Search

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

We examined quantitative (in terms of mtDNA/nuclear DNA) and structural (in terms of common deletions in the MT-ND4 gene region) characteristics of mitochondrial DNA (mtDNA) in varicose veins (VVs) and venous wall layers by comparing mitochondrial genome parameters, as well as mitochondrial function (in terms of mitochondrial membrane potential (MtMP)), in varicose vein (VV) vs. non-varicose vein (NV) tissue samples.We analyzed paired great saphenous vein samples (VV vs. NV segments from each patient left after venous surgery) harvested from patients with VVs. Relative mtDNA level and the proportion of no-deletion mtDNA were determined by a multiplex quantitative PCR (qPCR), confirming the latter with a more sensitive method - droplet digital PCR (ddPCR). Mitochondria's functional state in VVs was assessed using fluorescent (dependent on MtMP) live-staining of mitochondria in venous tissues.Total mtDNA level was lower in VV than in NV samples (predominantly in the t. media layer). ddPCR analysis showed lower proportion of no-deletion mtDNA in VVs. Because of the decrease in relative MtMP in VVs, our results suggest a possible reduction of mitochondrial function in VVs.Quantitative and structural changes (copy number and integrity) of mtDNA are plausibly involved in VV pathogenesis. Future clinical studies implementing the mitochondrial targeting may be eventually fostered after auxiliary mechanistic studies.

Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene. In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status. DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1. We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.

EGFR tyrosine-kinase inhibitors (TKIs) are used as targeted therapeutics for the treatment of advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. EGFR C797S is common causes of acquired resistance to third-generation TKIs. There is wide-spread opinion that resistance-conferring mutation present even in a small proportion of cancer cells before the start of therapy could potentially predict poor response to a targeted drug. In our study, we tested whether C797S can be found in previously untreated NSCLCs. We analyzed DNA samples extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue sections of 470 lung adenocarcinoma patients, including 235 samples with activating EGFR mutations. Screening was performed using highly sensitive droplet digital PCR assay. No tumor samples with baseline C797S were identified. C797S does not occur in TKI-naïve NSCLCs and provide evidence that screening for this mutation before TKIs administration may not be necessary.

To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpoB gene of Mycobacterium tuberculosis. 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes (p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpoB gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpoB gene, 10 RIFR isolates containing other mutations in the rpoB gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpoB gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants—urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined. 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. 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. 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.

3559 Background: Recently, conflicting evidence has emerged showing the association of ctDNA level and cancer progression. The aim of our study was the development of a method for detecting ctDNA in plasma and the investigation of the prognostic value of ctDNA retention after surgery in the prospective way. Methods: This prospective, single-center, sample collection study; pts with early-stage malignancies of the different origin were included. Tumor somatic mutations were determined by target sequencing of DNA from FFPE tumor blocks. Sequencing was performed using the custom NGS panel covering regions of frequent somatic mutations in 50 genes. Tumor-specific mutations were monitored in plasma samples taken before and after surgery. The median time between surgery and plasma collection was 7 days (5-15). Mutations of plasma ctDNA were determined by ddPCR. The plasma sample was considered "positive" if the content of ctDNA was more than 0.5 copies of mutant DNA in ml plasma. We needed 265 pts for improving 1-year disease free survival (DFS) from 60% to 80% with α=0.01, β=0.1, 10% loss of f.-up and duration of the study for 2 years. Results: The study comprised 271 pts with various cancers including colorectal – 91 (33,6%), pancreatic – 37 (13,7%), breast – 66 (24,4%), lung – 35 (12,9%) and gastric cancer – 42 (15,5%). Pts with stage I was 50 (18,5%), stage II – 118 (43,5%) and stage III – 103 (38%). The median time of the f.-up was 9 mos. (1-37). No significant association was found between the level of ctDNA before surgery and DFS either in the general group or in groups stratified by tumor sites (HR 2.4, 95%CI 0.8-7.1, р=0.12 and HR 1.5, 95%CI 0.4-6.3, р=0.5, correspondingly). ctDNA was detected in the plasma after surgery in 57 (10%) pts: 9 (9.9%) cases of colorectal, 10 (27%) - pancreatic, 9 (13.6%) - breast, 19 (54.3%) - lung, and 10 (23.8%) - gastric cancer. Progression of the disease was detected in 28/57 (49%) pts with ctDNA(+) and 17/214 (8%) - in ctDNA(-) pts (p

MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection. Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay. NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30 kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-70 °C, and both were inactivated via heating at 95 °C for 15 min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.