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.2015.08.007
E. Batorov, M. Tikhonova, I. Kryuchkova, V. Sergeevicheva, D. Batorova, S. Sizikova, G. Ushakova, A. Gilevich, A. Ostanin, E. Chernykh
Numerous studies have shown that high-dose chemotherapy and autologous hematopoietic stem cell transplantation (AHSCT) led to a profound and long-lasting state of immunodeficiency characterized by persisting low levels of T cells in hemoblastosis patients. Well-timed T-cell reconstitution is crucial for early restoration of anti-infectious and anti- tumor immune response. Lymphocyte recovery is mediated through the two main mechanisms – a homeostatic proliferation of T cells and generation of new naive T cells via thymopoiesis. It is known, that homeostatic proliferation is important for the restoration of T cell count in immune competent host during the 1st year following AHSCT. Thymus begins to fill up T cell repertoire approximately from the 6th month following AHSCT.
We have investigated dynamics of CD4+FOXP3+ Treg recovery following AHSCT and possible relationship between Tregs and clinical outcomes since the suppressive activity of Tregs under lymphopenic conditions may influence on peripheral expansion of T cells. Thymic activity following AHSCT has been evaluated by measuring amounts of CD4+ CD45RA+CD31+ naïve T cells, i.e. “recent thymic emigrants” (RTEs).109 patients with non-Hodgkin’s lymphomas, Hodgkin’s lymphoma and multiple myeloma underwent AHSCT in 2009–2014. The content of circulating CD4+FOXP3+ Tregs and CD4+CD45RA+CD31+ T cells was evaluated using flow cytometry before AHSCT, at the day of engraftment, and following 6 and 12 months.
Pre-transplant count of CD4+FOXP3+ Tregs was significantly higher compared to healthy controls (5.4 ± 2.9 vs 3.8 ± 1.9%; pU = 0.011; here and below data presented as Mean ± SD). Percentage of Tregs restored rapidly and reached initially high level at the time of engraftment, and then subsequently decreased within a year until it lowered to healthy donors‘ values. CD4+FOXP3+ Tregs at the time of engraftment were increased in patients with relapse or progression of disease within 6 and 12 months following AHSCT compared to non-relapsed patients (11.0 ± 6.1 vs 6.2 ± 3.0%; pU = 0.016, and 10.1 ± 5.2 vs 6.1 ± 3.8%; pU = 0.008). Pre-transplant count of CD4+CD45RA+CD31+ T cells was significantly lower compared to healthy controls (17.1 ± 11.4 vs 30.3 ± 11.2%, pU = 0.0005) and did not reach donors‘ values following 12 month (23.1 ± 13.5%, pU = 0.032). Relapsed patients had the same quantity of RTEs as the patients with remission within the 1st year following AHSCT. There was no any significant association between RTEs and Tregs counts.
Surprisingly, we have found high levels of circulating CD4+CD45RA- T cells co-expressing CD31 molecule i
大量研究表明，大剂量化疗和自体造血干细胞移植(AHSCT)导致造血细胞病患者持续低水平T细胞的长期免疫缺陷状态。适时的t细胞重建对于抗感染和抗肿瘤免疫应答的早期恢复至关重要。淋巴细胞的恢复是通过两种主要机制介导的——T细胞的稳态增殖和通过胸腺生成产生新的初始T细胞。已知，在AHSCT后的第一年，稳态增殖对于免疫能力宿主T细胞计数的恢复是重要的。大约从AHSCT后6个月胸腺开始填补T细胞库。我们研究了AHSCT后CD4+FOXP3+ Treg恢复的动态，以及Treg与临床结果之间的可能关系，因为淋巴细胞减少条件下Treg的抑制活性可能影响T细胞的外周扩增。AHSCT后胸腺活动通过测量CD4+ CD45RA+CD31+ naïve T细胞的数量来评估，即“近期胸腺移植物”(rte)。2009-2014年，109例非霍奇金淋巴瘤、霍奇金淋巴瘤和多发性骨髓瘤患者接受了AHSCT。流式细胞术检测AHSCT前、移植当日、移植后6个月和12个月循环CD4+FOXP3+ Tregs和CD4+CD45RA+CD31+ T细胞的含量。移植前CD4+FOXP3+ Tregs计数明显高于健康对照组(5.4±2.9 vs 3.8±1.9%;pU = 0.011;此处和以下数据以Mean±SD表示)。Tregs的百分比迅速恢复，并在移植时达到最初的高水平，随后在一年内下降，直到降至健康供体的值。移植时CD4+FOXP3+ Tregs在AHSCT后6个月和12个月内复发或疾病进展的患者中与非复发患者相比增加(11.0±6.1 vs 6.2±3.0%;pU = 0.016，和10.1±5.2 vs 6.1±3.8%;pU = 0.008)。移植前CD4+CD45RA+CD31+ T细胞计数明显低于健康对照组(17.1±11.4 vs 30.3±11.2%，pU = 0.0005)， 12个月后未达到供者水平(23.1±13.5%，pU = 0.032)。在AHSCT后的第一年内，复发患者的rte数量与缓解患者相同。rte和Tregs之间没有明显的联系。令人惊讶的是，我们在AHSCT患者中发现了高水平的循环CD4+CD45RA- T细胞共表达CD31分子，因为该分子在健康对照组的记忆亚群中并不常见(20.7±12.0 vs 8.2±2.1%，pU <0.00001)。CD4+CD45RA-CD31+ T细胞相对数量与CD4+CD45RO+CD31+人群高度相关(rS=0.72;p & lt;0.00001)。CD4+CD45RA-CD31+ T细胞计数在AHSCT术后1个月内迅速恢复，恢复到移植前水平，并在随访期间保持不变。移植后1年内早期复发和缓解患者的CD4+CD45RA-CD31+ T细胞相对计数无差异。我们关于Tregs重组的数据可能证实了之前的假设，即在免疫恢复期间Tregs的存在保持了最佳的T细胞受体多样性。然而，这些细胞的过量导致增殖活性和免疫反应的抑制，并与早期复发有关。相反，相对缓慢的rte恢复决定了它们在移植后第一年内对生存缺乏影响。CD31分子在T细胞记忆亚群(CD4+CD45RA-和/或CD4+CD45RO+)中的生物学作用和出现方式尚不清楚。CD31+记忆T细胞在淋巴增生性疾病发病机制中的作用有待进一步研究。
{"title":"P81","authors":"E. Batorov, M. Tikhonova, I. Kryuchkova, V. Sergeevicheva, D. Batorova, S. Sizikova, G. Ushakova, A. Gilevich, A. Ostanin, E. Chernykh","doi":"10.1016/j.ejcsup.2015.08.007","DOIUrl":"10.1016/j.ejcsup.2015.08.007","url":null,"abstract":"<div><p>Numerous studies have shown that high-dose chemotherapy and autologous hematopoietic stem cell transplantation (AHSCT) led to a profound and long-lasting state of immunodeficiency characterized by persisting low levels of T cells in hemoblastosis patients. Well-timed T-cell reconstitution is crucial for early restoration of anti-infectious and anti- tumor immune response. Lymphocyte recovery is mediated through the two main mechanisms – a homeostatic proliferation of T cells and generation of new naive T cells via thymopoiesis. It is known, that homeostatic proliferation is important for the restoration of T cell count in immune competent host during the 1st year following AHSCT. Thymus begins to fill up T cell repertoire approximately from the 6th month following AHSCT.</p><p>We have investigated dynamics of CD4+FOXP3+ Treg recovery following AHSCT and possible relationship between Tregs and clinical outcomes since the suppressive activity of Tregs under lymphopenic conditions may influence on peripheral expansion of T cells. Thymic activity following AHSCT has been evaluated by measuring amounts of CD4+ CD45RA+CD31+ naïve T cells, i.e. “recent thymic emigrants” (RTEs).109 patients with non-Hodgkin’s lymphomas, Hodgkin’s lymphoma and multiple myeloma underwent AHSCT in 2009–2014. The content of circulating CD4+FOXP3+ Tregs and CD4+CD45RA+CD31+ T cells was evaluated using flow cytometry before AHSCT, at the day of engraftment, and following 6 and 12<!--> <!-->months.</p><p>Pre-transplant count of CD4+FOXP3+ Tregs was significantly higher compared to healthy controls (5.4<!--> <!-->±<!--> <!-->2.9 vs 3.8<!--> <!-->±<!--> <!-->1.9%; <em>pU</em> <!-->=<!--> <!-->0.011; here and below data presented as Mean<!--> <!-->±<!--> <!-->SD). Percentage of Tregs restored rapidly and reached initially high level at the time of engraftment, and then subsequently decreased within a year until it lowered to healthy donors‘ values. CD4+FOXP3+ Tregs at the time of engraftment were increased in patients with relapse or progression of disease within 6 and 12<!--> <!-->months following AHSCT compared to non-relapsed patients (11.0<!--> <!-->±<!--> <!-->6.1 vs 6.2<!--> <!-->±<!--> <!-->3.0%; <em>pU</em> <!-->=<!--> <!-->0.016, and 10.1<!--> <!-->±<!--> <!-->5.2 vs 6.1<!--> <!-->±<!--> <!-->3.8%; <em>pU</em> <!-->=<!--> <!-->0.008). Pre-transplant count of CD4+CD45RA+CD31+ T cells was significantly lower compared to healthy controls (17.1<!--> <!-->±<!--> <!-->11.4 vs 30.3<!--> <!-->±<!--> <!-->11.2%, <em>pU</em> <!-->=<!--> <!-->0.0005) and did not reach donors‘ values following 12<!--> <!-->month (23.1<!--> <!-->±<!--> <!-->13.5%, <em>pU</em> <!-->=<!--> <!-->0.032). Relapsed patients had the same quantity of RTEs as the patients with remission within the 1st year following AHSCT. There was no any significant association between RTEs and Tregs counts.</p><p>Surprisingly, we have found high levels of circulating CD4+CD45RA- T cells co-expressing CD31 molecule i","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Page 4"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54308691","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.2015.08.010
N. Bgatova , I. Kulikova , I. Kachesov , R. Yui , M. Ergazina , S. Chepko , N. Isakova , Y. Borodin , V. Konenkov
Background
Squamous cell carcinoma is the most common malignancy in the lower lip. Although lower lip squamous cell carcinoma is slow growing, up to 29% of tumors develop metastases to the cervical lymph nodes. Thus, identification of biological markers that could provide prognostic information about the invasive or metastatic potential of these lesions is important. It is known that angiogenesis, lymphangiogenesis and proliferation play an important role in tumor progression. Therefore, the goal of this study was to analyze the immunohistochemical expression of Ki-67, CD34 and Podoplanin in hyperkeratosis and in squamous cell carcinoma of the lower lip. It was intended to assess the possibility of using such markers as indicators of morphological aggressiveness of squamous cell carcinoma of the lower lip (LLSCC).
Materials and methods
Seventy-one cases of the lower lip lesions, obtained from the files of Novosibirsk Regional Oncology Center were selected for this study. The specimens were divided into three groups: a lower lip hyperkeratosis group consisting of 22 cases; LLSCC with keratinization consisting of 34 cases and LLSCC without keratinization consisting of 15 cases. To analyze angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the lower lip biopsy material for the CD34, vascular marker, Podoplanin, lymphatic-specific markers and Ki-67, marker of proliferation. Tissue samples were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). The lymphatic and blood vessels volume density and Ki-67 cells numerical density were morphometrically analyzed in all groups and compared using the non-parametric Mann–Whitney test and the Wilcoxon signed rank test. A level of significance of 5% (p < 0.05) was adopted for all tests.
Results
All cases of a lower lip hyperkeratosis and LLSCC were positive for Ki-67, CD34 and Podoplanin. With respect to the pattern of staining, specimens exhibited a predominantly peripheral staining for CD34 and Podoplanin in inflammatory infiltrates and tumor sites. In contrast, staining for Ki-67 was predominantly central in inflammatory infiltrates and tumor sites in hyperkeratosis and LLSCC. When compared to lower lip hyperkeratosis, LLSCC (both with keratinization and without keratinization) showed a higher number of immunopositive Ki-67 cells (by 64% and 77%, respectively, p < 0.05). It was found that proliferative activity of tumor cells in LLSCC with keratinization was 2 times higher than that in LLSCC without keratinization. Comparison of the volume density of blood vessels showed that the density of CD34+ – blood vessels in hyperkeratosis was lower by 77% than in LLSCC without keratinization and lower by 64% than in
{"title":"A94","authors":"N. Bgatova , I. Kulikova , I. Kachesov , R. Yui , M. Ergazina , S. Chepko , N. Isakova , Y. Borodin , V. Konenkov","doi":"10.1016/j.ejcsup.2015.08.010","DOIUrl":"10.1016/j.ejcsup.2015.08.010","url":null,"abstract":"<div><h3>Background</h3><p>Squamous cell carcinoma is the most common malignancy in the lower lip. Although lower lip squamous cell carcinoma is slow growing, up to 29% of tumors develop metastases to the cervical lymph nodes. Thus, identification of biological markers that could provide prognostic information about the invasive or metastatic potential of these lesions is important. It is known that angiogenesis, lymphangiogenesis and proliferation play an important role in tumor progression. Therefore, the goal of this study was to analyze the immunohistochemical expression of Ki-67, CD34 and Podoplanin in hyperkeratosis and in squamous cell carcinoma of the lower lip. It was intended to assess the possibility of using such markers as indicators of morphological aggressiveness of squamous cell carcinoma of the lower lip (LLSCC).</p></div><div><h3>Materials and methods</h3><p>Seventy-one cases of the lower lip lesions, obtained from the files of Novosibirsk Regional Oncology Center were selected for this study. The specimens were divided into three groups: a lower lip hyperkeratosis group consisting of 22 cases; LLSCC with keratinization consisting of 34 cases and LLSCC without keratinization consisting of 15 cases. To analyze angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the lower lip biopsy material for the CD34, vascular marker, Podoplanin, lymphatic-specific markers and Ki-67, marker of proliferation. Tissue samples were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). The lymphatic and blood vessels volume density and Ki-67 cells numerical density were morphometrically analyzed in all groups and compared using the non-parametric Mann–Whitney test and the Wilcoxon signed rank test. A level of significance of 5% (<em>p</em> <!--><<!--> <!-->0.05) was adopted for all tests.</p></div><div><h3>Results</h3><p>All cases of a lower lip hyperkeratosis and LLSCC were positive for Ki-67, CD34 and Podoplanin. With respect to the pattern of staining, specimens exhibited a predominantly peripheral staining for CD34 and Podoplanin in inflammatory infiltrates and tumor sites. In contrast, staining for Ki-67 was predominantly central in inflammatory infiltrates and tumor sites in hyperkeratosis and LLSCC. When compared to lower lip hyperkeratosis, LLSCC (both with keratinization and without keratinization) showed a higher number of immunopositive Ki-67 cells (by 64% and 77%, respectively, <em>p</em> <!--><<!--> <!-->0.05). It was found that proliferative activity of tumor cells in LLSCC with keratinization was 2 times higher than that in LLSCC without keratinization. Comparison of the volume density of blood vessels showed that the density of CD34+ – blood vessels in hyperkeratosis was lower by 77% than in LLSCC without keratinization and lower by 64% than in ","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Page 6"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54308718","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.2015.08.022
N. Dementyeva , R. Derks , I. Kohler , N. Merzlikin , S. Shelepov , D. Kokova , A. Sazonov , O. Mayboroda , I. Saltikova
Background
Opisthorchiasis is a form of foodborne trematodiasis which is caused by liver flukes. It has been shown that a chronic Opisthorchiasis infection increases a risk of cholagiocarcinoma of liver. It is commonly believed that a gradual change of homeostasis in a parasite microenvironment (bile) leads to liver fluke-induced cancer. Nevertheless, no systematic, analytically driven studies confirming this hypothesis have been published yet. The restricted access to clinical material and extreme complexity of the biological matrix (bile) both are the important “rate limiting factors” for a progress in the field. Here we present for the first time a cross-platform mass spectrometric analysis of bile juice collected from the patients with cholangiocarcinoma-associated diseases. We show that an effective analysis of such complex biological matrix as bile juice requires a combination of orthogonal analytical platforms (e.g. RPLC–MS and HILIC–MS) maximizing coverage of the metabolic space.
Materials and methods
28 patients with O. felineus infection and 30 negative controls were included in the study. The infection status was confirmed using microscopy analysis of the bile. Bile samples were collected from the gallbladder using sterile puncture, directly frozen and stored at −80 °C until analysis. The samples were randomized and organized into the acquisition blocks consisting of the samples and quality controls (QC). Experiments were carried out with a Dionex Ultimate 3000 LC system (Thermo Scientific/Dionex, The Netherlands) equipped with a Dual Gradient Separation pump allowing for parallel LC analysis, and hyphenated to an Impact UHR-qTOF mass analyzer (Bruker Daltonics, Germany). Reversed-phase experiments (RPLC) were performed with an UHPLC BEH Shield RP18 column 100 × 2.1 mm, 1.7 μm (Waters) and HILIC experiments with a Luna HILIC column (Phenomenex, The Netherlands) of 100 2.00 mm, 3 μ m. RPLC data were acquired in ESI positive mode and HILIC in negative mode, respectively. The data acquisition rate was set to 1 Hz over a mass range of m/z 50–1000. The LC–MS data files were aligned by using the in-house developed alignment algorithm MS-Align 2 tool (www.ms-utils.org/msalign2).
Results
After the data prepressing, which includes alignment, noise filtering and peak picking two data matrixes costing of 412 features (metabolites) for RPLC and 428 ones for HILIC were generated. To evaluate a degree of similarity between the two data matrixes the RV coefficient (a multivariate extension of correlation coefficient) was used. The coefficient has flattened at 0.58 showing that despite a strong overlap between the datasets there is a substantial number of the
{"title":"P158","authors":"N. Dementyeva , R. Derks , I. Kohler , N. Merzlikin , S. Shelepov , D. Kokova , A. Sazonov , O. Mayboroda , I. Saltikova","doi":"10.1016/j.ejcsup.2015.08.022","DOIUrl":"10.1016/j.ejcsup.2015.08.022","url":null,"abstract":"<div><h3>Background</h3><p><em>Opisthorchiasis</em> is a form of foodborne trematodiasis which is caused by liver flukes. It has been shown that a chronic <em>Opisthorchiasis</em> infection increases a risk of cholagiocarcinoma of liver. It is commonly believed that a gradual change of homeostasis in a parasite microenvironment (bile) leads to liver fluke-induced cancer. Nevertheless, no systematic, analytically driven studies confirming this hypothesis have been published yet. The restricted access to clinical material and extreme complexity of the biological matrix (bile) both are the important “rate limiting factors” for a progress in the field. Here we present for the first time a cross-platform mass spectrometric analysis of bile juice collected from the patients with cholangiocarcinoma-associated diseases. We show that an effective analysis of such complex biological matrix as bile juice requires a combination of orthogonal analytical platforms (e.g. RPLC–MS and HILIC–MS) maximizing coverage of the metabolic space.</p></div><div><h3>Materials and methods</h3><p>28 patients with <em>O. felineus</em> infection and 30 negative controls were included in the study. The infection status was confirmed using microscopy analysis of the bile. Bile samples were collected from the gallbladder using sterile puncture, directly frozen and stored at −80<!--> <!-->°C until analysis. The samples were randomized and organized into the acquisition blocks consisting of the samples and quality controls (QC). Experiments were carried out with a Dionex Ultimate 3000 LC system (Thermo Scientific/Dionex, The Netherlands) equipped with a Dual Gradient Separation pump allowing for parallel LC analysis, and hyphenated to an Impact UHR-qTOF mass analyzer (Bruker Daltonics, Germany). Reversed-phase experiments (RPLC) were performed with an UHPLC BEH Shield RP18 column 100<!--> <!-->×<!--> <!-->2.1<!--> <!-->mm, 1.7<!--> <!-->μm (Waters) and HILIC experiments with a Luna HILIC column (Phenomenex, The Netherlands) of 100<!--> <span><math><mrow><mo>×</mo></mrow></math></span> <!-->2.00<!--> <!-->mm, 3<!--> <!-->μ<!--> <!-->m. RPLC data were acquired in ESI positive mode and HILIC in negative mode, respectively. The data acquisition rate was set to 1<!--> <!-->Hz over a mass range of <em>m</em>/<em>z</em> 50–1000. The LC–MS data files were aligned by using the in-house developed alignment algorithm MS-Align 2 tool (<span>www.ms-utils.org/msalign2</span><svg><path></path></svg>).</p></div><div><h3>Results</h3><p>After the data prepressing, which includes alignment, noise filtering and peak picking two data matrixes costing of 412 features (metabolites) for RPLC and 428 ones for HILIC were generated. To evaluate a degree of similarity between the two data matrixes the RV coefficient (a multivariate extension of correlation coefficient) was used. The coefficient has flattened at 0.58 showing that despite a strong overlap between the datasets there is a substantial number of the","PeriodicalId":11675,"journal":{"name":"Ejc Supplements","volume":"13 1","pages":"Pages 12-13"},"PeriodicalIF":0.0,"publicationDate":"2015-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ejcsup.2015.08.022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54308918","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号