Ribonucleic-acid-biomarker candidates for early-phase group detection of common cancers

Cancer is considered as a challenging lethal agent around the world and its detection at early stages would help prevention of the high mortality. Among the widely used biomarkers in clinical diagnosis of cancer, extracellular non-coding RNAs as ribonucleic acid biomarkers serve as state-of-the-art candidates for molecular diagnosis. In that regard, microRNAs are of great priority mainly because of high variety and stability in body fluids. Accordingly, common miRNAs among most prevalent cancers could help us (pre)diagnose cancer with high accuracy in target samples. In this study, common lethal cancers to humans were investigated in case of miRNA profiles to determine the possible common correlation between miRNA up-regulation or down-regulation (as a ribonucleic acid biomarker) and developing the cancers. It was shown that among the investigated miRNAs, five typical extracellular miRNAs (miR-18a, miR-21, miR-155, miR-221, and miR-375) dysregulation are predominant in most cancer varieties comprising breast, colon, lung, prostate, pancreas, gastric, ovarian, esophagus and liver. This could serve as an appropriate target site for developing point-of-care approaches for cancer detection e.g. designing diagnostic biosensor-based microarrays or kits for both quantification and qualification of the biomarkers. Besides, the miRNA candidates could be efficiently applied to cancer therapeutic approaches.     

Clinical proteomics: towards early detection of cancers

A key challenge in clinical proteomic of cancer is the identification of biomarkers that would allow early detection, diagnosis and monitor progression of the disease to improve long-term survival of patients. Recent advances in proteomic instrumentation and computational methodologies offer unique chance to rapidly identify these new candidate markers or pattern of markers. The combination of retentate affinity chromatography and surfaced-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry is one of the most interesting new approaches for cancer diagnostic using proteomic profiling. This review aims to summarize the results of studies that have used this new technology method for the early diagnosis of human cancer. Despite promising results, the use of the proteomic profiling as a diagnostic tool brought some controversies and technical problems and still requires some efforts to be standardised and validated.     

A suite of DNA methylation markers that can detect most common human cancers

Cancer-specific DNA methylation from the tumor derived fraction of cell free DNA found in blood samples could be used for minimally invasive detection and monitoring of cancer. The knowledge of marker regions with cancer-specific DNA methylation is necessary to the success of such a process. We analyzed the largest cancer DNA methylation dataset available—TCGA Illumina HumanMethylation450 data with over 8,500 tumors—in order to find cancer-specific DNA methylation markers for most common human cancers. First, we identified differentially methylated regions for individual cancer types and those were further filtered against data from normal tissues to obtain marker regions with cancer-specific methylation, resulting in a total of 1,250 hypermethylated and 584 hypomethylated marker CpGs. From hypermethylated markers, optimal sets of six markers for each TCGA cancer type were chosen that could identify most tumors with high specificity and sensitivity [area under the curve (AUC): 0.969-1.000] and a universal 12 marker set that can detect tumors of all 33 TCGA cancer types (AUC >0.84). In addition to hundreds of new DNA methylation markers, our approach also identified markers that are in current clinical use, SEPT9 and GSTP1, indicating the validity of our approach and a significant potential utility for the newly discovered markers. The hypermethylated markers are linked to polycomb associated loci and a significant fraction of the discovered markers is within noncoding RNA genes; one of the best markers is MIR129-2. Future clinical testing of herein discovered markers will confirm new markers that will improve minimally invasive diagnosis and monitoring for multiple cancers.     

Circulating tumor cells in the early detection of human cancers

Cancer is a severe disease with high morbidity and mortality globally. Thus, early detection is emerging as an important topic in modern oncology. Although the strategies for early detection have developed rapidly in recent decades, they remain challenging due to the subtle symptoms in the initial stage of the primary tumor. Currently, tumor biomarkers, imaging, and specific screening tests are widely used in various cancer types; however, each method has limitations. The harms are even overweight against the benefits in some cases. Therefore, early detection approaches should be improved urgently. Liquid biopsy, for now, is a convenient and non-invasive way compared to the traditional tissue biopsy in screening and early diagnosis. Circulating tumor cells (CTCs) are vital in liquid biopsy and play a central role in tumor dissemination and metastases. They have promising potential as cancer biomarkers in early detection. This review updates the knowledge of the biology of CTC; it also highlights the CTC enrichment technologies and their applications in the early detection of several human cancers.     

Biomarkers for early detection of high risk cancers: From gliomas to nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NpC) is a malignant disease associated with Epstein-Barr virus infection, and often diagnosed at an advanced stage. This significantly curtails patient survival. We hypothesize that a panel of biomarkers can be assembled to assess NpC incidence, early detection, and tumor progression during therapeutic intervention. Our thesis rests on a model of successfully predicting high-risk gliomas by means of a carefully crafted panel of molecular mitotic biomarkers (i.e., securin, survivin and MCM2). The strategy we propose holds strong promise for prevention and cure of NpC. The approach we propose seeks to identify certain biomarkers from viral materials, patient tissues and assessment of related diseases, whose signatures, taken together, will be endowed with some degree of congruency, or sense of a coordinated language (i.e., “votes”). Biomarker “voting” will then permit to outline a broad coordinated molecular map for the molecular and epigenetic characterization of each individual patient''s NpC tumor. We will draw on the process of contrasting biomarkers in health and disease, which rests on the auto-proteomic concept particularly relevant in high-risk cancer individuals, such as is the case for NpC. In brief we defend, current advances in human proteome profiling proffers the possibility of having individual baseline proteomic profiles using local body fluids (e.g., saliva, nasal secretions, sputum) or systemic fluids (e.g., plasma, serum, cerebrospinal fluid) to unravel a personalized molecular map for high-risk NpC individuals. Regular check-up will monitor for new or impending manifestations of NpC, and provide a secure assessment of incidence and early detection.     

Microwave early detection of breast cancers using a virtual-focus scanning method

In this work, we apply a virtual-focus scanning method to detect a tumor inside a simplified two-dimensional breast model via the finite difference time domain method. For the purpose of simplification, we construct a microwave detection system with an N-element infinitesimal dipole array, and excite the central element of the array while letting all elements receive the time-shifted scattered fields in order to filter out unwanted signals. With the application of the two-step, virtual-focus scanning method, our computerized prototype system has accurately detected randomly generated tumors in all cases investigated, including small tumors in early stages of development.     

DNA methylation signatures in circulating cell-free DNA as biomarkers for the early detection of cancer

Detecting cell-free DNA(cfDNA) or circulating tumor DNA(ctDNA) in plasma or serum could serve as a "liquid biopsy", which would be useful for numerous diagnostic applications. cfDNA methylation detection is one of the most promising approaches for cancer risk assessment. Here, we reviewed the literature related to the use of serum or plasma circulating cell-free DNA for cancer diagnosis in the early stage and their power as future biomarkers.     

TCF21 and PCDH17 methylation: An innovative panel of biomarkers for a simultaneous detection of urological cancers

The three main types of urological cancers are mostly curable by surgical resection, if early detected. We aimed to identify novel DNA methylation biomarkers common to these three urological cancers, potentially suitable for non-invasive testing. From a candidate list of markers created after gene expression assessment of pharmacologically treated cell lines and tissue samples, two genes were selected for further validation. Methylation levels of these genes were quantified in a total of 12 cancer cell lines and 318 clinical samples. PCDH17 and TCF21 methylation levels provided a sensitivity rate of 92% for bladder cancer, 67% for renal cell tumors and 96% for prostate cancer. Methylation levels were significantly different from those of cancer free individuals (n = 37) for all tumor types (p < 0.001), providing 83% sensitivity and 100% specificity for cancer detection. Although in urine samples the sensitivity was 60%, 32% and 26% for bladder, renal, and prostate tumors, respectively (39% overall), absolute specificity was retained. We identified novel and highly specific methylation markers common to the three main urological cancers. However, additional efforts are required to increase the assay’s sensitivity, enabling the simultaneous non-invasive screening of urological tumors in a single voided urine analysis.     

Immunological detection of changes in genomic DNA methylation during early zebrafish development.

DNA methylation reprogramming, the erasure of DNA methylation patterns shortly after fertilization and their reestablishment during subsequent early development, is essential for proper mammalian embryogenesis. In contrast, the importance of this process in the development of non-mammalian vertebrates such as fish is less clear. Indeed, whether or not any widespread changes in DNA methylation occur at all during cleavage and blastula stages of fish in a fashion similar to that shown in mammals has remained controversial. Here we have addressed this issue by applying the techniques of Southwestern immunoblotting and immunohistochemistry with an anti-5-methylcytosine antibody to the examination of DNA methylation in early zebrafish embryos. These techniques have recently been utilized to demonstrate that development-specific changes in genomic DNA methylation also occur in Drosophila melanogaster and Dictyostelium discoideum, both organisms for which DNA methylation was previously not thought to occur. Our data demonstrate that genome-wide changes in DNA methylation occur during early zebrafish development. Although zebrafish sperm DNA is strongly methylated, the zebrafish genome is not detectably methylated through cleavage and early blastula stages but is heavily remethylated in blastula and early gastrula stages.     

Do we use the appropriate controls for the identification of informative methylation markers for early cancer detection?

It is possible to miss potential DNA methylation markers of tumorigenesis because of the initial filtering of profiling results on the basis of inappropriate controls.     

Bisulfite modification of immobilized DNAs for methylation detection

We have developed a novel method for detecting DNA methylation status of multiple samples, in which the DNA samples were firstly immobilized on the slide and treated with bisulfite directly on the chip. In this experiment, DNAs of pUC19 plasmid were restricted by the enzymes, and ligated with a linker bearing 5'-terminal acrylamide group at the sticky ends. Using universal acrylamide gel polymerization technique, a large amount of DNAs could be immobilized on the slide. The immobilized DNAs were converted by soaking the chip in bisulfite reaction mixtures for 16 h. The probes for detection of the methylation patterns of CpG sites hybridized with the converted DNAs on the microarray, and non-specifically bound probes were cleaned by electrophoresis. We have optimized the experimental conditions of both bisulfite treatment and electrophoresis to increase sensitivity and specificity. The results were further validated by bisulfite DNA sequencing. The experiments show that the method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This novel method could be used as a convenient tool to detect the methylation status of the multiple genes for a large amount of samples in the future.     

Targeting protein lysine methylation and demethylation in cancers

During the last decade, we saw an explosion of studies investigating the role of lysine methylation/demethylation of histones and non-histone proteins, such as p53, NF-kappaB, and E2F1. These 'Ying-Yang' post-translational modifications are important to fine-tuning the activity of these proteins. Lysine methylation and demethylation are catalyzed by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). PKMTs, PKDMs, and their substrates have been shown to play important roles in cancers. Although the underlying mechanisms of tumorigenesis are still largely unknown, growing evidence is starting to link aberrant regulation of methylation to tumorigenesis. This review focuses on summarizing the recent progress in understanding of the function of protein lysine methylation, and in the discovery of small molecule inhibitors for PKMTs and PKDMs. We also discuss the potential and the caveats of targeting protein lysine methylation for the treatment of cancer.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.