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[{"key": "dc.contributor.advisor", "value": "Kononen, Juha (LT)", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Lahtinen, Laura (FT)", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Yl\u00e4nne, Jari (Prof., FT)", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Isomursu, Aleksi", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2015-04-14T19:28:51Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2015-04-14T19:28:51Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2015", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1472165", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/45629", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Epidermaalisen kasvutekij\u00e4n reseptorin (EGFR) monoklonaalisia vasta-aineita (mAb) k\u00e4ytet\u00e4\u00e4n apuna metastaattisen kolorektaalisy\u00f6v\u00e4n (mCRC) hoidossa. Aktivoivat mutaatiot KRAS- ja NRAS-onkogeeneiss\u00e4 tekev\u00e4t sy\u00f6p\u00e4solut vastustuskykyisiksi anti-EGFR-vasta-aineille, ja t\u00e4st\u00e4 syyst\u00e4 tuumorit tulee genotyypitt\u00e4\u00e4 ennen hoidon aloittamista. Kudosbiopsian ottaminen on kuitenkin invasiivinen toimenpide, eik\u00e4 n\u00e4yte v\u00e4ltt\u00e4m\u00e4tt\u00e4 edusta kattavasti sy\u00f6v\u00e4n kaikkien solupopulaatioiden geneettisi\u00e4 muutoksia. Verenkierron solunulkoisen DNA:n (cfDNA) on havaittu sis\u00e4lt\u00e4v\u00e4n maligneista soluista l\u00e4ht\u00f6isin olevia onkogeenisi\u00e4 mutaatioita, ja se voi olla hyv\u00e4 vaihtoehtoinen tuumoriper\u00e4isen DNA:n l\u00e4hde.\n\nKehitimme ja optimoimme l\u00e4mp\u00f6tilagradienttien ja laimennossarjojen avulla E-ice-COLD-PCR (engl. enhanced, improved and complete enrichment co-amplification at lower denaturation temperature PCR)-pohjaisen menetelm\u00e4n plasman cfDNA:ssa esiintyvien KRAS-geenin kodonien 12 ja 13 mutaatioiden selektiiviseen monistamiseen; n\u00e4m\u00e4 mutantit vastaavat yhdess\u00e4 noin 80 %:a kolorektaalisy\u00f6v\u00e4n kaikista RAS-mutaatioista. Monistetut mutantit osoitettiin mittaamalla PCR-tuotteista l\u00f6ytyvien alleelien m\u00e4\u00e4r\u00e4t kohdennetun pyrosekvensoinnin avulla. 16:sta mCRC-potilaasta ker\u00e4ttiin 4x3 ml perifeerist\u00e4 verta K2EDTA-putkiin. Sentrifugoinnin vaikutusta cfDNA:n erist\u00e4miseen ja sen j\u00e4lkeiseen mutaatiom\u00e4\u00e4ritykseen arvioitiin erottelemalla plasma soluista nelj\u00e4n erilaisen sentrifugointiprotokollan mukaan: 10 min/500 x g; 10 min/500 x g + 10 min/16000 x g; 10 min/1600 x g; sek\u00e4 10 min/1600 x g + 10 min/16000 x g. cfDNA eristettiin plasmasta kaupallisilla piidioksidikalvopylv\u00e4ill\u00e4, ja n\u00e4ytteet joissa havaittiin v\u00e4hiten kontaminoivaa genomista DNA:ta valittiin KRAS-mutaatiom\u00e4\u00e4ritykseen. Tuloksia verrattiin potilaiden tautimuutoksista aiemmin osoitettuihin mutaatioihin.\n\nTestin analyyttinen herkkyys KRAS-mutaatioita osoitettaessa oli 0,1 % (c.35G>A)-0,25 % (c.35G>C). Diagnostinen havaitsemisraja (LOD) oli vastaavasti 0,25 %-0,5 % (\u03b1 = 0,05). cfDNA:ta oli potilaiden veress\u00e4 keskim\u00e4\u00e4rin 59 \u00b1 50 ng/ml (x\u0304 \u00b1 sd), ja tulokset vastasivat t\u00e4lt\u00e4 osin aiemmin julkaistuja tutkimuksia. Konsentraatio vaihteli sentrifugointiprotokollan mukaan (P = 0,022): 10 min 500 x g:ll\u00e4 erotellusta plasmasta eristettiin yleisesti ottaen hieman v\u00e4hemm\u00e4n cfDNA:ta kuin saman potilaan muista n\u00e4ytteist\u00e4. Kahdenv\u00e4lisiss\u00e4 post-hoc-analyyseiss\u00e4 havaitut erot eiv\u00e4t kuitenkaan olleet tilastollisesti merkitsevi\u00e4. Kun n\u00e4ytteist\u00e4 m\u00e4\u00e4ritettiin KRAS-mutaatioita, menetelm\u00e4n herkkyys oli 17 % ja spesifisyys 89 %.\n\nE-ice-COLD-PCR-avusteinen kohdennettu pyrosekvensointi mahdollistaa KRAS-mutaatioiden c.35G>A ja c.35G>C herk\u00e4n havaitsemisen pienest\u00e4kin m\u00e4\u00e4r\u00e4st\u00e4 DNA:ta. Vaikka testin tarkkaa suorituskyky\u00e4 ei m\u00e4\u00e4ritetty erikseen muilla KRAS-geenin kodoneissa 12 ja 13 esiintyvill\u00e4 mutaatioilla (c.34G>T, c.34G>A, c.34G>C, c.35G>T ja c.38G>A), t\u00e4m\u00e4 on tarvittaessa helposti toteutettavissa. Testill\u00e4 voidaan havaita patogeenisi\u00e4 KRAS-mutaatioita my\u00f6s mCRC-potilaiden cfDNA:sta, mist\u00e4 voi olla hy\u00f6ty\u00e4 kun tavanomaisia kudosn\u00e4ytteit\u00e4 ei ole saatavilla tai kun halutaan suorittaa useita per\u00e4tt\u00e4isi\u00e4 mutaatiom\u00e4\u00e4rityksi\u00e4, esimerkiksi hankinnaisen resistenssin havaitsemiseksi. Diagnostisessa herkkyydess\u00e4 todettujen puutteiden vuoksi testin kliininen validointi edellytt\u00e4\u00e4 kuitenkin kattavia, prospektiivisia jatkotutkimuksia.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAb) are used for treating metastatic colorectal cancer (mCRC). Activating mutations in oncogenes KRAS and NRAS result in resistance to anti-EGFR mAbs, necessitating tumor genotyping prior to the therapy. However, tissue biopsy is an invasive procedure that may fail to address the spatiotemporal clonal heterogeneity of cancer. Circulating cell-free DNA (cfDNA) has\nbeen shown to contain oncogenic mutations originating from malignant cells, presenting a promising alternative source of tumor-derived genetic material.\n\nWe used temperature gradients and dilution series to develop and optimize an enhanced, improved and complete enrichment co-amplification at lower denaturation temperature PCR (E-ice-COLD-PCR) assay for the selective amplification of KRAS codon 12 and 13 mutations in plasma cfDNA; together these mutants comprise approximately 80% of all RAS mutations in colorectal cancer. The amplified variants were detected by subjecting the PCR products to allele quantification by targeted pyrosequencing. 16 mCRC patients were recruited and 4x3\nml of peripheral blood was drawn from each patient into K2EDTA tubes. To evaluate the effect of centrifugation on cfDNA extraction and subsequent mutation testing, plasma was separated according to four different centrifugation protocols: 10 min at 500 x g; 10 min at 500 x g + 10 min at 16,000 x g; 10 min at 1,600 x g; and 10 min at 1,600 x g + 10 min at 16,000 x g. cfDNA was extracted from the plasma using commercially available silica membrane columns and the sample set displaying the least amount of genomic DNA contaminants was tested for KRAS codon 12 and 13 mutations. The results were compared to previously detected mutations in the patients\u2019 tumors.\n\nThe maximum analytical sensitivity for calling KRAS mutations c.35G>A and c.35G>C was 0.1% and 0.25% mutant in a wild type background, respectively, while the diagnostic limit of detection (LOD) was 0.25%-0.5% (\u03b1 = 0.05). Concordant with previous studies, the average amount of cfDNA in the patients\u2019 blood was 59 \u00b1 50 ng/ml (x\u0304 \u00b1 sd). Concentrations varied according to the individual centrifugation protocol (P = 0.022). More specifically, plasma centrifuged for 10 min at 500 x g tended to yield slightly less cfDNA than any of the\ncorrelated samples, although the pairwise post-hoc comparisons did not reach statistical significance. When the samples were tested for KRAS mutations, the resulting sensitivity was 17% and the specificity 89%.\n\nE-ice-COLD-PCR-enhanced targeted pyrosequencing allows for robust and sensitive detection of KRAS c.35G>A and c.35G>C from minute amounts of DNA. Even though the test performance was not directly validated for other hotspot mutations in KRAS codons 12 and 13 (i.e. c.34G>T, c.34G>A, c.34G>C, c.35G>T and c.38G>A), this can be accomplished separately as needed. The method can be used for detecting pathogenic KRAS mutations in the cfDNA of mCRC patients. This may prove useful for tumor genotyping when conventional tissue samples\nare unavailable or repeated testing is required, e.g. in the early detection of acquired resistance. However, due to the observed sensitivity issues, comprehensive prospective studies are needed to confirm the viability of the test for clinical use.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Aleksi Isomursu (vealisom) on 2015-04-14 19:28:50.123818. Form: Pro gradu -lomake (https://kirjasto.jyu.fi/julkaisut/julkaisulomakkeet/pro-gradu-lomake). JyX data: [jyx_publishing-allowed (fi) =True]", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by jyx lomake-julkaisija (jyx-julkaisija@noreply.fi) on 2015-04-14T19:28:50Z\nNo. of bitstreams: 2\nURN:NBN:fi:jyu-201504141582.pdf: 1335473 bytes, checksum: dc70e82c27339faae1b500922ef171ae (MD5)\nlicense.html: 4816 bytes, checksum: a941d9f28eaf2aa13e47dadc2e4edd51 (MD5)", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2015-04-14T19:28:51Z (GMT). No. of bitstreams: 2\nURN:NBN:fi:jyu-201504141582.pdf: 1335473 bytes, checksum: dc70e82c27339faae1b500922ef171ae (MD5)\nlicense.html: 4816 bytes, checksum: a941d9f28eaf2aa13e47dadc2e4edd51 (MD5)\n Previous issue date: 2015", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "1 verkkoaineisto (50 sivua)", "language": null, "element": "format", "qualifier": "extent", "schema": "dc"}, {"key": "dc.format.mimetype", "value": "application/pdf", "language": null, "element": "format", "qualifier": "mimetype", "schema": "dc"}, {"key": "dc.language.iso", "value": "eng", "language": null, "element": "language", "qualifier": "iso", "schema": "dc"}, {"key": "dc.rights", "value": "In Copyright", "language": "en", "element": "rights", "qualifier": null, "schema": "dc"}, {"key": "dc.subject.other", "value": "metastatic colorectal cancer", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "personalized medicine", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "KRAS mutation testing", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "E-ice-COLD-PCR", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "pyrosequencing", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Plasma cell-free DNA in diagnostic KRAS mutation testing", "language": null, "element": "title", "qualifier": null, "schema": "dc"}, {"key": "dc.type", "value": "master thesis", "language": null, "element": "type", "qualifier": null, "schema": "dc"}, {"key": "dc.identifier.urn", "value": "URN:NBN:fi:jyu-201504141582", "language": null, "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.ontasot", "value": 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Jyv\u00e4skyl\u00e4", "language": "en", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Solu- ja molekyylibiologia", "language": "fi", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Cell and molecular biology", "language": "en", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.date.updated", "value": "2015-04-14T19:28:51Z", "language": null, "element": "date", "qualifier": "updated", "schema": "dc"}, {"key": "yvv.contractresearch.collaborator", "value": "public", "language": null, "element": "contractresearch", "qualifier": "collaborator", "schema": "yvv"}, {"key": "yvv.contractresearch.funding", "value": "5000", "language": null, "element": "contractresearch", 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