Molecular Biomarkers & Diagnosis

We have simultaneously determined the presence of 30 different trace metals in human seminal plasma by Inductively Coupled Plasma Mass Spectrometry (ICP−MS). To date, high concentrations of metals (e.g. Zn, Cu, Mg and Fe) in seminal plasma have been sporadically reported and only few reports have discussed the relationships between trace metals and human semen profiles. Ninety-four seminal plasma specimens were first classified in three groups (normozoospermia, oligozoospermia, and azoospermia) according to the WHO guideline for sperm concentration. No significant differences were found among the three groups for all the metals examined. The concentration of metals in the specimens were then compared with sperm concentration, motility, morphology, and volume using multivariate regression analysis, principal component analysis (PCA) and cluster analysis. The most cumulative contribution ratio and grouping were extracted for profile analysis. Zn showed a positive correlation with motility and morphology (r = 0.3142 and 0.6205), whereas trace metals such as Se and Tl showed positive correlations with all sperm properties. Heavy metals such as Cu, Cd, and Cr also showed high positive or negative correlations with sperm properties. In summary, this study is the first to report the simultaneous determination of metals (in addition to the commonly reported Zn), including trace and heavy metals, in human seminal plasma as analyzed by multivariate regression analysis

Pancreatic cancer is one of the most aggressive human malignancies and ninth leading cause of cancer death in the world. Most patients diagnosed with pancreatic cancer die within 6 months, and only 4% survive 5 years after diagnosis. Approximately one-fifth of patients with presumably ‘curable’ Pancreatic Ductal Adenocarcinoma (PDA) experience impending relapse and death, making surgical removal almost futile. Early diagnostic, prognostic and predictive biomarkers and better therapeutic options which could help personalize treatment regimens are desperately needed to improve the survival rate of pancreatic cancer patients. By employing BxPC-3 cell line model, high throughput comparative, quantitative and system proteomic analysis, we have been able to identify annexin-a4, annexin-a5 annexin a-11, caveolin-1, EGFR, H-RAS, Integrin-α6β4 proteins which were significantly upregulated in oxidatively stressed BxPC-3 cells. The present investigations have shown the presence of quite robust oxidative response in BxPC-3 cells as compared to HPDE control. The high throughput proteomic and bioinformatic analysis have shown the aberrantly regulated Cav-1-Fyn-SOS-cRAF-ERK (where Cav-1 is caveolin-1) signaling pathway, reveals that oxidative stress might activate Cav-1 protein in the membrane which in turn activate the downstream proto-oncogene tyrosine-kinase Fyn and triggers MAPK/ERK pathway. The data suggest that the above stated proteins could be used as diagnostic and prognostic biomarkers and using Cav-1-Fyn-SOS-cRAF-ERK pathway better therapeutic options could be explored.

Selenium (Se) is a trace element, essential for human health but it can be toxic at moderately higher intake levels. In this study biomarkers of Se toxicity after sub-acute intra-peritoneal exposure to Se (62.5 µg Se/kg bw/ day; 14 days) were investigated in mice. Such exposure corresponds to high human Se-intake levels. Focus was put on the biomarkers of systemic effects and on the toxicity in liver, kidney and brain. The sub-acute exposure to Se resulted in an increase in the concentrations of systemic inflammation biomarkers IL-6 (p=0.025) and resistin (p=0.049) and in a decrease of TNF-α (p=0.008). No effect on concentrations of MCP-1, tPAI-1, leptin and insulin was observed in serum. Also biomarkers of oxidative stress, anti-oxidant parameters and enzymes ALT and AST were not affected. In the tissue homogenates of liver, kidney and brain changes were observed in the activities of enzymes LDH (p=0.013), ALP (p=0.0009) and GGT (p=0.0047). In the brain homogenate an influx of TG (p=0.0044) and a decrease in the total GSH concentration (p=0.008) was observed. It was concluded that although the sub-acute exposure to a relatively high concentration of Se causes an increase in concentration of some biomarkers of intracellular processes, especially in the brain, the effect of Se can be considered as low toxicity.

Over-expression of the HER2/neu receptor occurs in 20 to 30 percent of breast tumors and is linked to poorer prognosis. The HER2/neu expression status determines whether or not patient will receive trastuzumab- based treatment. In clinical practice, over-expression of HER2/neu is routinely identified using Immunohistochemistry (IHC) or Fluorescence in Situ Hybridization (FISH), both of which are invasive approaches requiring tissue samples. Serum assays for the Extra Cellular Domain of HER2/neu receptor (HER2 ECD) have been reported but the use is very limited due to serum interference factors (e.g. human anti-animal immunoglobulin antibodies) that lead to false test results and inconsistency with tissue Her2 status. We have developed an ELISA based approach using an MBB buffer to eliminate false results and to obtain more accurate assessment of HER2 ECD levels. Using this refined assay we retroactively measured HER2/neu levels from breast cancer patients and controls. Abnormal HER2 ECD levels were detected in about 32% of invasive breast cancer patients but not in controls or patients with benign diseases. In addition, we also showed that patients with elevated serum HER2 levels appeared to have worse survival regardless of treatments. In a small group of 12 Ductal Carcinoma in situ (DCIS) patients who received HER2/neu peptide vaccination and surgery, only one patient showed constantly rising HER2 levels after treatment and this patient had recurrence of HER2 positive tumor within 5 years. Our studies indicate that once the serum interference issue is resolved, serum HER2 ECD can have potential clinical utility to supplement the tissue based tests.

Background: Immunological abnormalities have been identified in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients fulfilling the 1994 Centers for Disease Control diagnostic criteria. Significant developments have been made to diagnostic criteria, but potential immunological markers have not been assessed in patients fulfilling these latest clinical requirements. Therefore, this study evaluated immunological parameters in patients that also fulfill the latest diagnostic criteria available known as the International Consensus Criteria.

Methods: The Immunological investigations including Natural Killer cell activity and phenotyping studies for dendritic cells, neutrophils, B cells and regulatory T cells were performed on whole blood samples collected from all participants using flow cytometric protocols. The physical functioning of all participants was also evaluated using scores from the Short Form Health Survey, and the World Health Organization Disability Adjustment Schedule. Results were compared according 1994 Centers of Disease Control and Prevention defined patients, and International Consensus Criteria defined patients, and healthy controls.

Results: Natural killer cell activity was consistently and significantly decreased, and regulatory T cells were significantly increased in both patient groups compared to healthy controls. Differences were found in human neutraphil antigens and expression of natural killer cell receptors between patient groups. Highly significant correlations were also found between physical status and some immune parameters in International Consensus Criteria defined patients.

Conclusion: This preliminary investigation on different diagnostic criteria suggests that the International Consensus Criteria may be more effective a detecting salient differences in the immune system.

Background: Patients with advanced metastatic cancer often have elevated levels of circulating transforming growth factor beta (TGF-β) that is thought to stimulate receptor mediated signaling through phosphorylated SMAD transcription factors in peripheral blood mononuclear cells.

Methods: To identify this TGF-β-dependent gene expression profile in cancer patients, we first evaluated a multi-gene expression profile from ex-vivo treated PBMCs with TGF-β1 stimulation. Change in expression, when challenged with a specific TGF-β receptor type kinase inhibitor was then derived to be ligand and inhibitor specific. Once this profile was established, we examined its role in identifying the activation of a canonical TGF-β1 signaling pathway in patient samples.

Results: We discovered a 37 gene sub-set where the expression profile from ex-vivo treated PBMCs was significantly associated with SMAD phosphorylation in cancer patients. We found significant correlation between the ex-vivo derived expression signature and circulating levels of TGF-β1 in patient samples. Additionally, we report association between the expression profile and the presence of several plasma proteins in disease samples that are known to be concomitantly present with TGF-β- dependent pathway activation.

Conclusion: An expression profile for TGF-β1 cytokine mediated-signaling in cancer patients is identified, which may serve as a biomarker to measure the pharmacodynamic effect of TGF-β inhibitors during clinical drug development and as marker of disease diagnosis.

Toxicity of nanoparticles on the human health is one main feature for successful application of nanoparticles in medicine. In this paper, the cytotoxicity effects of CZF-MNPs and CZF-MNPs @ DMSA were evaluated. For this purpose, at first the characterization of Cobalt-Zinc Ferrite Magnetic Nanoparticles (CZF-MNPs) coated with biocompatible Dimercaptosuccinic Acid (DMSA) was investigated using of Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR) and Atomic Absorption Spectrophotometer (AAS). In following, the cytotoxicity effects of CZF-MNPs and CZF-MNPs @ DMSA were investigated on human prostate cancer cell lines, HPCs, (PC3 and DU145). The results showed that the average size of bare and coated nanoparticles was about 16 and 40 nm. The FTIR spectra results showed the presence of DMSA cover on the surface of nanoparticles. Furthermore, in vitro MTT assay CZF-MNPs @ DMSA at high concentrations (1.2 and 1.5 mM Fe) study results showed that they have some cytotoxicity on HPCs (PC3 and DU145).

Background: Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes. All of them differ from each other resulting in a wide array of clinical pictures, risks of recurrence and response to treatment. In recent years, tumor biomarkers have changed the way breast cancers are diagnosed and treated. In this paper we have sought to investigate the differential expression of ANXA1, a multifunctional calcium binding protein, among various molecular subtypes of breast cancers and in particular triple negative tumors.

Methods: ANXA1 was first studied using in-silico analysis on available DNA microarray and RNA sequencing data of human breast tissues. Next we ascertained ANXA1 expression on cell lines and breast carcinoma tissue microarrays along with cognate normal breast tissue.

Results: Whereas ANXA1 expression is normally restricted to the normal myoepithelial cell layer it becomes ectopically and aberrantly expressed in tumor cells of a significant minority of aggressive breast cancers. Specifically, we found that ANXA1 expression is severely deregulated in high-grade breast cancers that comprise clinically aggressive tumors such as triple-negative and, to some extent, HER2-positive breast cancers.

Conclusion: Our results indicate that ANXA1 is a valuable breast cancer biomarker that can help to segregate and dissect out subsets of high histological grade breast cancers paving the way to a better understanding of breast cancer progression and metastasis.