Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.049
S. Kovalenko , G. Paul , N. Matyash , A. Kozyakov
Mutations in BRCA1 and CHEK2 genes associated with hereditary breast cancer were tested in 7920 randomly selected individuals of Novosibirsk (Russia). Mutations BRCA1 5382insC and CHEK2 1100delC were the most frequent, they were found in 0.25% and 0.4% of the general population respectively. We suggested to find mutations carriers by the screening of all breast/ovary cancer patients for the most frequent mutations (BRCA1 5382insC and CHEK2 1100delC) with subsequent analysis of the first-line relatives of cancer patients if one of the mutations was found.
From June 2013 till January 2015, all patients from Novosibirsk regional oncology hospital with the diagnosis of breast cancer and some patients with the diagnosis of ovary cancer were tested for mutations BRCA1 5382insC and CHEK2 1100delC. A total of 2655 cancer patients were analyzed independently of their family history. We found 122 mutations carriers, among them 99 patients with mutations in BRCA1 gene and 23 patients with mutation CHEK2 1100delC. Among mutation carriers, 105 patients agreed to have a medical genetic counseling and after pedigree analysis 193 first-line relatives aged above 25 years were elucidated. One hundred ten first-line relatives of mutation carriers were analyzed for the mutations presence and 40 mutations carriers were found among relatives.
From September 2013 till December 2013, 32 relatives of BRCA mutation carriers underwent breast MRI. In 5 cases, breast cancer was detected by MRI and all cancers except one were confirmed histologically with biopsy analysis. Importantly, all tumors were 5 mm and less in size, stage I cancer was detected in all cases.
At a follow-up of 1.5 years, all 105 mutation carrier probands were interviewed by phone regarding possible relapse and/or possible primary cancer in their relatives. Five of 105 probands lost to follow-up may have died. Among responding 100 patients, 2 died as reported by relatives, relapse was reported in 7 probands – mutation carrier probands, primary tumors were reported in 8 relatives of probands.
Mutation carrier probands reported one bilateral breast cancer, four ovary cancers, one bladder cancer and one non-specified oncogynecological tumor.
There were five cases of primary breast cancer, one ovary cancer, one colon cancer, one lung cancer among relatives of breast cancer patients with mutations. The frequency of tumors found in mutation carriers exceeded the average frequencies of cancer for this population.
The economic value of the regional genetic screening can be easily estimated according to the data obtained in this study and data on treatment cost for stage I and stage IV breast cancer. To summarize briefly, the screening of hot-spot mutations provides not only increase of lifespan expectancy and life quality for mutation carriers, but can be also a tool for financial saving of medical system due to the increase of early stage breast can
{"title":"P107","authors":"S. Kovalenko , G. Paul , N. Matyash , A. Kozyakov","doi":"10.1016/j.ejcsup.2015.08.049","DOIUrl":"10.1016/j.ejcsup.2015.08.049","url":null,"abstract":"<div><p>Mutations in BRCA1 and CHEK2 genes associated with hereditary breast cancer were tested in 7920 randomly selected individuals of Novosibirsk (Russia). Mutations BRCA1 5382insC and CHEK2 1100delC were the most frequent, they were found in 0.25% and 0.4% of the general population respectively. We suggested to find mutations carriers by the screening of all breast/ovary cancer patients for the most frequent mutations (BRCA1 5382insC and CHEK2 1100delC) with subsequent analysis of the first-line relatives of cancer patients if one of the mutations was found.</p><p>From June 2013 till January 2015, all patients from Novosibirsk regional oncology hospital with the diagnosis of breast cancer and some patients with the diagnosis of ovary cancer were tested for mutations BRCA1 5382insC and CHEK2 1100delC. A total of 2655 cancer patients were analyzed independently of their family history. We found 122 mutations carriers, among them 99 patients with mutations in BRCA1 gene and 23 patients with mutation CHEK2 1100delC. Among mutation carriers, 105 patients agreed to have a medical genetic counseling and after pedigree analysis 193 first-line relatives aged above 25 years were elucidated. One hundred ten first-line relatives of mutation carriers were analyzed for the mutations presence and 40 mutations carriers were found among relatives.</p><p>From September 2013 till December 2013, 32 relatives of BRCA mutation carriers underwent breast MRI. In 5 cases, breast cancer was detected by MRI and all cancers except one were confirmed histologically with biopsy analysis. Importantly, all tumors were 5<!--> <!-->mm and less in size, stage I cancer was detected in all cases.</p><p>At a follow-up of 1.5<!--> <!-->years, all 105 mutation carrier probands were interviewed by phone regarding possible relapse and/or possible primary cancer in their relatives. Five of 105 probands lost to follow-up may have died. Among responding 100 patients, 2 died as reported by relatives, relapse was reported in 7 probands – mutation carrier probands, primary tumors were reported in 8 relatives of probands.</p><p>Mutation carrier probands reported one bilateral breast cancer, four ovary cancers, one bladder cancer and one non-specified oncogynecological tumor.</p><p>There were five cases of primary breast cancer, one ovary cancer, one colon cancer, one lung cancer among relatives of breast cancer patients with mutations. The frequency of tumors found in mutation carriers exceeded the average frequencies of cancer for this population.</p><p>The economic value of the regional genetic screening can be easily estimated according to the data obtained in this study and data on treatment cost for stage I and stage IV breast cancer. To summarize briefly, the screening of hot-spot mutations provides not only increase of lifespan expectancy and life quality for mutation carriers, but can be also a tool for financial saving of medical system due to the increase of early stage breast can","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Page 28"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.049","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.050
A. Kozlov
The hypothesis of the possible evolutionary role of tumors suggests that hereditary tumors may supply evolving multicellular organisms with extra cell masses for the expression of newly evolving genes (Kozlov, 2014). After expression of novel genes in tumor cells, tumors may differentiate in new directions and give rise to new cell types, tissues and organs.
In the presentation, the bulk of data supporting the positive evolutionary role of tumors will be reviewed, obtained both in the lab of the author and from the literature sources.
The following issues will be addressed: the widespread occurrence of tumors in multicellular organisms; features of tumors that could be used in evolution; the relationship of tumors to evo-devo; examples of recapitulation of some tumor features in recently evolved organs; the types of tumors that might play the role in evolution; examples of tumors that already have played the role in evolution.
The discussion of experimental confirmation of nontrivial predictions of the hypothesis will include the analysis of evolutionary novelty of tumor-specifically expressed EST sequences; ELFNI – AS1, a human gene with possible microRNA function expressed predominantly in tumors and originated in primates; PBOV1, a human gene of the recent de novo origin with predicted highly tumor-specific expression profile; and the evolutionary novelty of human cancer/testis antigen genes; the data obtained on transgenic fish tumors regression model; and other data.
It can be concluded that expression of protogenes, evolutionarily young and/or novel genes in tumors might be a new biological phenomenon, a phenomenon of carcino-evo-devo genes, predicted by the hypothesis of evolution by tumor neofunctionalization.
{"title":"A118","authors":"A. Kozlov","doi":"10.1016/j.ejcsup.2015.08.050","DOIUrl":"10.1016/j.ejcsup.2015.08.050","url":null,"abstract":"<div><p>The hypothesis of the possible evolutionary role of tumors suggests that hereditary tumors may supply evolving multicellular organisms with extra cell masses for the expression of newly evolving genes (Kozlov, 2014). After expression of novel genes in tumor cells, tumors may differentiate in new directions and give rise to new cell types, tissues and organs.</p><p>In the presentation, the bulk of data supporting the positive evolutionary role of tumors will be reviewed, obtained both in the lab of the author and from the literature sources.</p><p>The following issues will be addressed: the widespread occurrence of tumors in multicellular organisms; features of tumors that could be used in evolution; the relationship of tumors to evo-devo; examples of recapitulation of some tumor features in recently evolved organs; the types of tumors that might play the role in evolution; examples of tumors that already have played the role in evolution.</p><p>The discussion of experimental confirmation of nontrivial predictions of the hypothesis will include the analysis of evolutionary novelty of tumor-specifically expressed EST sequences; ELFNI – AS1, a human gene with possible microRNA function expressed predominantly in tumors and originated in primates; PBOV1, a human gene of the recent de novo origin with predicted highly tumor-specific expression profile; and the evolutionary novelty of human cancer/testis antigen genes; the data obtained on transgenic fish tumors regression model; and other data.</p><p>It can be concluded that expression of protogenes, evolutionarily young and/or novel genes in tumors might be a new biological phenomenon, a phenomenon of carcino-evo-devo genes, predicted by the hypothesis of evolution by tumor neofunctionalization.</p></div>","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Pages 28-29"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.050","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.052
M. Kruchinina , A. Starikov , S. Kurilovich , V. Kruchinin , V. Volodin , S. Rykhlitskii , A. Gromov , S. Peltec , S. Shehovtsov , V. Generalov
Background
The aim of this work was to assess the potential of the optical methods for studying erythrocytes (Er) and blood serum (BS) of patients with colorectal cancer (CC).
Methods
A total of 26 persons (52 + 8 years old) with CC (histologically – adenocarcinoma) in the T1–2 stage (the 1st group consisting of 10 patients) and in the terminal stage T3–4 (the 2nd group involving 16 patients) were examined. The metastases (in the liver area) were detected in 6 patients; the remaining patients had no metastases. The degree of lymph node involvement in most patients was not determined, the ten corresponding N1. The control group consisted of 16 healthy people (50 + 6 years old). Electric and viscoelastic Er parameters were investigated by dielectrophoresis, their membrane structure – by TLC and gas chromatography. The optical properties of BS were studied by the methods of ellipsometry. The reaction of the monoclonal antibody CD 24 with BS antigens of CC patients was studied by spectroscopic ellipsometry close to the conditions of surface plasmon resonance (SPR) (ProteOn XPR36 (BioRad).
Results
We observed significant differences in Er parameters, associated with the CC stage. Given in the 2nd group (T3–4) summarized rigidity, viscosity, electrical conductivity, the relative polarizability, indexes of aggregation and destruction were significantly higher than those in the 1st (T1–2) and in the control group (p < 0.001–0.05). At the same time the patients of the 2nd group had marked disturbances of Er deformability, leading to the development of microcirculatory disorders and tissue hypoxia with the expressed deficit of intracellular macroergs. We observed high levels of cholesterol fraction, oleic, stearic acids, high index of cholesterol/phospholipids (PHL) and low levels of total lipids, easily oxidable PHL, arachidonic acid, omega-3 index in Er membranes in the 2nd group in comparison with those in the 1st group of patients (p < 0.0001–0.03). Scanning ellipsometry showed marked heterogeneity in thickness and composition, the abundance of discontinuities in thin films of BS of patients in the 2nd group compared to the 1st one (p < 0.001). Increasing the refractive index in combination with the reduction in film thickness as CC stage was weighting has been observed (p < 0.01–0,.05). The concentration of the antigens to the CD24 in the BS of patients (obtained by SPR) in the terminal stages of CC was higher than that in the T1–2 (p < 0.001). We revealed correlations between Er parameters, BS ellipsometry characteristics and biochemical parameters, which reflected the interaction between these components depending on the CC stage.
{"title":"P41","authors":"M. Kruchinina , A. Starikov , S. Kurilovich , V. Kruchinin , V. Volodin , S. Rykhlitskii , A. Gromov , S. Peltec , S. Shehovtsov , V. Generalov","doi":"10.1016/j.ejcsup.2015.08.052","DOIUrl":"10.1016/j.ejcsup.2015.08.052","url":null,"abstract":"<div><h3>Background</h3><p>The aim of this work was to assess the potential of the optical methods for studying erythrocytes (Er) and blood serum (BS) of patients with colorectal cancer (CC).</p></div><div><h3>Methods</h3><p>A total of 26 persons (52<!--> <!-->+<!--> <!-->8<!--> <!-->years old) with CC (histologically – adenocarcinoma) in the T1–2 stage (the 1st group consisting of 10 patients) and in the terminal stage T3–4 (the 2nd group involving 16 patients) were examined. The metastases (in the liver area) were detected in 6 patients; the remaining patients had no metastases. The degree of lymph node involvement in most patients was not determined, the ten corresponding N1. The control group consisted of 16 healthy people (50<!--> <!-->+<!--> <!-->6<!--> <!-->years old). Electric and viscoelastic Er parameters were investigated by dielectrophoresis, their membrane structure – by TLC and gas chromatography. The optical properties of BS were studied by the methods of ellipsometry. The reaction of the monoclonal antibody CD 24 with BS antigens of CC patients was studied by spectroscopic ellipsometry close to the conditions of surface plasmon resonance (SPR) (ProteOn XPR36 (BioRad).</p></div><div><h3>Results</h3><p>We observed significant differences in Er parameters, associated with the CC stage. Given in the 2nd group (T3–4) summarized rigidity, viscosity, electrical conductivity, the relative polarizability, indexes of aggregation and destruction were significantly higher than those in the 1st (T1–2) and in the control group (<em>p</em> <!--><<!--> <!-->0.001–0.05). At the same time the patients of the 2nd group had marked disturbances of Er deformability, leading to the development of microcirculatory disorders and tissue hypoxia with the expressed deficit of intracellular macroergs. We observed high levels of cholesterol fraction, oleic, stearic acids, high index of cholesterol/phospholipids (PHL) and low levels of total lipids, easily oxidable PHL, arachidonic acid, omega-3 index in Er membranes in the 2nd group in comparison with those in the 1st group of patients (<em>p</em> <!--><<!--> <!-->0.0001–0.03). Scanning ellipsometry showed marked heterogeneity in thickness and composition, the abundance of discontinuities in thin films of BS of patients in the 2nd group compared to the 1st one (<em>p</em> <!--><<!--> <!-->0.001). Increasing the refractive index in combination with the reduction in film thickness as CC stage was weighting has been observed (<em>p</em> <!--><<!--> <!-->0.01–0,<span><math><mrow><mo>⧹</mo></mrow></math></span>.05). The concentration of the antigens to the CD24 in the BS of patients (obtained by SPR) in the terminal stages of CC was higher than that in the T1–2 (<em>p</em> <!--><<!--> <!-->0.001). We revealed correlations between Er parameters, BS ellipsometry characteristics and biochemical parameters, which reflected the interaction between these components depending on the CC stage.</p></div><div>","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Pages 29-30"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.052","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.080
T. Prudnikova , N. Litvyakov , N. Domanitskaya , N. Cherdyntseva , V. Belyavskaya
Background
Heparansulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. d-glucuronyl C5-epimerase (GLCE) is a crucial enzyme in HS synthesis, converting d-glucuronic acid (GlcA) to l-iduronic acid (IdoA) to increase HS flexibility. Aberrant modification may result in wrong structure of polysaccharide chains of HS and defects of microenvironment associated with malignant transformation.We previously experimentally identified the GLCE polymorphism Ile597Val. Its localization close to the activity center of the enzyme and different physical parameters of the involved amino acids suggest that the polymorphism is functional. So, three different variants of GLCE dimers with different enzymatic activity may exist in heterozygous carriers. Bioinformatics’ search (PubMed resource) revealed interracial variations in allele frequency distribution. Unusual high frequency of allele G was shown for black race (45%) compared with white race (17%). Taking into account the increased resistance of negroid race to breast cancer, we assume a potential involvement of the GLCE polymorphism in breast cancer.
Aim
The estimation of effects of GLCE functional polymorphism A2017G (Ile597Val) on the gene expression levels in normal and breast cancer cells and LOH in breast tumors.
Materials and methods
Breast cancer patients (n = 144.) had histologically verified diagnoses. Blood and breast cancer tissue samples as well as matched control tissues were collected from each patient during surgery. Genomic DNA was isolated by phenol extraction. Total RNA was isolated by TRIZol, RNA quantity was accessed by Qubit instrument with appropriate reagents and cDNA was obtained using First Strand cDNA Synthesis kit. SNP A2017G (rs3865014) was analyzed by Custom Real-Time SNP Array and GLCE expression levels were determined using Taq-Man-based Real-Time PCR (Applied Biosystems). Statistical analysis was carried out using a Statistika 9.0 software.
Results
AA genotype carriers had a 2-fold increase in GLCE mRNA levels in tumors compared with control surrounding tissues (0.37 ± 0.77 versus 0.17 ± 0.16, respectively, p < 0.05). Oppositely, AG genotype carriers had a 1.5-fold decrease in GLCE mRNA levels in tumors compared with control surrounding tissues (0.39 ± 0.29 versus 0.58 ± 0.33, respectively, p < 0.05 ). However, in any case,the GLCE expression in both normal tissues and breast tumors was more active in AG genotype carriers than in AA carriers. It is known that LOH is often associated
{"title":"P113","authors":"T. Prudnikova , N. Litvyakov , N. Domanitskaya , N. Cherdyntseva , V. Belyavskaya","doi":"10.1016/j.ejcsup.2015.08.080","DOIUrl":"10.1016/j.ejcsup.2015.08.080","url":null,"abstract":"<div><h3>Background</h3><p>Heparansulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. <span>d</span>-glucuronyl C5-epimerase (GLCE) is a crucial enzyme in HS synthesis, converting <span>d</span>-glucuronic acid (GlcA) to <span>l</span>-iduronic acid (IdoA) to increase HS flexibility. Aberrant modification may result in wrong structure of polysaccharide chains of HS and defects of microenvironment associated with malignant transformation.We previously experimentally identified the GLCE polymorphism Ile597Val. Its localization close to the activity center of the enzyme and different physical parameters of the involved amino acids suggest that the polymorphism is functional. So, three different variants of GLCE dimers with different enzymatic activity may exist in heterozygous carriers. Bioinformatics’ search (PubMed resource) revealed interracial variations in allele frequency distribution. Unusual high frequency of allele G was shown for black race (45%) compared with white race (17%). Taking into account the increased resistance of negroid race to breast cancer, we assume a potential involvement of the GLCE polymorphism in breast cancer.</p></div><div><h3>Aim</h3><p>The estimation of effects of GLCE functional polymorphism A2017G (Ile597Val) on the gene expression levels in normal and breast cancer cells and LOH in breast tumors.</p></div><div><h3>Materials and methods</h3><p>Breast cancer patients (n<!--> <!-->=<!--> <!-->144.) had histologically verified diagnoses. Blood and breast cancer tissue samples as well as matched control tissues were collected from each patient during surgery. Genomic DNA was isolated by phenol extraction. Total RNA was isolated by TRIZol, RNA quantity was accessed by Qubit instrument with appropriate reagents and cDNA was obtained using First Strand cDNA Synthesis kit. SNP A2017G (rs3865014) was analyzed by Custom Real-Time SNP Array and GLCE expression levels were determined using Taq-Man-based Real-Time PCR (Applied Biosystems). Statistical analysis was carried out using a Statistika 9.0 software.</p></div><div><h3>Results</h3><p>AA genotype carriers had a 2-fold increase in GLCE mRNA levels in tumors compared with control surrounding tissues (0.37<!--> <!-->±<!--> <!-->0.77 versus 0.17<!--> <!-->±<!--> <!-->0.16, respectively, <em>p</em> <!--><<!--> <!-->0.05). Oppositely, AG genotype carriers had a 1.5-fold decrease in GLCE mRNA levels in tumors compared with control surrounding tissues (0.39<!--> <!-->±<!--> <!-->0.29 versus 0.58<!--> <!-->±<!--> <!-->0.33, respectively, <em>p</em> <!--><<!--> <!-->0.05 ). However, in any case,the GLCE expression in both normal tissues and breast tumors was more active in AG genotype carriers than in AA carriers. It is known that LOH is often associated ","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Page 45"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.080","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.081
D. Pszczółkowska, A. Ellert-Miklaszewska, A. Gieryng, M. Kijewska, P. Wiśniewski, B. Kamińska
Malignant gliomas are fast-growing, heterogeneous and invasive brain tumors strongly infiltrated by non-tumor cells. Glioma attracts variety of immune cells, in particular microglia/macrophages and re-program these cells into immunosuppressive, tumor-supporting cells. Factors responsible for pro-invasive macrophage polarization and shaping tumor microenvironment in tumor-supporting manner are poorly known. We analyzed glioma secretome using proteomical approach and identified lactadherin (Mfge8) and osteopontin (Spp1) in microglia-activating fractions. Both osteopontin and lactadherin are αvβ3/αvβ5 integrin ligands able to interact with receptors present on microglia and macrophages and thus could be involved in pro-invasive polarization of microglia/macrophages. Moreover, both Spp1 and Mfge8 are overexpressed in glioma cells, but not in non-transformed astrocytes. C6 glioma cells stably expressing shRNA specific to lactadherin (shMfge8), osteopontin (shSpp1) and negative shRNA (shNeg) were implanted into striatum of Wistar rats. There was no difference in proliferation and viability of C6 glioma cells, cells stably expressing shRNA specific to lactadherin, ostopontin and negative shRNA in vitro, that demonstrates the negligible effect of autocrine production of both protein on tumor cell growth. Knockdown of Spp1 and Mfge8 resulted in significant reduction of tumor volume in rat model of glioma. Immunochemical analysis of brain sections revealed similar numbers of infiltrating microglia/macrophages (Iba1 staining), but the reduced number of ameboid, arginase 1 expressing cells in Mfge8 – depleted tumor. Treatment of endothelial cells with rhMFGE8 revealed significant effect of that protein on angiogenesis in vitro, however lactadherin-depleted tumors do not exhibit reduced blood vessel density in rat glioma model. FACS analysis showed that silencing of Spp1 does not affect total number of CD11b-positive cells, but strongly modulates microenvironment by leading to significant changes in percentage of Tc and Treg cells infiltrating tumor-bearing hemisphere. Our results suggest that glioma-derived integrin ligands are important factor in polarization of glioma infiltrating microglia/macrophages into the pro-invasive phenotype and its targeting could be a new therapeutic strategy.
{"title":"P142","authors":"D. Pszczółkowska, A. Ellert-Miklaszewska, A. Gieryng, M. Kijewska, P. Wiśniewski, B. Kamińska","doi":"10.1016/j.ejcsup.2015.08.081","DOIUrl":"10.1016/j.ejcsup.2015.08.081","url":null,"abstract":"<div><p>Malignant gliomas are fast-growing, heterogeneous and invasive brain tumors strongly infiltrated by non-tumor cells. Glioma attracts variety of immune cells, in particular microglia/macrophages and re-program these cells into immunosuppressive, tumor-supporting cells. Factors responsible for pro-invasive macrophage polarization and shaping tumor microenvironment in tumor-supporting manner are poorly known. We analyzed glioma secretome using proteomical approach and identified lactadherin (Mfge8) and osteopontin (Spp1) in microglia-activating fractions. Both osteopontin and lactadherin are <em>αvβ</em>3/<em>αvβ</em>5 integrin ligands able to interact with receptors present on microglia and macrophages and thus could be involved in pro-invasive polarization of microglia/macrophages. Moreover, both Spp1 and Mfge8 are overexpressed in glioma cells, but not in non-transformed astrocytes. C6 glioma cells stably expressing shRNA specific to lactadherin (shMfge8), osteopontin (shSpp1) and negative shRNA (shNeg) were implanted into striatum of Wistar rats. There was no difference in proliferation and viability of C6 glioma cells, cells stably expressing shRNA specific to lactadherin, ostopontin and negative shRNA in vitro, that demonstrates the negligible effect of autocrine production of both protein on tumor cell growth. Knockdown of Spp1 and Mfge8 resulted in significant reduction of tumor volume in rat model of glioma. Immunochemical analysis of brain sections revealed similar numbers of infiltrating microglia/macrophages (Iba1 staining), but the reduced number of ameboid, arginase 1 expressing cells in Mfge8 – depleted tumor. Treatment of endothelial cells with rhMFGE8 revealed significant effect of that protein on angiogenesis in vitro, however lactadherin-depleted tumors do not exhibit reduced blood vessel density in rat glioma model. FACS analysis showed that silencing of Spp1 does not affect total number of CD11b-positive cells, but strongly modulates microenvironment by leading to significant changes in percentage of Tc and Treg cells infiltrating tumor-bearing hemisphere. Our results suggest that glioma-derived integrin ligands are important factor in polarization of glioma infiltrating microglia/macrophages into the pro-invasive phenotype and its targeting could be a new therapeutic strategy.</p></div>","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Pages 45-46"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.081","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/J.EJCSUP.2015.08.099
S. A. Solodskikh, V. Bashmakov, T. Gorbacheva, A. V. Panevina, A. Maslov, Andrey A. Mikhailov, I. Moshurov, V. Popov
{"title":"P48: Integrated analysis of genomic and transcriptomic data in clear-cell renal cell carcinoma","authors":"S. A. Solodskikh, V. Bashmakov, T. Gorbacheva, A. V. Panevina, A. Maslov, Andrey A. Mikhailov, I. Moshurov, V. Popov","doi":"10.1016/J.EJCSUP.2015.08.099","DOIUrl":"https://doi.org/10.1016/J.EJCSUP.2015.08.099","url":null,"abstract":"","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"55"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/J.EJCSUP.2015.08.099","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54310189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.125
A. Zhabina
{"title":"A72: Particular qualities of the expression of markers of sensitivity to cytostatics at patients various solid tumours","authors":"A. Zhabina","doi":"10.1016/j.ejcsup.2015.08.125","DOIUrl":"https://doi.org/10.1016/j.ejcsup.2015.08.125","url":null,"abstract":"","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"71"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.125","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54310720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.076
E. Pisareva , N. Gutkina , S. Kovalenko , V. Shamanin
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 wild-type 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 oligonucleotide 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 detectio
{"title":"P65","authors":"E. Pisareva , N. Gutkina , S. Kovalenko , V. Shamanin","doi":"10.1016/j.ejcsup.2015.08.076","DOIUrl":"10.1016/j.ejcsup.2015.08.076","url":null,"abstract":"<div><p>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.</p><p>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.</p><p>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.</p><p>In this study we developed and compared 3 new KRAS assays (1) real-time PCR with allele-specific primers; (2) real-time wild-type 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 oligonucleotide 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.</p><p>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 detectio","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Pages 42-43"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54309876","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-11-01DOI: 10.1016/J.EJCSUP.2015.08.126
G. Zhukova, A. Shikhliarova, T. Gudtskova, M. Bragina, I. Novikova, V. Zernov, T. Barteneva, O. Polozhentsev, A. Soldatov, M. Rudenko, E. Shirnina
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Pub Date : 2015-11-01DOI: 10.1016/j.ejcsup.2015.08.042
K. Kirsanov , E. Lesovaya , N. Shalginskikh , D. Naberezhnov , V. Glazunov , G. Belitsky , M. Yakubovskaya
Purpose
DNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases.
Results
We revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action.
Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs.
After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line.
Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histon
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