therefore combined functional CRISPR screening with lncRNA expression profiling of PC patients. Methods: Total RNA sequencing (RNAseq) data was generated from 31 adjacent normal (AN) and 125 tumor samples from 141 clinically localized PC patients, along with 17 primary tumor samples from metastatic PC patients. Raw reads were mapped to the hg38 reference genome and kallisto was used for quantification. CRISPR activating (CRISPRa) and CRISPR interference (CRISPRi) screens were performed in the LNCaP PC cell line stably expressing either dCas9-VP64 or dCas9-KRAB, respectively. Cells were transduced in duplicate with custom single guide RNA (sgRNA) libraries targeting 20,306 and 20,474 lncRNA transcripts of interest using 72,281 and 72,360 sgRNAs (CRISPRa and CRISPRi, respectively). Cells were harvested and DNA extracted from an early (day 4 post-transduction) and a late (day 17-21) timepoint and next-generation sequenced. MAGeCK was used for data analysis. Results: To identify lncRNAs with biomarker potential in PC, we analyzed lncRNA expression in total RNAseq data from 158 PC patients. Using differential expression analysis and cox regression analysis with biochemical recurrence as endpoint, we identified 6,928 lncRNAs with biomarker potential. To investigate if any of these had a functional role in driving PC progression, we performed CRISPRa and CRISPRi screens to assess how lncRNA activation/inhibition affected PC cell proliferation. Based on the screens, lncRNA candidates with the most prominent phenotypes (normalized read count difference &amp;gt;200 and log-fold change &amp;gt;33% between the early and late timepoint for ≥3 sgRNAs in both replicates) were selected for individual validation. This identified 7 (CRISPRa) and 8 (CRISPRi) negative hits (decreased cell proliferation) along with 5 (CRISPRa) and 2 (CRISPRi) positive hits (increased cell proliferation). Individually activated/inhibited LNCaP cell lines have been established for the 22 candidate lncRNAs and proliferation assays are performed to validate their functional role in PC progression. Conclusion: We identified numerous lncRNAs with biomarker potential and a possible driver role in PC progression. Citation Format: Simone Weiss, Allegra Lord, Bernhard Schmierer, Anne B. Rovsing, Emil A. Thomsen, Jacob G. Mikkelsen, Benedicte Ulhøi, Jakob S. Pedersen, Michael Borre, Karina D. Sørensen. Genome-Scale CRISPRa and CRISPRi screening for lncRNA drivers of prostate cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3778.

MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE

You have accessJournal of UrologyCME1 Apr 2023MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE Akinaru Yamamoto, Atsunari Kawashima, Kentaro Jingushi, Yuichi Motoyama, Satoshi Nojima, Sassi Nesrine, Yohei Okuda, Toshiki Oka, Toshihiro Uemura, Gaku Yamamichi, Eisuke Tomiyama, Kosuke Nakano, Makoto Matsushita, Yu Ishizuya, Yoshiyuki Yamamoto, Taigo Kato, Koji Hatano, Tsujikawa Kazutake, and Norio Nonomura Akinaru YamamotoAkinaru Yamamoto More articles by this author , Atsunari KawashimaAtsunari Kawashima More articles by this author , Kentaro JingushiKentaro Jingushi More articles by this author , Yuichi MotoyamaYuichi Motoyama More articles by this author , Satoshi NojimaSatoshi Nojima More articles by this author , Sassi NesrineSassi Nesrine More articles by this author , Yohei OkudaYohei Okuda More articles by this author , Toshiki OkaToshiki Oka More articles by this author , Toshihiro UemuraToshihiro Uemura More articles by this author , Gaku YamamichiGaku Yamamichi More articles by this author , Eisuke TomiyamaEisuke Tomiyama More articles by this author , Kosuke NakanoKosuke Nakano More articles by this author , Makoto MatsushitaMakoto Matsushita More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Taigo KatoTaigo Kato More articles by this author , Koji HatanoKoji Hatano More articles by this author , Tsujikawa KazutakeTsujikawa Kazutake More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003332.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper urinary tract (UT) and bladder are known to differ from the embryology stage. There have been numerous reports of mouse models of bladder carcinogenesis, but there are few reports of mouse models of upper tract urothelial carcinogenesis. The aim of this study is to establish a novel mouse model of upper tract urothelial carcinogenesis. METHODS: We used two strains including C57/BL6 of male and female mice. We fed 0.05% N-butyl-N-(4-hydro-oxybutyl) nitorosamine (BBN) from 6 weeks old to the harvest day. The harvested organs were diagnosed by two different pathologists with histopathological evaluation using Hematoxylin and Eosin staining. Both sides of UT from mouse drinking BBN were subjected to whole transcriptome sequencing (WTS). In human samples, 74 upper tract urothelial carcinoma (UTUC) tissues and 73 adjacent normal (AN) tissues also subjected to WTS from 74 UTUC patients. RESULTS: Female mice of certain strain showed early weight loss compared to the others. They showed high incidence of UTUC with less incidence of bladder cancer (Figure 1), while the others rarely had UTUC. The total incidence of UTUC in this mouse strain was 83.3%, with the earliest UTUC observed at 8 weeks after the start of BBN drinking. The incidence of bilateral UTUC was 22.2%, and these individuals showed weight loss relatively early after the start of BBN drinking. The cancerous side of UT expressed a basal type of mRNA expression profile, with increased expression of Cd44, Cdh3, and Krt14, similar to previous reports of bladder carcinogenesis mouse model (Figure 2). We detected 593 differential expressed genes between cancerous side and non-cancerous side of UT in this mouse model, which could also classify human UTUC and AN samples. In pathway enrichment analysis of these genes, several pathways associated with DNA replication were commonly upregulated in cancer tissues between mouse and human samples. CONCLUSIONS: We have established the novel animal model developing UTUC with high probability. This model could reflect the human UTUC. Source of Funding: This work was supported by a KAKENHI grant (21K20968, 22K09523) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e963 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akinaru Yamamoto More articles by this author Atsunari Kawashima More articles by this author Kentaro Jingushi More articles by this author Yuichi Motoyama More articles by this author Satoshi Nojima More articles by this author Sassi Nesrine More articles by this author Yohei Okuda More articles by this author Toshiki Oka More articles by this author Toshihiro Uemura More articles by this author Gaku Yamamichi More articles by this author Eisuke Tomiyama More articles by this author Kosuke Nakano More articles by this author Makoto Matsushita More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Taigo Kato More articles by this author Koji Hatano More articles by this author Tsujikawa Kazutake More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

Single-Cell Heterogeneity of the Liver-Infiltrating Lymphocytes in Individuals with Chronic Echinococcus multilocularis Infection.

Human alveolar echinococcosis (AE) is a tumor-like disease predominantly located in the liver. The cellular composition and heterogeneity of the lesion-infiltrating lymphocytes which produce an "immunosuppressive" microenvironment are poorly understood. Here, we profiled 83,921 CD45+ lymphocytes isolated from the peripheral blood (PB), perilesion (PL), and adjacent normal (AN) liver tissue of four advanced-stage AE patients using single-cell RNA and T-cell receptor (TCR) sequencing technology. We identified 23 large clusters, and the distributions and transcriptomes of these cell clusters in the liver and periphery were different. The cellular proportions of exhausted CD8+ T cells and group 2 innate lymphoid cells (ILC2s) were notably higher in PL tissue, and the expression features of these cell subsets were related to neoplasm metastasis and immune response suppression. In the 5 CD8+ T-cell populations, only CD8+ mucosa-associated invariant T (MAIT) cells were enriched in PL samples and the TRAV1-2_TRAJ33_TRAC TCR was clonally expanded. In the 11 subsets of CD4+ T cells, Th17 cells and induced regulatory T cells (iTregs) were preferentially enriched in PL samples, and their highly expressed genes were related to cell invasion, tumor metastasis, and inhibition of the inflammatory immune response. Exhaustion-specific genes (TIGIT, PD-1, and CTLA4) were upregulated in Tregs. Interestingly, there was a close contact between CD8+ T cells and iTregs or Th17 cells, especially for genes related to immunosuppression, such as PDCD1-FAM3C, which were highly expressed in PL tissue. This transcriptional data set provides valuable insights and a rich resource for deeply understanding the immune microenvironment in AE, which could provide potential target signatures for AE diagnosis and immunotherapies.

Glycogen synthase kinase-3β inactivation promotes cervical cancerprogression, invasion, and drug resistance.

Human papillomavirus (HPV) infection-dependent cervical cancer is one of the most common gynecological cancers and often becomes aggressive, with rapid proliferation, invasion/migration, and drug resistance. Here, 135 fresh human cervical squamous cell carcinoma (CSCC) tissue specimens, comprising 21 adjacent normal (AN), 30 cervical intraepithelial neoplasia (CIN1-3 ), 45 CSCC, and 39 drugs (chemo-radiation)-resistant cervical tumor (DRCT) tissues were included. HPV-positive (HeLa, SiHa), HPV-negative (C33A), and cisplatin-resistant (CisR-HeLa/-SiHa/-C33A) cell lines were used for in vitro studies. HPV16/18 oncoproteins E6/E7, pERK1/2, and glycogen synthase kinase-3 (GSK3) and the matrix metalloproteinases (MMPs) MMP-9/-2 were assessed using immunohistochemistry, WB, and gelatin zymography. HPV16/18 infection was observed in 16.7% of the CIN1-3 , 77.8% of the CSCC, and 89.7% of DRCT samples. Total and inactive GSK3β expressions were associated with overall CSCC progression (p=0.039 and p=0.024, respectively) and chemoresistance (p=0.004 and p=0.014, respectively). Positive correlations were observed, between the expression of E6 and pGSK3β expression (p=0.013); E6 and CSCC progression (p<0.0001)/drug resistance (p=0.0001). CisR-HeLa/-SiHa was more dependent on pGSK3β, and activation of GSK3 by SMIs (iAkt), treatment with nimbolide, or knockdown of E6/E7 reduced cisplatin resistance and promoted apoptosis. Hence, the activation of GSK3β with nimbolide and iAkt can be exploited for therapeutic interventions of cervical cancer.

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis.

Despite having numerous advances in therapeutics, mortality and morbidity due to oral cancer incidence are still very high. Early detection can improve the chances of survival in most patients. However, diagnosis at early stages can be challenging as premalignant conditions are usually asymptomatic. Currently, histological assessment remains the gold standard for diagnosis. Early diagnosis poses challenges to pathologists due to less severe morphological changes associated with early stages. Therefore, a fast and robust method of detection based on molecular changes is needed for early diagnosis. © 2021 Wiley Periodicals LLC. In the present study, Fourier transform infrared (FTIR) spectroscopic imaging has been used to differentiate early-stage oral hyperplasia from adjacent normal (AN) and oral squamous cell carcinoma (OSCC). Hyperplasia is often considered as an initial event in the pathogenesis of oral cancer and OSCC is the most common advanced stage of malignancy. Differentiating normal versus hyperplasia and hyperplasia versus OSCC can remain quite challenging on occasion using conventional staining as the histological assessment is based on morphological changes. Unsupervised hierarchical cluster analysis (UHCA) has been performed on FTIR images of multiple tissues together that provided some degree of classification among tissue groups. The AN epithelium clustered distinctively using UHCA from both hyperplasia and grades 1 and 2 of OSCC. An increase in the content of DNA, denaturation of protein, and altered lipid structures were more clearly elucidated with spectral analysis. This study demonstrates a simple strategy to differentiate early-stage oral hyperplasia from ANand OSCC using UHCA. This study also proposes a future alternative method where FTIR imaging can be used as a diagnostic tool for cancer at early stages.

Th1/Th2/Th17 cytokine profile in hepatic cystic Echinococcosis patients with different cyst stages.

The study aimed to investigate possible correlation between expression level of Th1/Th2/Th17-type profile and cyst viability in the systemic and local immunity of hepatic cystic Echinococcosis (CE) patients. Expression of Th1-type interleukin (IL)-2, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, Th2-type IL-4, IL-6, IL-10 and Th17-type IL-17A was examined in the serum and liver samples of hepatic CE patients with different cyst stages. Compared with healthy controls, Th1/Th2/Th17-type cytokines were significantly increased in the serum of hepatic CE patients. Moreover, expression of these cytokines was also at higher level in the inflammatory cell band of peri-lesion liver (PL) tissues than that in the adjacent normal (AN) liver tissues. Interestingly, elevation of Th1-type and Th17-type cytokines was more evident in PL tissues of patients with inactive cysts. Relatively, Th2-type cytokines were predominant in PL tissues of patients with active cysts. Our findings provide new insights that Th1/Th2/Th2-type cytokine profile was associated with cyst stages. In hepatic CE patients with inactive cysts, Th1 and Th17-type cytokines were predominant. Comparatively, Th2-type cytokines were more evident in hepatic CE patients with active cysts, which may provide basis for the immune response diversity in hepatic CE patients with different cyst stages.

Epigenetic silencing of MEIS2 in prostate cancer recurrence

BackgroundCurrent diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, resulting in overdiagnosis and overtreatment of clinically insignificant tumors. Thus, to improve the management of PC, novel biomarkers are urgently needed.ResultsIn this study, we integrated genome-wide methylome (Illumina 450K DNA methylation array (450K)) and RNA sequencing (RNAseq) data performed in a discovery set of 27 PC and 15 adjacent normal (AN) prostate tissue samples to identify candidate driver genes involved in PC development and/or progression. We found significant enrichment for homeobox genes among the most aberrantly methylated and transcriptionally dysregulated genes in PC. Specifically, homeobox gene MEIS2 (Myeloid Ecotropic viral Insertion Site 2) was significantly hypermethylated (p < 0.0001, Mann-Whitney test) and transcriptionally downregulated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue in our discovery sample set, which was also confirmed in an independent validation set including > 500 PC and AN tissue samples in total (TCGA cohort analyzed by 450K and RNAseq). Furthermore, in three independent radical prostatectomy (RP) cohorts (n > 700 patients in total), low MEIS2 transcriptional expression was significantly associated with poor biochemical recurrence (BCR) free survival (p = 0.0084, 0.0001, and 0.0191, respectively; log-rank test). Next, we analyzed another RP cohort consisting of > 200 PC, AN, and benign prostatic hyperplasia (BPH) samples by quantitative methylation-specific PCR (qMSP) and found that MEIS2 was significantly hypermethylated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue samples (AN and BPH) with an AUC > 0.84. Moreover, in this cohort, aberrant MEIS2 hypermethylation was significantly associated with post-operative BCR (p = 0.0068, log-rank test), which was subsequently confirmed (p = 0.0067; log-rank test) in the independent TCGA validation cohort (497 RP patients; 450K data).ConclusionsTo the best of our knowledge, this is the first study to investigate, demonstrate, and independently validate a prognostic biomarker potential for MEIS2 at the transcriptional expression level and at the DNA methylation level in PC.

The clinical significance of CCBE1 expression in human colorectal cancer.

PurposeThe identification and discovery of prognostic markers for colorectal cancer (CRC) are of great clinical significance. CCBE1 is expressed in various tumors and its expression correlates with lymphangiogenesis and angiogenesis. However, the association between CCBE1 expression and CRC outcome has not been reported. The aim of this study was to investigate clinical significance of CCBE1 expression in CRC.Patients and methodsCCBE1 expression was examined in 30 pairs of fresh CRC tissues and compared with adjacent normal (AN) tissues using quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry (IHC) staining. Tissue microarray immunohistochemical staining was used to study the CCBE1 expression characteristics of 204 CRC patient samples collected from January 2002 to December 2007, and the relationship of CCBE1 with clinicopathological features and prognosis of CRC was analyzed.ResultsCCBE1 was highly expressed in CRC tissues compared with matched AN tissues (P=0.001). Moreover, high expression of CCBE1 was significantly associated with tumor differentiation, lymph node metastasis, vascular invasion, liver metastasis and TNM stage in CRC patients (P≤0.01). Kaplan–Meier survival analysis revealed that high CCBE1 expression, poor tumor differentiation, lymph node metastasis and vascular invasion were significantly associated (all P<0.001) with poor prognosis for patients. Furthermore, univariate and multivariate Cox analysis revealed that high CCBE1 expression, poor tumor differentiation, lymph node metastasis and vascular invasion were independent risk factors for both overall survival (OS) and disease-free survival (DFS) of CRC patients (all P<0.05). OS and DFS of 267 CRC patients from The Cancer Genome Atlas (TCGA) database showed the same trend (log-rank P=6e-04, HR [high] =2.4; log-rank P=0.0081, HR [high] =1.9).ConclusionHigh levels of CCBE1 contribute to the aggressiveness and poor prognosis of CRC. CCBE1 can serve as a novel potential biomarker to predict CRC patients’ prognosis.

Increased expression of antisense lncRNA SPINT1-AS1 predicts a poor prognosis in colorectal cancer and is negatively correlated with its sense transcript.

PurposeColorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Natural antisense transcripts (NATs) are pervasively expressed in human genome and have been confirmed to contribute to cancer progression. In our study, we aimed to investigate the expression and clinical pertinence of serine peptidase inhibitor, Kunitz type 1 antisense RNA1 (SPINT1-AS1) in CRC.Materials and methodsThe expression levels of SPINT1-AS1 and the corresponding sense transcript SPINT1 mRNA were analyzed in 150 pairs of CRC tissues and adjacent normal (AN) tissues, along with 45 pairs of preoperative and postoperative serum exosome samples by the strand-specific real-time quantitative polymerase chain reaction.ResultsCompared with AN tissues, the expression of SPINT1-AS1 was increased (P<0.001, 3.771 vs 0.980) in CRC tissues, while SPINT1 mRNA expression was decreased in CRC (P<0.001, 0.927 vs 1.165), and there was an obviously negative correlation between SPINT1-AS1 expression and its sense transcript (r=−0.701, P<0.001). SPINT1-AS1 yielded an area under the receiver operating characteristic curve value of 0.865 (95% confidence interval, 0.821–0.902) for discriminating CRC tissues from AN tissues. Moreover, high SPINT1-AS1 expression was correlated with regional lymph node metastasis (P<0.001), distant metastasis (P<0.001), and shorter relapse-free survival (RFS) time (P<0.001), and Cox regression analysis indicated that SPINT1-AS1 was an independent prognostic factor for RFS. Meanwhile, significant reduction of SPINT1-AS1 expression level (P=0.001) was observed in CRC serum exosomes after surgical resection.ConclusionSPINT1-AS1 is upregulated in CRC tissues and plays an essential role in CRC progression and prognosis. Thereby, SPINT1-AS1 may serve as a candidate prognostic biomarker and molecular therapy target for CRC.

The elevated activation of NFκB and AP-1 is correlated with differential regulation of Bcl-2 and associated with oral squamous cell carcinoma progression and resistance.

Oral cancer is the sixth most common cancer in the world. Failure of chemoradiation therapy is a major concern for treating oral cancer patients. The objective of this study is to determine the B cell lymphoma-2 (bcl-2) expression and its regulation by nuclear factor κB (NFκB) and activator protein 1 (AP-1) in oral cancer progression and chemoradiation resistance. In the present study, a total of 123 (n=123) human samples were included. Briefly, 64 fresh samples were from adjacent normal (AN), primary oral tumors without treatment (PT), and tumors with resistance to chemoradiation therapy with local recurrence (RCRT). Fifty-nine samples were human tongue cancers and normal samples (TMA). Messenger RNA (mRNA) expression levels of bcl-2 and protein levels of bcl-2, NFκB, AP-1, and inactive GSK3α/β were measured by semiquantitative RT-PCR, immunohistochemistry, Western blot, and ChIP analysis. Increased bcl-2 expression was observed in PT compared to AN. The RCRT tumors showed maximum expression of bcl-2 mRNA and protein over the PT and AN groups. Bcl-2 protein and mRNA expression were positively correlated with NFκB and AP-1 expression. AP-1 expression was strongly correlated with bcl-2 in the RCRT group of tumors. Further, inactive GSK3α/β showed a positive trend with bcl-2 expression in oral tongue cancer specimens. Collectively, our results demonstrated cumulative effect of AP-1 and NFĸB for bcl-2 gene regulation in overall PT progression and chemoradiation resistance. The study provides evidence of increased bcl-2 mRNA/protein fueled by NFĸB in PT and AP-1 in RCRT. These regulations of bcl-2 by NFκB and AP-1 are important in OSCC progression and chemoradiation resistance.

HLA-DRB1 and HLA-DQB1 methylation changes promote the occurrence and progression of Kazakh ESCC

Human leukocyte antigen II (HLA-II) plays an important role in host immune responses to cancer cells. Changes in gene methylation may result in aberrant expression of HLA-II, serving a key role in the pathogenesis of Kazakh esophageal squamous cell carcinoma (ESCC). We analyzed the expression level of HLA-II (HLA-DP, -DQ, and -DR) by immunohistochemistry, as well as the methylation status of HLA-DRB1 and HLA-DQB1 by MassARRAY spectrometry in Xinjiang Kazakh ESCC. Expression of HLA-II in ESCC was significantly higher than that in cancer adjacent normal (ACN) samples (P < 0.05). Decreased HLA-II expression was closely associated with later clinical stages of ESCC (P < 0.05). Hypomethylation of HLA-DRB1 and hypermethylation of HLA-DQB1 was significantly correlated with occurrence of Kazakh ESCC (P < 0.01), and mainly manifested as hypomethylation of CpG9, CpG10-11, and CpG16 in HLA-DRB1 and hypermethylation of CpG6-7 and CpG16-17 in HLA-DQB1 (P < 0.01). Moreover, hypomethylation of HLA-DQB1 CpG6-7 correlated with poor differentiation in ESCCs, whereas hypermethylation of HLA-DRB1 CpG16 and hypomethylation of HLA-DQB1 CpG16-17 were significantly associated with later stages of ESCC (P < 0.05). A significant inverse association between HLA-DRB1 CpG9 methylation and HLA-II expression was found in ESCC (P < 0.05). These findings suggest aberrant HLA-DRB1 and HLA-DQB1 methylation contributes to the aberrant expression of HLA-II. These molecular changes may influence the immune response to specific tumor epitopes, promoting the occurrence and progression of Kazakh ESCC.

Abstract 659: Genome-wide profiling of the prostate cancer methylome for biomarker discovery.

Abstract Prostate cancer (PC) is the most common malignancy, and the second most common cause of cancer related death amongst men in the Western world. The PC biomarkers currently available are unable to distinguish between aggressive and indolent PC, causing significant over-diagnosis and over-treatment. Here, by genomewide profiling of the DNA methylome, we aimed to identify new molecular markers that can improve the accuracy of diagnosis and prognosis of PC. In total, 21 PC, 9 normal (N), and 12 adjacent normal (AN) prostate tissue samples, as well as 15 prostate (cancer) cell lines, were run on the Illumina Infinium HumanMethylation450 BeadChip, which investigates &amp;lt;485,000 CpG sites. The methylation data was analyzed using the Mann-Whitney rank-sum test. Identification of differential methylation between N and AN samples could potentially aid correct diagnosis of patients with false negative biopsies. However, only 16 probes showed significant differential methylation in N vs. AN samples (p-value &amp;lt;0.05, Benjamini-Hochberg-adjusted, Δβ≥0.2), none of which corresponded to the same genomic locus. Also, multi-dimensional scaling, using the 10,000 most variable CpG sites, showed that N and AN samples clustered very tightly together, whereas PC samples showed great heterogeneity. Due to this similarity in methylation between N and AN samples, these were pooled into one control group for further analyses. We applied a Δβ cutoff value of 0.2 and a p-value of 0.05 (adjusted), which revealed 26,286 significantly differentially methylated CpG sites between PC and controls (3076 hypomethylated and 23,210 hypermethylated in PC). Global analysis showed that PC samples were hypermethylated in CpG islands (CGIs), a trend which was even more pronounced in CGI shores and shelves. Although less common overall, hypomethylation was also more prominent in shores and shelves compared to CGIs. Applying a strict Δβ cutoff value of 0.6, only 69 significantly differentially methylated CpG sites remained. Of these, 55 sites were gene associated (41 different genes), all were hypermethylated in PC samples, and all but one were located in CGIs. Out of these 41 genes, we selected 5 candidates for validation. In-house and publicly available gene expression datasets showed that at least three of the genes were downregulated in PC, consistent with silencing caused by aberrant promoter methylation. Initial validation was carried out in PC cell lines by bisulphite sequencing, which confirmed the array results for all 5 genes. We are currently validating these findings by methylation-specific qPCR analysis of a large independent patient cohort with long clinical follow-up, in order to assess the diagnostic/prognostic biomarker potential of these 5 novel candidate methylation markers. These results will be presented at the conference. Citation Format: Siri H. Strand, Michal Switnicki, Philippe Lamy, Soeren Hoeyer, Michael Borre, Jakob S. Pedersen, Torben Oerntoft, Karina D. Soerensen. Genome-wide profiling of the prostate cancer methylome for biomarker discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2013-659

Predictive Genes in Adjacent Normal Tissue Are Preferentially Altered by sCNV during Tumorigenesis in Liver Cancer and May Rate Limiting

BackgroundIn hepatocellular carcinoma (HCC) genes predictive of survival have been found in both adjacent normal (AN) and tumor (TU) tissues. The relationships between these two sets of predictive genes and the general process of tumorigenesis and disease progression remains unclear.Methodology/Principal FindingsHere we have investigated HCC tumorigenesis by comparing gene expression, DNA copy number variation and survival using ∼250 AN and TU samples representing, respectively, the pre-cancer state, and the result of tumorigenesis. Genes that participate in tumorigenesis were defined using a gene-gene correlation meta-analysis procedure that compared AN versus TU tissues. Genes predictive of survival in AN (AN-survival genes) were found to be enriched in the differential gene-gene correlation gene set indicating that they directly participate in the process of tumorigenesis. Additionally the AN-survival genes were mostly not predictive after tumorigenesis in TU tissue and this transition was associated with and could largely be explained by the effect of somatic DNA copy number variation (sCNV) in cis and in trans. The data was consistent with the variance of AN-survival genes being rate-limiting steps in tumorigenesis and this was confirmed using a treatment that promotes HCC tumorigenesis that selectively altered AN-survival genes and genes differentially correlated between AN and TU.Conclusions/SignificanceThis suggests that the process of tumor evolution involves rate-limiting steps related to the background from which the tumor evolved where these were frequently predictive of clinical outcome. Additionally treatments that alter the likelihood of tumorigenesis occurring may act by altering AN-survival genes, suggesting that the process can be manipulated. Further sCNV explains a substantial fraction of tumor specific expression and may therefore be a causal driver of tumor evolution in HCC and perhaps many solid tumor types.

Quantitative evaluation of DNA hypermethylation in malignant and benign breast tissue and fluids

The assessment of DNA had demonstrated altered methylation in malignant compared to benign breast tissue. The purpose of our study was to (i) confirm the predictive ability of methylation assessment in breast tissue, and (ii) use the genes found to be cancer predictive in tissue to evaluate the diagnostic potential of hypermethylation assessment in nipple aspirate fluid (NAF) and mammary ductoscopic (MD) samples. Quantitative methylation specific (qMS)-PCR was conducted on three specimen sets: 44 malignant (CA) and 34 normal (NL) tissue specimens, 18 matched CA, adjacent normal (ANL) tissue and NAF specimens, and 119 MD specimens. Training and validation tissue sets were analyzed to determine the optimal group of cancer predictive genes for NAF and MD analysis. NAF and MD cytologic review were also performed. Methylation of CCND-2, p16, RAR-beta and RASSF-1a was significantly more prevalent in tumor than in normal tissue specimens. Receiver operating characteristic curve analysis demonstrated an area under the curve of 0.96. For the 18 matched CA, ANL and NAF specimens, the four predictive genes identified in cancer tissue contained increased methylation in CA vs. ANL tissue; NAF samples had higher methylation than ANL specimens. Methylation frequency was higher in MD specimens from breasts with cancer than benign samples for p16 and RASSF-1a. In summary, i) routine quantitative DNA methylation assessment in NAF and MD samples is possible, and ii) genes hypermethylated in malignant breast tissue are also altered in matched NAF and in MD samples, and may be useful to assist in early breast cancer detection.

Regional three-dimensional geometry and function of left ventricles with fibrous aneurysms. A cine-computed tomography study.

To assess the extent and nature of the dysfunction surrounding aneurysms of the left ventricle (LV), we examined the parameters of local and global three-dimensional shape, size, and function of LVs of eight patients with histologically confirmed anterior fibrous aneurysms. Three-dimensional reconstructions of each LV were made from 10-12 short-axis fast cine-angiographic computed tomography (cine-CT) slices encompassing the entire heart at end diastole and end systole. Regional three-dimensional wall thickness, thickening, motion, curvature, and stress index were calculated for 84 elements encompassing the entire LV. The aneurysmal border was defined by a sharp decrease in end-diastolic wall thickness and separated the LV into an aneurysmal zone and a normal zone that was further divided into adjacent normal (AN) and remote normal (RN) zones. As expected, thickening was negligible in both the aneurysmal and the border zones. Although both the AN and the RN zones had normal wall thickness (1.05 +/- 0.20 and 1.09 +/- 0.20 cm, respectively), thickening was depressed in the AN (0.22 +/- 0.08 cm) but not the RN (0.44 +/- 0.19 cm) zones. The size of the dysfunction zone (defined as less than 2 mm thickening) was found to be considerably greater than the anatomic size of the aneurysm (60.9 +/- 13.7% versus 33.6 +/- 7.6% of the left ventricular endocardial area, respectively; p less than 0.001). In addition, the AN zone had a smaller curvature and a higher stress index than the RN zone. LVs with fibrous aneurysms are characterized by a relatively large region of nonfunction that encompasses the thin aneurysmal area and its transitional border zone, a normally functioning remote zone, and an intermediate region of normal wall thickness but with reduced function, which may be attributed to its low curvature and high stress index.