Multidimensional Analysis of a Cell-Free DNA Whole Methylome Sequencing Assay for Early Detection of Gastric Cancer: Protocol for an Observational Case-Control Study.

BACKGROUND: Commonly used noninvasive serological indicators serve as a step before endoscope diagnosis and help identify the high-risk gastric cancer (GC) population. However, they are associated with high false positives and high false negatives. Alternative noninvasive approaches, such as cancer-related features in cell-free DNA (cfDNA) fragments, have been gradually identified and play essential roles in early cancer detection. The integrated analysis of multiple cfDNA features has enhanced detection sensitivity compared to individual features. OBJECTIVE: This study aimed to develop and validate an assay based on assessing genomic-scale methylation and fragmentation profiles of plasma cfDNA for early cancer detection, thereby facilitating the early diagnosis of GC. The primary objective is to evaluate the overall specificity and sensitivity of the assay in predicting GC within the entire cohort, and subsequently within each clinical stage of GC. The secondary objective involved investigating the specificity and sensitivity of the assay in combination with possible serological indicators. METHODS: This is an observational case-control study. Blood samples will be prospectively collected before gastroscopy from 180 patients with GC and 180 nonmalignant control subjects (healthy or with benign gastric diseases). Cases and controls will be randomly divided into a training and a testing data set at a ratio of 2:1. Plasma cfDNA will be isolated and extracted, followed by bisulfite-free low-depth whole methylome sequencing. A multidimensional model named Thorough Epigenetic Marker Integration Solution (THEMIS) will be constructed in the training data set. The model includes features such as the methylated fragment ratio, chromosomal aneuploidy of featured fragments, fragment size index, and fragment end motif. The performance of the model in distinguishing between patients with cancer and noncancer controls will then be evaluated in the testing data set. Furthermore, GC-related biomarkers, such as pepsinogen, gastrin-17, and Helicobacter pylori, will be measured for each patient, and their predictive accuracy will be assessed both independently and in combination with the THEMIS model. RESULTS: Recruitment began in November 2022 and will be ended in April 2024. As of August 2022,250 patients have been enrolled. The final data analysis is anticipated to be completed by September 2024. CONCLUSIONS: This is the first registered case-control study designed to investigate a stacked ensemble model integrating several cfDNA features generated from a bisulfite-free whole methylome sequencing assay. These features include methylation patterns, fragmentation profiles, and chromosomal copy number changes, with the aim of identifying the GC population. This study will determine whether multidimensional analysis of cfDNA will prove to be an effective strategy for distinguishing patients with GC from nonmalignant individuals within the Chinese population. We anticipate the THEMIS model will complement the standard-of-care screening and aid in identifying high-risk patients for further diagnosis. TRIAL REGISTRATION: ClinicalTrial.gov NCT05668910; https://www.clinicaltrials.gov/study/NCT05668910. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48247.

P-score in preoperative biopsies accurately predicts P-score in final pathology at radical prostatectomy in patients with localized prostate cancer.

BACKGROUND: Prostate cancer (PCa) is a highly heterogeneous, multifocal disease, and identification of clinically significant lesions is challenging, which complicates the choice of adequate treatment. The Prostatype® score (P-score) is intended to guide treatment decisions for newly diagnosed PCa patients based on a three-gene signature (IGFBP3, F3, and VGLL3) and clinicopathological information obtained at diagnosis. This study evaluated association of the P-score measured in preoperative magnetic resonance imaging/transrectal ultrasound fusion-guided core needle biopsies (CNBs) and the P-score measured in radical prostatectomy (RP) specimens of PCa patients. We also evaluated the P-score association with the pathology of RP specimens. Furthermore, concordance of the P-score in paired CNB and RP specimens, as well as in index versus concomitant nonindex tumor foci from the same RP was investigated. METHODS: The study included 100 patients with localized PCa. All patients were diagnosed by CNB and underwent RP between 2015 and 2018. Gene expression was assessed with the Prostatype® real-time quantitative polymerase chain reaction kit and the P-score was calculated. Patients were categorized into three P-score risk groups according to previously defined cutoffs. RESULTS: For 71 patients, sufficient CNB tumor material was available for comparison with the RP specimens. The CNB-based P-score was associated with the pathological T-stage in RP specimens (p = 0.02). Moreover, the CNB-based P-score groups were in substantial agreement with the RP-based P-score groups (weighted κ score: 0.76 [95% confidence interval, 95% CI: 0.60-0.92]; Spearman's rank correlation coefficient r = 0.83 [95% CI: 0.74-0.89]; p < 0.0001). Similarly, the P-score groups based on paired index tumor and concomitant nonindex tumor foci (n = 64) were also in substantial agreement (weighted κ score: 0.74 [95% CI: 0.57-0.91]; r = 0.83 [95% CI: 0.73-0.89], p < 0.0001). CONCLUSIONS: Our findings suggest that the P-score based on preoperative CNB accurately reflects the pathology of the whole tumor, highlighting its value as a decision support tool for newly diagnosed PCa patients.

Validation of the prognostic value of a three-gene signature and clinical parameters-based risk score in prostate cancer patients.

BACKGROUND: The study aimed to validate the prognostic value of the Prostatype® risk score (P-score), which includes a three-gene signature and conventional risk factors, in a retrospective cohort. METHODS: All 716 patients diagnosed with prostate cancer from 2008 to 2010 at Skåne University Hospital, Sweden, were included. After excluding patients based on pathological and clinical eligibility criteria, RNA quality, and presence of metastases at diagnosis, a final cohort comprising 316 patients was further analyzed. Expression levels of three genes (IGFBP3, F3, and VGLL3) were measured in archived formalin-fixed paraffin-embedded core needle biopsies. The gene expression data were combined with clinical parameters (Gleason score, prostate-specific antigen, and clinical tumor stage) to calculate the P-score for each patient. Predictive performance of the P-score in terms of prostate cancer-specific mortality (PCSM), distant metastasis and adverse pathological outcomes were investigated. RESULTS: The P-score predicted both PCSM (hazard ratio [HR] = 1.6) and metastasis (HR = 1.46). The P-score had an area under curve (AUC) of 0.93 when predicting the PCSM risk at 10 years (95% confidence interval [CI]: 0.89-0.98), which was significantly better than both D'Amico (AUC: 0.81, 95% CI: 0.72-0.90, p < 0.001) and UCSF-CAPRA (AUC: 0.88, 95% CI: 0.80-0.96, p < 0.05). Decision curve analysis showed a higher net benefit of the P-score compared to both D'Amico and CAPRA. All three risk scores performed similarly in the prediction of distant metastases. For patients who underwent radical prostatectomy (RP), a higher P-score correlated with adverse pathological features such as pathologic tumor stage T3-4 (p < 0.0001) and ≥International Society of Urological Pathology grade group 3 (p < 0.0001). CONCLUSIONS: Our findings provide evidence for the prognostic value of the P-score. The P-score predicted the risk for PCSM more accurately than the D'Amico and CAPRA scores. Performance was similar when predicting the risk for development of distant metastases within 10 years. Moreover, the P-score correlated with adverse pathological outcomes in RP specimens. Thus, the P-score could provide useful information for patients and their doctors to make informed decisions at the time of diagnosis.

A Novel Risk Score (P-score) Based on a Three-Gene Signature, for Estimating the Risk of Prostate Cancer-Specific Mortality.

Purpose: To develop and validate a risk score (P-score) algorithm which includes previously described three-gene signature and clinicopathological parameters to predict the risk of death from prostate cancer (PCa) in a retrospective cohort. Patients and Methods: A total of 591 PCa patients diagnosed between 2003 and 2008 in Stockholm, Sweden, with a median clinical follow-up time of 7.6 years (1-11 years) were included in this study. Expression of a three-gene signature (IGFBP3, F3, VGLL3) was measured in formalin-fixed paraffin-embedded material from diagnostic core needle biopsies (CNB) of these patients. A point-based scoring system based on a Fine-Gray competing risk model was used to establish the P-score based on the three-gene signature combined with PSA value, Gleason score and tumor stage at diagnosis. The endpoint was PCa-specific mortality, while other causes of death were treated as a competing risk. Out of the 591 patients, 315 patients (estimation cohort) were selected to develop the P-score. The P-score was subsequently validated in an independent validation cohort of 276 patients. Results: The P-score was established ranging from the integers 0 to 15. Each one-unit increase was associated with a hazard ratio of 1.39 (95% confidence interval: 1.27-1.51, p < 0.001). The P-score was validated and performed better in predicting PCa-specific mortality than both D'Amico (0.76 vs 0.70) and NCCN (0.76 vs 0.71) by using the concordance index for competing risk. Similar improvement patterns are shown by time-dependent area under the curve. As demonstrated by cumulative incidence function, both P-score and gene signature stratified PCa patients into significantly different risk groups. Conclusion: We developed the P-score, a risk stratification system for newly diagnosed PCa patients by integrating a three-gene signature measured in CNB tissue. The P-score could provide valuable decision support to distinguish PCa patients with favorable and unfavorable outcomes and hence improve treatment decisions.

Spatiotemporal characteristics and impact mechanism of high-quality development of cultural tourism in the Yangtze River Delta urban agglomeration.

The Yangtze River Delta urban agglomeration is the leading and demonstration area for the high-quality development of culture tourism (HDCT) in China. It is of great significance to study the spatiotemporal characteristics and impact mechanism of the HDCT for revealing the internal law of HDCT and promoting the collaborative innovation of culture tourism among cities. Based on the scientific construction of the evaluation system of HDCT, this paper made a quantitative analysis of 26 cities' HDCT by using coupling coordination degree model, Lisa spatiotemporal transition and spatial Durbin model (SDM). The results show that: The overall level of 26 cities' HDCT shows a fluctuating upward trend, and presents a "Z" pattern in space. More than 80% of the cities are at the medium and high level. Shanghai has obvious advantages in the primacy degree. There is a significant positive spatial autocorrelation among cities with high-quality of culture tourism development. The spatial clustering and proximity of the same kind are increasing, and the radiation effect is gradually obvious. The local spatial association patterns are mainly HH and LL agglomeration, and the characteristics of polarization are gradually prominent. The local spatial correlation structure of HDCT has strong stability, the transfer inertia between types is prominent, and the overall spatial evolution is lack of integration with obvious path dependence and lock-in effect. The spatiotemporal evolution of the HDCT is a complex process under the interaction of multiple factors, and there is a significant spatial spillover effect (0.256). The level of economic development, technological innovation, professional talent allocation are the three main factors. According to the dominant factor, it can be divided into economy stabilizing type, industry optimizing type, innovation driving type and traffic impacting type. These findings have implications for local governments and tourism management departments to achieve high-quality innovative development of cultural tourism.

Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries.

Adenosine to inosine editing is common in the human transcriptome and changes of this essential activity is associated with disease. Children with ADAR1 mutations develop fatal Aicardi-Goutières syndrome characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy of breast, lung and liver cancer. ADAR1 silencing in breast cancer cells leads to increased apoptosis, suggesting an anti-apoptotic function that promotes cancer progression. Yet, suitable high-throughput editing assays are needed to efficiently screen chemical libraries for modifiers of ADAR1 activity. We describe the development of a bioluminescent reporter system that facilitates rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter-transcript editing. Stably introduced into cancer cell lines, the system reports on elevated endogenous ADAR1 editing activity induced by interferon as well as knockdown of ADAR1 and ADAR2. In a single-well setup we used the reporter in HeLa cells to screen a small molecule library of 33 000 compounds. This yielded a primary hit rate of 0.9% at 70% inhibition of editing. Thus, we provide a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.

ADAR1 Editing and its Role in Cancer.

It is well established that somatic mutations and escape of immune disruption are two essential factors in cancer initiation and progression. With an increasing number of second-generation sequencing data, transcriptomic modifications, so called RNA mutations, are emerging as significant forces that drive the transition from normal cell to malignant tumor, as well as providing tumor diversity to escape an immune attack. Editing of adenosine to inosine (A-to-I) in double-stranded RNA, catalyzed by adenosine deaminases acting on RNA (ADARs), is one dynamic modification that in a combinatorial manner can give rise to a very diverse transcriptome. Since the cell interprets inosine as guanosine (G), A-to-I editing can result in non-synonymous codon changes in transcripts as well as yield alternative splicing, but also affect targeting and disrupt maturation of microRNAs. ADAR-mediated RNA editing is essential for survival in mammals, however, its dysregulation causes aberrant editing of its targets that may lead to cancer. ADAR1 is commonly overexpressed, for instance in breast, lung, liver and esophageal cancer as well as in chronic myelogenous leukemia, where it promotes cancer progression. It is well known that ADAR1 regulates type I interferon (IFN) and its induced gene signature, which are known to operate as a significant barrier to tumor formation and progression. Adding to the complexity, ADAR1 expression is also regulated by IFN. In this review, we discussed the regulatory mechanisms of ADAR1 during tumorigenesis through aberrant editing of specific substrates. Additionally, we hypothesized that elevated ADAR1 levels play a role in suppressing an innate immunity response in cancer cells.

ADARs and editing: The role of A-to-I RNA modification in cancer progression.

Cancer arises when pathways that control cell functions such as proliferation and migration are dysregulated to such an extent that cells start to divide uncontrollably and eventually spread throughout the body, ultimately endangering the survival of an affected individual. It is well established that somatic mutations are important in cancer initiation and progression as well as in creation of tumor diversity. Now also modifications of the transcriptome are emerging as a significant force during the transition from normal cell to malignant tumor. Editing of adenosine (A) to inosine (I) in double-stranded RNA, catalyzed by adenosine deaminases acting on RNA (ADARs), is one dynamic modification that in a combinatorial manner can give rise to a very diverse transcriptome. Since the cell interprets inosine as guanosine (G), editing can result in non-synonymous codon changes in transcripts as well as yield alternative splicing, but also affect targeting and disrupt maturation of microRNA. ADAR editing is essential for survival in mammals but its dysregulation can lead to cancer. ADAR1 is for instance overexpressed in, e.g., lung cancer, liver cancer, esophageal cancer and chronic myoelogenous leukemia, which with few exceptions promotes cancer progression. In contrast, ADAR2 is lowly expressed in e.g. glioblastoma, where the lower levels of ADAR2 editing leads to malignant phenotypes. Altogether, RNA editing by the ADAR enzymes is a powerful regulatory mechanism during tumorigenesis. Depending on the cell type, cancer progression seems to mainly be induced by ADAR1 upregulation or ADAR2 downregulation, although in a few cases ADAR1 is instead downregulated. In this review, we discuss how aberrant editing of specific substrates contributes to malignancy.

Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis.

RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3'UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression.

Expression of the p53 target Wig-1 is associated with HPV status and patient survival in cervical carcinoma.

The p53 target gene WIG-1 (ZMAT3) is located in chromosomal region 3q26, that is frequently amplified in human tumors, including cervical cancer. We have examined the status of WIG-1 and the encoded Wig-1 protein in cervical carcinoma cell lines and tumor tissue samples. Our analysis of eight cervical cancer lines (Ca Ski, ME-180, MS751, SiHa, SW756, C-4I, C-33A, and HT-3) by spectral karyotype, comparative genomic hybridization and Southern blotting revealed WIG-1 is not the primary target for chromosome 3 gains. However, WIG-1/Wig-1 were readily expressed and WIG-1 mRNA expression was higher in the two HPV-negative cervical cell lines (C33-A, HT-3) than in HPV-positive lines. We then assessed Wig-1 expression by immunohistochemistry in 38 cervical tumor samples. We found higher nuclear Wig-1 expression levels in HPV-negative compared to HPV positive cases (p = 0.002) and in adenocarcinomas as compared to squamous cell lesions (p<0.0001). Cases with moderate nuclear Wig-1 staining and positive cytoplasmic Wig-1 staining showed longer survival than patients with strong nuclear and negative cytoplasmic staining (p = 0.042). Nuclear Wig-1 expression levels were positively associated with age at diagnosis (p = 0.023) and histologic grade (p = 0.034). These results are consistent with a growth-promoting and/or anti-cell death function of nuclear Wig-1 and suggest that Wig-1 expression can serve as a prognostic marker in cervical carcinoma.