Scholarly article on topic 'P90'

P90 Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — A. Ponomaryova, E. Rykova, N. Cherdyntseva, E. Morozkin, I. Zaporozhchenko, et al.

Background The analysis of circulating tumor nucleic acids (DNAs and RNAs) in the blood seems to be a promising approach for the development of the low-invasive methods of tumor detection, valuable for clinical practice. Detection of oncogenic and tumor suppressor miRNAs in the blood plasma/serum is evidence of their participation in pathogenesis and suggest the possibility of their use as tumor markers and targets for therapy. Thus, the determination of expression level of tumor-associated miRNAs in plasma can lead to significant progress in understanding the tumor development and treatment. Aim Estimation the changes in expression level of miRNAs (miR-19b, miR-25, miR-125b, miR-126, miR-205) in blood plasma from lung cancer patients during combined therapy and to estimate their value as disease monitoring markers. Materials and methods Blood samples were taken from patients (n=23) with non-small cell lung cancer treated at the Tomsk Cancer Research Institute. These samples were stabilized and fractionated into plasma and blood cells. MicroRNA was isolated from blood plasma using single-phase phenol-free extraction protocol and purified on silica-based spin columns (BioSilica Ltd, Novosibirsk, Russia). Concentration of five above mentioned miRNAs was measured by quantitative RT-PCR and normalized to miR-16 using dCt method. Results In this study we analyzed the dynamic expression changes of circulating DNA in blood plasma from lung cancer patients during the combined therapy. Circulating miRNAs were isolated from plasma samples of non-small cell lung cancer patients before treatment, within 30days after completing chemotherapy and 15days after surgery, by using developed methodological approach. In case of miR-19b and miR-125b analysis was found that the miRNA expression level correlates with clinical response to chemotherapy and surgery. Increasing level of miR-19b and decreasing level of miR-125b were associated with therapeutic response. Using Repeated measures ANOVA analysis we demonstrated that the miR-19b and miR-125b expression levels changes throughout three check-up points during the combined treatment are characterized by the significant cubic trend (P =0.00284 and P =0.029, respectively). Changes of miRNA-126 expression level during post-treatment follow-up were not characterized by the definite trend and not correlated with changes of other miRNAs expression level. It was shown the significant correlation between miRNA-25 and miRNA-205 expression levels, but was not found the trend of these miRNAs level changes. Conclusion The clinical utility of the circulating miR-19b and miR-125b expression analysis from this study remains to be validated in large cohorts of patients with different histological types of tumors, at the different stage of disease and outcomes. One of the criteria for inclusion in the group must be susceptibility and/or resistance to therapy. The research has been carried out with support of the grant from the Russian Science Support Foundation 14-04-01881 , Post-doctorate program in TPU.

Academic research paper on topic "P90"

KRAS is component of Ras/MAPK signaling cascade that regulates cell proliferation and cell survival. Somatic mutations in KRAS gene are often found in tumors and affect the sensitivity of tumors to target therapy. Mutations in codons 12 or 13 of KRAS gene in colorectal cancer (CRC) are associated with resistance to anti-EGFR antibodies Cetuximab and Panitumumab. The objectives of this work were to develop PCR tests for detection mutations in KRAS gene and analyze the frequency of mutations in KRAS gene in CRC in Russia.

DNA sequencing by Sanger is the most common method for mutation analysis. However, the method has a sensitivity of 20% mutant allele, which is often not sufficient for the analysis of somatic mutations in tumors.

One of the most sensitive mutation analysis methods is allele-specific real-time PCR. This method allows to detect 1% of mutated DNA in the sample. This sensitivity is sufficient for analysis of mutations in tumor samples containing 2-5% or more of tumor cells in normal tissue.

In this study we developed and compared 3 new KRAS assays (1) real-time PCR with allele-specific primers; (2) real-time wildtype blocking PCR with LNA (locked nucleic acid) blocker; and (3) Sanger sequencing with LNA-blocker. First assay is a PCR test with seven reactions using allele-specific primers for detection and genotyping 7 mutations in 12 and 13 codons of KRAS gene. Second assay is real-time PCR with only a single pair of primers and LNA oligonucleotide blocker. LNA-blocker is an oligonu-cleotide which has a wild-type sequence of codons 12 and 13 of KRAS gene. LNA-blocker binds strongly to wild-type KRAS DNA and suppresses its amplification, but does not block amplification of mutant DNA. The real-time PCR with LNA -blocker can detect mutant DNA but does not genotype mutation. Such assay can be used as a simple and sensitive screening test for mutant KRAS cases if exact genotyping of mutation is not required. We also used LNA-blocker to increase sensitivity of Sanger sequencing. To evaluate sensitivity and specificity of new tests DNA standards were prepared with different ratios of normal and mutant alleles using normal human DNA without mutation and recombinant plasmids with mutations in KRAS (G12C, G12S, G12R, G12V, G12D, G12A, G13D). After optimization all three assays had sensitivity 5% of mutant alleles for the detection of mutations in KRAS gene, using 2.5-40 ng of human DNA.

Performance of new assays for KRAS mutations was compared using 81 colorectal tumor samples. Before analysis relative content of tumor cells in the samples was evaluated by pathologist. If tumor content was less than 20% in the sample then regions with a maximum number of tumor cells were manually macrodissected before the DNA extraction. DNA was purified from formalin fixed paraffin embedded (FFPE) tissue using ''FFPE-DNA Kit" (Biolink). All three assays had high sensitivity (95-100%) and specificity (100%) for detection of KRAS mutations in clinical tumor samples. Mutations of the KRAS gene were found in 37 of 81 cases (46%) of CRC including 12 cases of mutation G13D, 11 cases of G12D, 5 cases of mutation G12V, 4 cases G12C, 3 cases of mutation G12A, 2 cases G12S, 1 case G13R. A single case with mutation G13R was missed by allele-specific PCR but was detected by real-time PCR with LNA-blocker and confirmed by sequencing.

New assays have high sensitivity and specificity and are suitable for detection of KRAS mutations in clinical FFPE tumor samples.

http://dx.doi.org/10.1016/j.ejcsup.2015.08.076

Dynamic changes of circulating microRNA expression in response to the lung cancer combined therapy

A. Ponomaryovaa,c'*, E. Rykovab, N. Cherdyntsevaa,d, E. Morozkinb, I. Zaporozhchenkob, T. Skvortsovab, A. Dobrodeeva, A. Zav'yaloV, S. TuzikoV, V. Vlassovb, P. Laktionovb. a Tomsk Cancer Research Institute, Tomsk, Russian Federation, b Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russian Federation, c National Research Tomsk Polytechnic University, Tomsk, Russian Federation, d National Research Tomsk State University, Tomsk, Russian Federation * Corresponding author.

Background: The analysis of circulating tumor nucleic acids (DNAs and RNAs) in the blood seems to be a promising approach for the development of the low-invasive methods of tumor detection, valuable for clinical practice. Detection of oncogenic and tumor suppressor miRNAs in the blood plasma/serum is evidence of their participation in pathogenesis and suggest the possibility of their use as tumor markers and targets for therapy. Thus, the determination of expression level of tumor-associated miRNAs in plasma can lead to significant progress in understanding the tumor development and treatment.

Aim: Estimation the changes in expression level of miRNAs (miR-19b, miR-25, miR-125b, miR-126, miR-205) in blood plasma from lung cancer patients during combined therapy and to estimate their value as disease monitoring markers. Materials and methods: Blood samples were taken from patients (n = 23) with non-small cell lung cancer treated at the Tomsk Cancer Research Institute. These samples were stabilized and fractionated into plasma and blood cells. MicroRNA was isolated from blood plasma using single-phase phenol-free extraction protocol and purified on silica-based spin columns (BioSilica Ltd, Novosibirsk, Russia). Concentration of five above mentioned miRNAs was measured by quantitative RT-PCR and normalized to miR-16 using dCt method.

Results: In this study we analyzed the dynamic expression changes of circulating DNA in blood plasma from lung cancer patients during the combined therapy. Circulating miRNAs were isolated from plasma samples of non-small cell lung cancer patients before treatment, within 30 days after completing chemotherapy and 15 days after surgery, by using developed methodological approach. In case of miR-19b and miR-125b analysis was found that the miRNA expression level correlates with clinical response to chemotherapy and surgery. Increasing level of miR-19b and decreasing level of miR-125b were associated with therapeutic response. Using Repeated measures ANOVA analysis we demonstrated that the miR-19b and miR-125b expression levels changes throughout three check-up points during the

combined treatment are characterized by the significant cubic trend (P = 0.00284 and P = 0.029, respectively). Changes of miRNA-126 expression level during post-treatment follow-up were not characterized by the definite trend and not correlated with changes of other miRNAs expression level. It was shown the significant correlation between miRNA-25 and miRNA-205 expression levels, but was not found the trend of these miRNAs level changes.

Conclusion: The clinical utility of the circulating miR-19b and miR-125b expression analysis from this study remains to be validated in large cohorts of patients with different histological types of tumors, at the different stage of disease and outcomes. One of the criteria for inclusion in the group must be susceptibility and/or resistance to therapy.

The research has been carried out with support of the grant from the Russian Science Support Foundation 14-04-01881, Post-doctorate program in TPU.

http://dx.doi.org/10.1016/j.ejcsup.2015.08.077

Tumour secreted factors cathepsins D and L induce pro-angiogenic changes in human omental microvascular endothelial cells (HOMECs) in ovarian cancer metastasis

Z. Pranjola'*, N. Gutowskia, M. Hannemannb, J. Whatmorea. a University of Exeter Medical School, Exeter, UK, b Royal Devon & Exeter NHS Foundation Trust, Exeter, UK * Corresponding author.

Background: Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of HOMECs by tumour -secreted factors in their microenvironment. We have previously shown that ovarian cancer cells secrete a range of factors with possible roles in metastatic angio-genesis including the lysosomal proteases cathepsin D (CD) and cathepsin L (CL). However, the role of these proteases in ovarian cancer metastasis to the omentum is not fully understood. Aim: To investigate whether proliferative effects of CD and CL are dependent on their catalytic activity in HOMECs.

To investigate the intracellular signalling kinases activated by CD and CL.

Method: HOMEC proliferation was assessed by using a colori-metric (WST1) assay. Potential signalling pathways were examined by phosphokinase array and ELISAs. pH experiments were carried out examine whether the observed effects were due to the catalytic activity of CD and CL.

Result: CD and CL (50 ng/ml) significantly increased HOMEC proliferation to 141 ± 27% (p = 0.001, n = 50) and 151% ± 34% (p = 0.001, n = 45) respectively vs. control (100%) 72 h post-treatment. Inhibitors of CD and CL enzyme activity had no effect on HOMEC proliferation and subsequent pH data suggest a non-proteolytic mitogenic activity of these cathepsins. Both proteins induced phosphorylation of ERK1/2, AKT and p38a to ~2, ~1.5 and ~1.5 folds respectively relative to total levels (compared to control).

Conclusion: CD and CL induced proliferation in HOMECs. CD and CL may non- proteolytically contribute to pro-angiogenic

responses of the omental microvasculature. Induction of phos-phorylation of proliferative kinases ERK1/2, AKT and p38a suggest possible downstream signalling cascades of these proteins.

http://dx.doi.org/10.1016/j.ejcsup.2015.08.078

Expression analysis 20 miRNAs in the clear cell renal cell carcinomas and surrounding tissues

I. Proninaa'*, V. Loginova,b, T. Kazubskayac, A. Karpukhina, E. Bragaa,b. a FSBSI Research Center of Medical Genetics, Moscow, Russian Federation, b FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation, c Blokhin Russian Cancer Research Center, Moscow, Russian Federation

* Corresponding author.

RCC is one of the main problems in oncourology. MiRNA expression profiles are highly specific for malignant tumors of different locations, and can be used to detect pathological molecular biomarkers and to develop the optimal treatment for each patient. Selection of miRNAs associated with the formation and development of malignant tumors in the kidney was performed using computer databases miRWalk (http://www.ma.uni-hei-del-berg.de/apps/zmf/mirwalk/) and miRBase (http://www.mir-base. org/). Paired samples of tumor and histologically normal tissue from 46 patients with RCC were studied. RNA was isolated by phenol-chloroform extraction and treated with RNase-free DNase after isolation. MiRNA expression analysis was performed by RT-qPCR using Applied Biosystems (USA) kits: TaqMan® Micro-RNA Assays, TaqMan® MicroRNA Reverse Transcription Kit, TaqMan® Fast Universal PCR Master Mix (2x). RNU6B was used as reference miRNA. Quantity alterations of 20 microRNAs (hsa-miR-219, -203, -148a, -129, -9, -34a, -34b, -34c-3p, -127, -193a-5p, -191, -17, -24 -2*, -339 -3p, -212, -375, -125b, -124a, -132, -137) were determined in samples of tumor compared to normal tissue biopsy from the kidney of each patient. It was shown that the expression of studied miRNAs usually decreased in RCC tumors. The largest decrease of expression was observed for miR-129, which expression was10-350-fold reduced in 93% of the samples (P < 0.05) with no change of expression in the remaining 7% of cases. MiR-375 (80% of cases,10-270-fold decrease), miR-34b (79% of cases, 10-60-fold decrease), miR-124a (70% of cases, 10200-fold decrease) also frequently showed reduced expression in tumors. In addition to the aforementioned ones, it had been shown that expression in the tumor compared with histologically normal tissue from the same patient was significantly decreased in more than half of the cases for miR-125b, miR-127, miR-203, miR-34c-3p, miR-9. Despite the fact that in 50% of cases decreased expression of miR-9 was detected, it was the only of studied microRNA with a significant increase in the expression in 21% of cases in 10-160 times (P < 0.05). Associations between the expression of investigated miRNAs and gender and age were not noted. Dealing with each miRNA alone, we could not detect the dependence of expression alteration of 20 selected miRNAs from the stage or degree of tumor differentiation of the studied RCC samples. However, the analysis of the expression profile of