Journal of Molecular Biomarkers & Diagnosis

DNA markers offer a useful tool in human clinical medicine to predict disease susceptibility and drug response. In animal science, they can be applied in marker-assisted selection, gender determination, species identification or disease diagnosis. By combining DNA microarray technology and SNP (Single Nucleotide Polymorphism) genotyping, many novel genetic markers in candidate genes have been identified. These markers offer more advantages in that they can be used for accurate animal selection and to rapidly to improve the reproductive performance over traditional breeding methods. Modified Random Amplified Polymorphic DNA (RAPD) or Random Amplified Microsatellite Polymorphism (RAMPO) fingerprinting were used for the study of DNA polymorphisms, and the results demonstrated that novel and stable markers can be used for animal gender determination and species identification. This paper presents effective strategies for identifying novel and stable markers based on DNA polymorphisms.

Circulating Tumor Cells (CTCs), circulating tumor DNA (ctDNA) and Extracellular Vesicles (ECVs) hold the key to understanding the biology of tumor metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. CTCs can be detected in blood from patients with metastatic and primary carcinomas. Over the past decade, the development of immune-magnetic platforms has permitted accurate enumeration of CTCs at extremely low frequencies. In several case reports, the presence of CTCs has been associated with shortened survival times. Circulating-mutant DNA in blood is believed to originate from apoptotic and necrotic cells of the primary tumor, which discharge their DNA early during tumorigenesis. It is conceivable that cell-free tumor-specific DNA could also be related to the rate of turnover of CTCs. Extracellular Vesicles (ECVs) are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell–cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bio-reactive macromolecules such as nucleic acids, proteins, and lipids. Improvements in technologies to isolate purer CTCs, ECVs and ctDNAs will help in a better characterization and enable a broad range of clinical applications, including early detection of disease and the discovery of personalized biomarkers to predict treatment responses and disease progression. This review highlights the progress made in the development of noninvasive biomarkers using CTCs, ctDNA and ECVs.

Background: Heparin-binding EGF-like Growth Factor (HB-EGF), a member of the EGF family, exerts its biological activity through activation of the EGF receptors. HB-EGF is initially synthesized as a membrane-anchored precursor protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF plays pivotal roles in many physiologic and pathologic processes such as development and cell proliferation. In this study, we measured soluble HB-EGF concentrations in serum samples obtained from healthy volunteers and patients of various cancer types.

Materials and methods: Soluble HB-EGF levels in human serum samples were quantified by the immuno-PCR method.

Results: The mean soluble HB-EGF levels of the 20 healthy volunteers and 10 colon, breast, ovarian, head and neck, non-small cell lung, pancreatic, and small cell lung cancer patients were 5.04, 18.0, 15.1, 10.4, 6.69, 9.78, 23.6, and 5.60 pg/mL, respectively. There was a statistically significant difference between the HB-EGF concentrations of healthy volunteers and patients in 5 out of 7 cancer types. Furthermore, a trend for HB-EGF levels to increase along with disease stage was observed.

Conclusion: Soluble HB-EGF may be a useful diagnostic serological biomarker for various cancer types, and a predictive and/or pharmacodynamic biomarker for HB-EGF-targeted therapeutics.

Background: We investigated the usefulness of bone markers, respectively Alkaline Phosphatase (AP) and Tartrat Resistant Acid Phosphatase 5b (TRACP 5b) for diagnosis, treatment and monitoring of patients with carcinoma of different origin. AP is a marker of bone formation, while TRACP 5b is a marker of bone resorption. The isoform 5b of the enzyme TRACP is expressed by osteoclasts and can be measured in blood. Objectives: The aim of this study was to evaluate the prognostic value of the bone markers AP and TRACP 5b to detect bone metastasis and pathological bone metabolism. Materials and

Methods: Our study comprised 101 patients with positive tumor markers. Sera of these patients were collected and the bone markers AP and TRACP 5b were determind. TRACP 5b was measured by a colorimetric test which determines the TRACP 5b by using the phophatase activity of this enzyme by dephosphorylation of p-Nitrophenylphosphate (pNPP). The test is a two site immunoassay by Medac Diagnostika, Germany.

Results: The sensitivity of AP in our study to detect bone metastasis is 52.9%, the specificity is 53.9%. TRACP 5b shows a sensitivity of 64.7% and a specificity of 70.9%. An elevated TRACP 5b activity is associated significantly with bone metastasis in our study groups (p=0.01). In patients with chronic elevated levels of liver enzymes we could see a significant elevation of TRACP 5b (p=0.005).

Conclusion: In conclusion TRACP 5b is more sensitive and specific to detect bone metastasis and bone turnover than the Alkaline Phosphatase. In patients with multimorbidity the origin of AP is not clear due to its multiorganic appearance. The levels of TRACP 5b are elevated in patients with bone metastasis and in patients with chronical dysfunction of the liver. TRACP 5b might be helpful in the diagnostic procedure of tumor-patients to detect bone metastasis. Moreover TRACP 5b seems to be helpful to indicate oncological patients with early dysfunctions in bone metabolism and helps to induce early treatment to these patients.

TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALBLps-d) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-d over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-d BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHTtreated BALBLps-d BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening.

Progressive Retinal Atrophy is a group of genetically inherited diseases in various canine breeds. The disease begins with retinal damage and progresses to blindness. Abnormalities in various genes are linked with different disease variants, and in most cases, involvement of one gene towards PRA is specific to one breed. However, Progressive Rod Cone Degeneration (PRCD) is an outlier. PRCD anomaly is associated with PRA in more than 20 breeds. The same gene mutation which causes PRA-prcd in dogs causes a form of Retinitis Pigmentosa in human. X-Linked Progressive Retinal Atrophy (XLPRA); a type of PRA, is a result of deletion in Retinitis Pigmentosa GTPase Regulator (RPGR). RPGR is a locus homologue of human Retinitis pigmentosa (RP3). We analyzed 22 clinically PRA positive dog samples for PRCD and RPGR association with PRA. We employed PCR-RFLP, capillary gel electrophoresis and sequencing. Experiment data suggests that tested mutation of PRCD has no association with PRA in all 15 Pomeranian and 1 Mongrel dogs which are locally bred by Indian breeders. In contrary, all English Cocker spaniel and Labrador Retriever samples showed PRCD association with PRA. Furthermore, accountability of tested mutations in RPGR has been concluded to be nil in all of the test samples.