Serum circulating cell free DNA as potential diagnostic and prognostic biomarker in non small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is leading cause of cancer related death and the survival rate for patients with NSCLC remain poor so early diagnosis of NSCLC represents the best opportunity for cure. Cell-free DNA (cf-DNA) is extracellular nucleic acids found in cell-free plasma/serum of humans, given the recent approval of a liquid biopsy in lung cancer, the use of circulating tumor DNA as a novel non-invasive diagnostic and prognostic biomarker is promising.ObjectivesStudying whether the concentrations of circulating Cell Free DNA in serum can be used as a diagnostic and prognostic biomarker for NSCLC patients.MethodThis study was carried out on 140 subjects included 60 patients with non small cell lung cancer,40 patients with Chronic Obstructive Pulmonary Disease (COPD) and 40 healthy controls. Quantitative analysis of serum circulating cf-DNA was done b y AlU-based quantitative real time PCR. Serum level of CEA was measured by ELISA.ResultsNSCLC patients demonstrated significantly higher values of each of ALU 215, ALU 247, and DNA integrity than both COPD patients and controls. On ROC curve analysis, the total accuracy of ALU 247, ALU 115, DNA integrity (92.1%, 83.6%, 56.4%) at cutoff points (325, 565 & 0.48) respectively. On combining both DNA integrity and CEA, improved sensitivity to 93.3% was noted. For NSCLC patients, ALU 115 & ALU 247 increased significantly with more advanced stage and highest level was noticed in metastatic patients. Regarding survival there was better overall survival among patients with low DNA integrity.ConclusionSerum cf-DNA concentrations and integrity index may be valuable tool in early diagnosis of NSCLC and prediction of prognosis of those patients.     

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.     

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

Expression of Androgen Receptor and Cancer Stem Cell Markers (CD44+/CD24− and ALDH1+): Prognostic Implications in Invasive Breast Cancer

BACKGROUND: Androgen receptor (AR) has emerged as a significant prognostic marker in early breast cancer (BCa). Association of AR with cancer stem cell (CSC) markers in BCa is unknown. Aim of the present study was to evaluate the immunohistochemical expression of AR, CD44, CD24 and ALDH1 in a cohort of Pakistani patients diagnosed with invasive BCa and to correlate the expression with 5- year disease free survival. PATIENTS AND METHODS: We evaluated immunohistochemical expression AR, CD44, CD24 and ALDH1 in formalin fixed paraffin embedded archival blocks of 166 cases of primary invasive BCa (stage I-III) and correlated the expression with clinicopathological variables and outcome using univariable and multivariable analysis. Survival data was computed by Kaplan Meier curves. RESULTS: Expression of AR was observed in 62.7% tumors whereas CD44, CD24 and ALDH1 were expressed in 61.4%, 44% and 30.1% tumors, respectively. AR expression was significantly associated with T1-T2 tumors, lower grade, estrogen and progesterone receptor expression (P < .05) and remained an independent prognostic indicator in multivariable analysis (adjusted HR 0.33, 95% CI 0.13–0.81; P = .016). Significant association was observed between concordant expression of AR and CD24 (P = .001) with a favorable impact on survival (P = .007) whereas expression of CSC phenotypes (CD44⁺, CD44⁺/CD24^? and ALDH1⁺) did not correlate with adverse outcome (P > .05). However, AR expression retained the association with better prognosis even in patients whose tumors exhibited a CSC phenotype. CONCLUSIONS: Expression of AR and CD24 in stage I-III invasive BCa correlates with favorable clinicopathological features and delineates a subgroup of patients with better disease-free survival.     

Molecular mechanisms by which aerobic exercise induces insulin sensitivity

Insulin resistance is a key feature of Type 2 diabetes and an important therapeutic target to address glycemic control to prevent diabetic complications. Lifestyle advice is the first step in the ADA/EASD consensus guidelines followed by metformin therapy. Aerobic exercise (AE) can increase insulin sensitivity by several molecular pathways including upregulation of insulin transporters in the cellular membrane of insulin-dependent cells. In addition, AE improves insulin sensitivity by amelioration of the pathophysiologic pathways involved in insulin resistance such as the reduction of adipokines, inflammatory and oxidative stress responses, and improvement of insulin signal transduction via different molecular pathways. This review details the molecular pathways by which AE induces beneficial effects on insulin resistance