The expression and clinical applications of Wnt-signaling molecules in breast and colorectal cancers

Cancer is a heterogeneous disease of an array of etiology. In the last two decades, wide range of evidence has converged to a common theme of neoplasia that normal cell proliferation controls are lost at the level of cell signaling, cell-cycle arrest, differentiation or apoptosis. The diagnosis, prognosis and management of cancer patients must be based on a thorough understanding of its molecular constitutes of the disease. Therefore, analysis of proteins involved in any of these deregulated processes is essential for us to elucidate the cause and progression of individual tumors. In the present thesis study, the expressions of various cell cycle regulators and their relationships to cell proliferation, apoptosis and tumor progression were investigated in invasive breast cancer using immunohistochemical methods. The study reported the identification of apoptotic index, Ki-67, c-myc, cyclin D3 and p21 were positively correlated, while p16, p27 and cyclin D1 were negatively correlated to tumor progression. Moreover, a clear decoupling between p21, p27 and p16 as well as between cyclin D1 and cyclin D3 to tumor progression was found, indicating their differential roles in tumor progression. We furthered our investigation by studying the expression of the frizzled related protein (Frp) and its Wnt-signaling molecules in the same tumor system, as Frp is a new family of secreted proteins involved both in apoptosis and Wnt-signaling pathway. Using in-situ hybridization and immunohistochemical methods, we demonstrated that Frp mRNA was down-regulated in tumor areas in most of the cases examined and negatively correlated to both Wnt-1 and β-catenin. β-catenin, a key transcriptional factor responsible for the activation of both c-myc and cyclin D1 in colorectal cancer, was found in the cytoplasm, but not in the nuclei of breast tumor masses, despite the fact that both c-myc and cyclin D1 were elevated in all tumor tissues. The role of β-catenin in the progression of colorectal cancer is a well recognized, but unsettled issue. To re-evaluate the prognostic and diagnostic significance of β-catenin in colorectal cancer, we examined β-catenin expression in more than 300 cases of specimens of polyps, adenomas, and adenocarcinomas using immunohistochemical method by using colorectal tumor specimens. The results showed that β-catenin nuclear signal was statistically correlated not only with the purported sequential stages in colorectal carcinogenesis, but also positively correlated with lymph node metastasis and survival of colorectal cancer patients. Moreover, adenomas associated with synchronous carcinomas showed significantly higher levels of nuclear β-catenin than adenomas without associated carcinomas. In our study, β-catenin nuclear expression was rather unique to colorectal adenocarcinomas, but rare or absent in other gastric-intestinal adenocarcinomas. In this regard, nuclear β-catenin may serve as an additional parameter to aid the distinction of colorectal adenocarcinomas from adenocarcinomas of other sites. To explore the potential of β-catenin as a suitable marker for colorectal cancer screening, we then investigated the possibility of detecting β-catenin DNA & RNA in sera/plasma of colorectal carcinoma and adenoma patients. The results were striking. In both β-catenin DNA & RNA were detected in the blood of 100% of patients with colorectal carcinomas, 79 to 90% of patients with adenomas, but none in normal volunteer controls using PCR and RT-PCR analysis. Finally, in order to have a global understanding of genes that participate in colorectal tumor progression, we analyzed the gene profiles of a benign polyp, adenoma and carcinoma compared to normal colonic tissues using 2,400 genes cDNA microarray. The study provides valuable new information on genes whose expressions were found altered in the process of colorectal cancer carcinogenesis. Among the deregulated genes, three genes, Wnt-5a, cyclin D1, and c-myc showed significant correlation with tumor progression. This finding provides a strong independent evidence that Wnt-signaling pathway plays a crucial role in colon carcinogenesis

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann–Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal–Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples

Expression of frizzled-related protein and Wnt-signalling molecules in invasive human breast tumours

Frizzled-related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine-rich domain (CRD) of the frizzled family proteins. The role of Frp protein is far from clear. To explore the role of Frp and its relationship to the Wnt-signalling pathway in breast cancer, in situ hybridization and immunohistochemical analyses of Frp, Wnt-1, APC, beta-catenin, and its target genes c-myc and cyclin D1 were conducted in 70 specimens of invasive ductal carcinomas of the human breast. Frp mRNA was down-regulated in 62 and elevated in eight tumour specimens, compared with adjacent normal tissues. In the course of tumour progression, however, Frp mRNA steadily increased in both tumour and the adjacent tissues. Interestingly, the number of cases with axillary lymph node metastasis was significantly lower in the group with elevated Frp than in the group with decreased Frp, suggesting that Frp may contribute as a prognostic factor in invasive breast cancer. Wnt-1, a gene implicated in human breast cancer, was markedly elevated in grade 1 tumours, but declined as tumour grade declined. The level of Wnt-1 was linearly correlated with its downstream target beta-catenin (p<0.05), but was inversely correlated with Frp (p<0.05), suggesting a possible negative regulatory role of Frp with regard to Wnt-1. APC was inversely correlated with beta-catenin (p<0.05). beta-catenin, a key transcriptional activator responsible for the activation of both c-myc and cyclin D1 in colorectal tumours, was detected at high levels in the plasma membranes of cells in normal tissue. In tumour masses, however, beta-catenin lost its tight association with the membrane and diffused into the cytoplasm. Surprisingly, it clearly did not penetrate the nuclei, despite the fact that both c-myc and cyclin D1 were markedly elevated in all tumour tissues. As revealed in this study, Wnt-1/beta-catenin plays very different roles in the oncogenesis of breast and colon cancers. This first systemic analysis of the Frp and the Wnt-signalling pathway in human breast cancer provides a springboard for further work on the role of Frp in the development of breast cancer. Copyright (C) 2001 John Wiley Sons, Ltd

Targeted Sequencing Approach and Its Clinical Applications for the Molecular Diagnosis of Human Diseases

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases

Targeted Sequencing Approach and Its Clinical Applications for the Molecular Diagnosis of Human Diseases

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases

Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum ▿

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases

The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing

In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R2 = 0.9471, P &lt; 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future

Disease-Specific Target Gene Expression Profiling of Molecular Imaging Probes: Database Development and Clinical Validation

Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2- deoxy-2-D-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/