Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

Background

Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.

Materials and methods

We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.

Results

We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).

Conclusion

The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.     

Serum prolidase activity and oxidative-antioxidative status in Legg-Calve-Perthes disease

We aimed to find out that whether collagen turnover is altered in the context of Legg-Calve-Perthes disease (LCPD) by evaluating serum prolidase activity. We also investigated the correlation between collagen turnover and oxidative-antioxidative status in LCPD. Plasma prolidase activity, total oxidant status (TOS), total antioxidant capacity, and oxidative stress index (OSI) were determined for 39 patients with LCPD and 40 healthy controls. Serum prolidase activity, TOS, and OSI were higher, but TAC was lower in patients with LCPD compared with controls. Prolidase activity was positively correlated with TOS and OSI levels. Serum prolidase activity is significantly associated with LCPD.     

Study of flow-induced hemolysis using novel Couette-type blood-shearing devices

To assist the development and application of blood-contacting medical devices, two novel flow-through Couette-type blood-shearing devices have been developed to study the quantitative relationship between blood damage indexes and flow-dependent parameters. One device is an axial flow-through Couette-type device supported by a pair of pin bearings adapted from the adult Jarvik 2000 blood pump. The other is a centrifugal flow-through Couette-type device supported with magnetic bearings adapted from the CentriMag blood pump. In both devices, a rotor spindle was used to replace the original impeller blades so that a small gap was created between the housing and the rotating spindle surface. Computational fluid dynamics simulations have shown that a uniform, high shear stress region can be generated inside the small gap while the shear stresses elsewhere are relatively low. The possibility of secondary blood damage caused by mechanical seals was eliminated due to the use of a magnetic rotor system. Blood flow through the gap was driven by an externally pressurized reservoir. By adjusting the rotational speed and blood flow rate, shear-induced hemolysis was quantified at a matrix of exposure time (0.039 to 1.48 s) and shear stress (50 to 320 Pa). All of the experiments were conducted at room temperature using heparinized ovine blood with a hematocrit value of 30%. The measured hemolysis levels were much lower than those published in the literature, and the overestimation of those earlier studies may be attributable to device-related secondary blood-damaging effects. A new set of coefficients for the power law model was derived from the regression of the experimental data.     

Serum brain-derived neurotrophic factor levels in conversion disorder: Comparative study with depression

The aim of the present study was to compare serum brain-derived neurotrophic factor (BDNF) levels of patients with major depressive disorder (MDD) and conversion disorder (CD). Serum BDNF levels were measured in the following three groups: 15 CD patients without any comorbid diagnosis of psychiatric disorder, 24 patients with MDD, and 26 healthy subjects without any psychiatric diagnosis or psychiatric treatment. The serum BDNF level of the healthy control group (31.4 +/- 8.8 ng/mL) was statistically higher than the level of the MDD group (21.2 +/- 11.3 ng/mL) and the CD group (24.3 +/- 9.0 ng/mL; P = 0.008). This suggests that BDNF level may play a similar role in the pathophysiology of MDD and CD.     

Inhibitory impact of subliminal electrical finger stimulation on SI representation and perceptual sensitivity of an adjacent finger

Simultaneous stimulation of two adjacent fingers above sensory perception threshold (supraliminal stimulation) leads to an inhibitory interaction effect on responses in primary somatosensory cortex (SI). Moreover, during electrical finger stimulation closely below threshold for conscious perception (subliminal stimulation) inhibitory interneurons in cortical layer 4 are assumed to be activated preferentially as compared to excitatory interneurons. Using fMRI in humans, here we show that interspersed subliminal electrical stimulation of an adjacent finger reduces the response to target finger stimulation in contralateral SI. This effect was shown in a complementary study to be associated behaviorally with a diminished detectability of test pulses on the target finger. We propose the mechanism underlying this lateral inhibitory effect to be related to a representational overlap of inhibitory interneurons in SI based on the divergence of thalamocortical feedforward projections, or to intracortical lateral inhibitory projections targeting juxtaposed receptive fields, or both.     

The use of computational fluid dynamics in the development of ventricular assist devices

Progress in the field of prosthetic cardiovascular devices has significantly contributed to the rapid advancements in cardiac therapy during the last four decades. The concept of mechanical circulatory assistance was established with the first successful clinical use of heart-lung machines for cardiopulmonary bypass. Since then a variety of devices have been developed to replace or assist diseased components of the cardiovascular system. Ventricular assist devices (VADs) are basically mechanical pumps designed to augment or replace the function of one or more chambers of the failing heart. Computational Fluid Dynamics (CFD) is an attractive tool in the development process of VADs, allowing numerous different designs to be characterized for their functional performance virtually, for a wide range of operating conditions, without the physical device being fabricated. However, VADs operate in a flow regime which is traditionally difficult to simulate; the transitional region at the boundary of laminar and turbulent flow. Hence different methods have been used and the best approach is debatable. In addition to these fundamental fluid dynamic issues, blood consists of biological cells. Device-induced biological complications are a serious consequence of VAD use. The complications include blood damage (haemolysis, blood cell activation), thrombosis and emboli. Patients are required to take anticoagulation medication constantly which may cause bleeding. Despite many efforts blood damage models have still not been implemented satisfactorily into numerical analysis of VADs, which severely undermines the full potential of CFD. This paper reviews the current state of the art CFD for analysis of blood pumps, including a practical critical review of the studies to date, which should help device designers choose the most appropriate methods; a summary of blood damage models and the difficulties in implementing them into CFD; and current gaps in knowledge and areas for future work.