BACKGROUND:Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined.METHODS:We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19. We also compared the therascreen amplification refractory mutation system assay with a droplet digital polymerase chain reaction assay for the detection of all the deletions in our study.RESULTS:The droplet digital polymerase chain reaction assay demonstrated 100% sensitivity against polymerase chain reaction fragment length analysis and detected all possible types of deletions revealed in our study (22 types). At the same time, the therascreen EGFR RGQ PCR Kit was not able to detect deletions c.2252-2276>A and c.2253-2276 and showed low performance for another long deletion.CONCLUSION:Thus, we can conclude that the extraordinary length of deletions and their atypical locations (shift at the 3'-region compared to known deletions) could be problematic for the therascreen EGFR RGQ PCR Kit and should be taken into account during targeted mutation test development. However, droplet digital polymerase chain reaction is a promising and reliable assay that can be used as a diagnostic tool to genotype formalin-fixed paraffin-embedded cancer samples for EGFR or another clinically relevant somatic mutation.

One of the hotspots in clinical research today is molecular genetic analysis of structural DNA alterations. Working with DNA obtained from formalin-fixed and paraffin-embedded (FFPE) tissue specimens is particularly challenging, due to cross-linking and fragmentation of DNA. We performed a comparative analysis of DNA extraction methods from FFPE tissue using two in-house protocols and Qiagen (QIAamp DNA FFPE Tissue Kit) and Roche (High Pure FFPET DNA Isolation Kit) commercial kits presented in the Russian market and used in clinical practice. To assess the quantity and quality of the isolated DNA, we used the real-time PCR to rate DNA yield, the inhibited impurity content and the degree of fragmentation. Our findings may be useful for a medical laboratory that performs testing of somatic mutations for the targeted therapy selection, and researchers who specialized in tumor genome structure studies in respect to patient prognosis and prediction of the sensitivity or resistance of tumor cells to therapy.

We studied angiogenic effects of two variants of indirect revascularization (transplantation of mononuclear fraction cells from autologous bone marrow alone or in combination with laser tunneling) after modeled ischemia of the lower limbs in rats. Doppler sonography and immunofl uorescence studies showed that intramuscular implantation of isolated fraction of autologous bone marrow mononuclear cells in combination with laser tunneling of the muscles is most effective and can be recommended as a method of angiogenesis stimulation in nonreconstructable distal vascular pathology of the extremities.

Studies of myocardial autopsy specimens from infants (0-12 months) with transposition of the main vessels showed the formation of a complex of compensatory adaptive, degenerative, and destructive changes, manifesting at a tissue level in cardiomyocyte heterogeneity and formation of cardiosclerosis zones. Cardiac myosin synthesis was replaced by synthesis of skeletal myosin, which was detected at the molecular level. Clinically it manifested in the progress of heart failure. Hyperplastic processes (intensive polyploid transformation of the nuclei) play an important role in heart remodeling in patients aged over 6 months. The findings of immunohistochemical and fluorescent studies seem to be prognostically important and provide more accurate data on the pathological processes in the myocardium at the initial stages of heart disease development starting from birth.

We studied the peculiarities of angiogenesis in the postinfarction period after transmyocardial laser revascularization and intramyocardial implantation of mononuclear bone marrow cells into the pericicatrical zone of the left ventricular myocardium in dogs. Morphological manifestation of angiogenesis in the myocardium after application of laser and cell technologies are angiomatosis, formation of large thin-wall vessels and sinusoids. The angiogenic effect of implanted mononuclear bone marrow cells is determined by high content (43-47%) of CD31+ cells in both adherent and nonadherent fractions. More pronounced angiogenic potential of nonadherent cells is determined by intensive expression of cytokine VEGF-B and D mRNA essential for arterial vessels growth. Immunohistochemical studies showed that about 90% cells of the nonadherent fraction are endothelial precursors expressing endothelial cell markers isolectin B4 and VEGF-R2. It was found that the use of adherent mononuclear bone marrow cells during the postinfarction period induces ossification of the epicardium and subepicardial myocardium layer, formation of cartilage plates, and focal calcification. Implantation of nonadherent mononuclear bone marrow cells into transmyocardial laser channels did not induce ectopic ossification of the myocardium.

We performed a fluorescent microscopic examination of human and animal aortic grafts at different stages of decellularization. Treatment of aortic grafts with trypsin/EDTA solution for 48 h leads to their complete decellularization and preserved the connective tissue fiber backbone, which gains a netlike structure. The use of this protocol of decellularization leads to disappearance of subintimal calcium deposits in human aortic grafts. Differences in laserinduced fluorescence spectra of aortas before decellularization and at different stages of this process were revealed. Our findings suggest that the use of fluorescence induced by excimer lasers is promising for identification of the composition of biological tissues, analysis of their state, and the presence of changes.

Disseminated ossification of the myocardium and severe angiomatosis were detected in sites of implantation of unseparated bone marrow mononuclear fraction cells in dogs with experimental chronic coronary disease. Minor immunocytochemical differences in cells of the mononuclear fraction adhering and not adhering to plastic were found. Significant differences in the expression of mRNA of chondro-osteogenesis genes (aggrecan, lumican, and osteopontin) in adherent and nonadherent cells were detected. The expression of aggrecan gene mRNA was 3-fold lower, of lumican gene 6-fold, and of osteopontin gene 11-fold lower in nonadherent cells compared to adherent fraction.