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Pharmacogenetic Studies of Paclitaxel in Ovarian Cancer: focus on interindividual differences in pharmacodynamics and pharmacokinetics
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
2007 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Ovarian cancer is one of the most common female cancer diseases in the world today and in Sweden more than 800 new cases are diagnosed every year. The standard treatment consists of chemotherapy with paclitaxel in combination with carboplatin after initial cytoreductive surgery. The response to treatment and the severity of adverse drug reactions after chemotherapy varies greatly among individuals, and one of the most important factors responsible for these differences is now recognized to be the genetic variability. One of the major obstacles to successful treatment is drug resistance. Several potential mechanisms have been suggested for the resistance to paclitaxel, such as mutations in the target protein β-tubulin, single nucleotide polymorphisms (SNPs) in the gene ABCB1, which encodes the transport protein P-glycoprotein. P-glycoprotein can mediate efflux of various drugs from cancer cells as well as from the circulation into the intestinal lumen, and overexpression and/or high activity leads to drug resistance and/or increased elimination. Another reason might be the high interindividual variability of paclitaxel plasma concentrations, which has been suggested to be influenced by variability in metabolic enzymes, such as CYP2C8 and CYP3A4, and transport proteins e.g. P-glycoprotein.

In the studies constituting this thesis we have investigated the possibilities of predicting the pharmacokinetics of paclitaxel as well as the tumor response and adverse drug reactions after chemotherapy in the preparation of personalized chemotherapy. We studied the correlation between the response and the presence of mutations in the dominant β-tubulin gene and SNPs in ABCB1. DNA from 40 ovarian tumors was screened for sequence variations in the β-tubulin gene without finding any, showing that β-tubulin mutations are rare and unlikely to be a clinically relevant resistance mechanism for paclitaxel. The SNPs G2677T/A and C3435T in the ABCB1 gene were determined in 53 ovarian cancer tumors from patients with poor (progressive disease or relapse within one year) or good (disease-free survival of more than one year) response to paclitaxel-carboplatin chemotherapy. Patients homozygously mutated for G2677T/A had a higher probability of responding to chemotherapy. There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment. No correlation was found for the C3435T variant.

By using a newly developed quantitative LC/MS method for the simultaneous determination of paclitaxel and its hydroxymetabolites in human plasma we assessed the individual elimination of paclitaxel in 33 ovarian cancer patients. The patients were genotyped for SNPs in the ABCB1, CYP2C8 and CYP3A4 genes and their in vivo CYP3A4 enzyme activity, tumor response and toxicity, especially the neurotoxicity, were determined. Patients heterozygous for G/A in position 2677 in ABCB1 had a significantly higher clearance of paclitaxel than patients with the wild type or homozygously mutated, but not compared to patients carrying the G/T alleles. A lower clearance of paclitaxel was also found for patients heterozygous for CYP2C8*3 when stratified according to the ABCB1 G2677T/A genotype. The CYP3A4 enzyme activity in vivo affected the relative influence of CYP2C8 and CYP3A4 on the metabolism, but not the total clearance of paclitaxel. The exposure to paclitaxel was correlated to the neurotoxicity, but not to the treatment response. In conclusion, our findings suggest that the SNP G2677T/A in the ABCB1 gene, but not β-tubulin mutations, might be a predictor for paclitaxel response and that the interindividual variability in paclitaxel pharmacokinetics might be predicted by ABCB1 and CYP2C8 genotypes and provide useful information for individualized chemotherapy.

Abstract [sv]

Ovarialcancer (äggstockscancer) är en av de vanligaste cancerformerna hos kvinnor i Sverige idag. Behandlingen består vanligen av tumörreducerande kirurgi följd av kemoterapi med paklitaxel och karboplatin. Målsättningen med detta avhandlingsarbete har varit att förbättra cytostatikabehandlingen (cellgiftsbehandlingen) med framförallt paklitaxel vid ovarialcancer genom att lägga grunden för individualisering av doser och förutsäga tumörsvaret vid behandlingen. Ett problem med dagens cancerbehandling är att många cancerceller så småningom blir resistenta mot olika cytostatika. För att angripa den mest resistenta cellen innan den induceras att öka uttrycket av, eller utveckla, fler resistensmekanismer vore det en fördel om vi före behandlingen kunde prediktera vilken dos av cytostatika som är bäst lämpad för individen samt om tumören kommer att reagera på behandlingen eller ej. En av de viktigaste faktorerna för skillnader i behandlingseffekt tros vara genetiska variationer mellan olika individer.

I våra studier har vi använt genetiska metoder för att studera om vi kan prediktera tumörsvaret vid behandlingen genom att bestämma mutationer i genen för paklitaxels målprotein, β-tubulin, samt bestämma genetiska variationer i ABCB1-genen, kodande för transportproteinet P-glykoprotein. Tanken är att ett förändrat målprotein eller en förändrad förmåga hos cancercellerna eller kroppen att transportera ut paklitaxel skulle leda till en skillnad i påverkan på tumören. DNA från 40 ovarialtumörer analyserades utan att en enda sekvensvariation hittades i genen för β-tubulin, vilket tyder på att genetiska förändringar i genen för β-tubulin sannolikt inte är en klinisk relevant resistensmekanism. De normalt förekommande genetiska variationerna G2677T/A och C3435T i ABCB1-genen bestämdes i DNA från 53 ovarialtumörer där behandlingen endera givit en bra (tumörfri minst ett år) eller dålig (progression av tumören eller tumörfri mindre än ett år) anti-tumöreffekt. Patienter som var dubbelmuterade i position 2677 dvs hade endera T/T eller T/A (A/A hittades inte i materialet) i denna position hade en högre sannolikhet att få ett bra anti-tumörsvar vid behandlingen. Även antalet muterade baser påverkade utfallet, ju fler muterade baser i position 2677, desto högre sannolikhet att få ett bra svar på behandlingen. Andelen T eller A var också högre i den grupp av patienter som fått en lyckad behandling.

För att kunna prediktera patientens individuella förmåga att bryta ner paklitaxel studerade vi inverkan av sekvensvariationer i generna för de nedbrytande enzymerna, CYP2C8 och CYP3A4, och transportproteinet P-glykoprotein (genen ABCB1) på eliminationen av läkemedlet i kroppen. Vi utvecklade en metod för att mäta paklitaxelkoncentrationerna i blodet och använde den för att studera hur snabbt 33 ovarialcancer patienter eliminerade cytostatikat från blodbanan. Hos dessa patienter bestämde vi förekomsten av kända genetiska variationer i generna ABCB1, CYP2C8 och CYP3A4 samt deras CYP3A4 enzymaktivitet i kroppen. Biverkningarna och tumörsvaret vid behandlingen utvärderades också. Eliminationen av paklitaxel hos dessa patienter var beroende av vilken bas som fanns i position 2677 i ABCB1-genen och förekomsten av den genetiska varianten CYP2C8*3. Enzymaktiviteten hos CYP3A4 kunde inte påvisas påverka eliminationen av paklitaxel utan snarare vilket enzym, CYP2C8 eller CYP3A4, som var relativt dominant i respektive patient. Exponeringen av paklitaxel korrelerade till den neurologiska påverkan som patienten orsakades av cytostatikat, men kunde inte korreleras till tumörsvaret vid slutet av cytostatikabehandlingen.

Sammanfattningsvis ger patientens genetiska variationer i ABCB1, men inte β-tubulin, information om behandlingsutfallet. Genetiska variationer i CYP2C8 och ABCB1 påverkar patientens förmåga att eliminera paklitaxel och kan förhoppningsvis användas för att individualisera doserna. Vår förhoppning är att resultaten i denna avhandling skall kunna användas för att individualisera och ytterligare förbättra cytostatikabehandlingen vid ovarialcancer.

sted, utgiver, år, opplag, sider
Institutionen för medicin och vård , 2007. , s. 100
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 982
Emneord [en]
pharmacogenetics, paclitaxel, LC/MS, Taxol, ovarian cancer, pharmacokinetics, CYP2C8, CYP3A4, ABCB1
Emneord [sv]
äggstockscancer, paklitaxel
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-8134ISBN: 978-91-85715-84-8 (tryckt)OAI: oai:DiVA.org:liu-8134DiVA, id: diva2:22995
Disputas
2007-03-02, Berzeliussalen, HU-entren, Hälsouniversitetet, Linköping, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2007-01-26 Laget: 2007-01-26 Sist oppdatert: 2018-01-13
Delarbeid
1. β-tubulin mutations in ovarian cancer using single strand conformation analysis – risk of false positive results from paraffin embedded tissues
Åpne denne publikasjonen i ny fane eller vindu >>β-tubulin mutations in ovarian cancer using single strand conformation analysis – risk of false positive results from paraffin embedded tissues
Vise andre…
2006 (engelsk)Inngår i: Cancer Letters, ISSN 0304-3835, Vol. 236, nr 1, s. 148-154Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Mutations in the β-tubulin gene have been proposed as a resistance mechanism to paclitaxel. We therefore investigated the presence of mutations in the β-tubulin M40 gene in 40 ovarian tumours (16 paraffin-embedded and 24 freshly frozen) selected for good or poor response to chemotherapy with paclitaxel or non-tubulin-affecting regimens. The presence of mutations was investigated using single strand conformation analysis followed by sequencing of the products with altered mobility. No sequence variants in the exons of the β-tubulin M40 gene were detected. Non-reproducible shifts were identified, in eight out of 16 paraffin embedded samples. This may explain some of the previously published discrepancies. In conclusion, sequence variants in the β-tubulin M40 gene are rare and are unlikely to be a clinically relevant explanation of resistance to paclitaxel.

Emneord
β-tubulin; Paclitaxel; Ovarian cancer; Mutation analysis; SSCA
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-14242 (URN)10.1016/j.canlet.2005.05.025 (DOI)
Merknad
Original Publication: Henrik Green, Per Rosenberg, Peter Söderkvist, György Horvath and Curt Peterson, β-tubulin mutations in ovarian cancer using single strand conformation analysis – risk of false positive results from paraffin embedded tissues, 2006, Cancer Letters, (236), 1, 148-154. http://dx.doi.org/10.1016/j.canlet.2005.05.025 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/ Tilgjengelig fra: 2007-01-26 Laget: 2007-01-26 Sist oppdatert: 2010-05-28
2. mdr-1 single nucleotide polymorphisms in ovarian cancer tissue – G2677T/A correlates with response to paclitaxel chemotherapy
Åpne denne publikasjonen i ny fane eller vindu >>mdr-1 single nucleotide polymorphisms in ovarian cancer tissue – G2677T/A correlates with response to paclitaxel chemotherapy
Vise andre…
2006 (engelsk)Inngår i: Clinical Cancer Research, ISSN 1078-0432, Vol. 12, nr 3 pt 1, s. 854-859Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Purpose: P-glycoprotein, encoded by the mdr-1 gene, confers multidrug resistance to a variety of antineoplastic agents, e.g., paclitaxel. Recently, different polymorphisms in the mdr-1 gene have been identified and their consequences for the function of P-glycoprotein, as well as for the treatment response to P-glycoprotein substrates, are being clarified. We analyzed the allelic frequencies at polymorphic sites G2677T/A and C3435T in ovarian cancer patients with good or poor response to treatment with paclitaxel in combination with carboplatin in order to evaluate their predictive values.

Experimental Design: Fifty-three patients were included in the study; 28 of them had been relapse-free for at least 1 year and 25 had progressive disease or relapsed within 12 months. A reference material consisting of 200 individuals was also analyzed. The genotypes of each single nucleotide polymorphism (SNP) were determined using Pyrosequencing.

Results: The G2677T/A SNP was found to significantly correlate with treatment outcome. The probability of responding to paclitaxel treatment was higher in homozygously mutated patients (T/T or T/A; Fisher's exact test; P < 0.05). The frequency of the T or A alleles was also higher in the group of patients who had a good response (P < 0.05). There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment (Χ2 test for linear-by-linear association; P = 0.03). However, the C3435T SNP was not found to correlate to treatment outcome.

Conclusions: The mdr-1 polymorphism G2677T/A in exon 21 correlates with the paclitaxel response in ovarian cancer and may be important for the function of P-glycoprotein and resistance to paclitaxel and provide useful information for individualized therapy.

HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-14243 (URN)10.1158/1078-0432.CCR-05-0950 (DOI)
Merknad
Original Publication: Henrik Green, Peter Söderkvist, Per Rosenberg, György Horvath and Curt Peterson, mdr-1 single nucleotide polymorphisms in ovarian cancer tissue – G2677T/A correlates with response to paclitaxel chemotherapy, 2006, Clinical Cancer Research, (12), 3 pt 1, 854-859. http://dx.doi.org/10.1158/1078-0432.CCR-05-0950 Copyright: American Association for Cancer Research, Inc. http://www.aacr.org/ Tilgjengelig fra: 2007-01-26 Laget: 2007-01-26 Sist oppdatert: 2010-05-28
3. Measurement of paclitaxel and its metabolites in human plasma using liquid chromatography/ion trap mass spectrometry with a sonic spray ionization interface
Åpne denne publikasjonen i ny fane eller vindu >>Measurement of paclitaxel and its metabolites in human plasma using liquid chromatography/ion trap mass spectrometry with a sonic spray ionization interface
2006 (engelsk)Inngår i: Rapid Communications in Mass Spectrometry, ISSN 1097-0231, Vol. 20, nr 14, s. 2183-2189Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

A quantitative liquid chromatography/ion trap mass spectrometry method for the simultaneous determination of paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel in human plasma has been developed and validated. 6α-,p-3'-Dihydroxypaclitaxel was also quantified using paclitaxel as a reference and docetaxel as an internal standard. The substances were extracted from 0.500 mL plasma using solid-phase extraction. The elution was performed with acetonitrile and the samples were reconstituted in the mobile phase. Isocratic high-performance liquid chromatography analysis was performed by injecting 50 µL of reconstituted material onto a 100 × 3.00 mm C12 column with a methanol:1% trifluoroacetic acid/ammonium trifluoroacetate in H2O 70:30 mobile phase at 350 µL/min. The [M+H]+ ions generated in the sonic spray ionization interface were isolated and fragmented using two serial mass spectrometric methods: one for paclitaxel (transition 854 → 569 & 551) and the dihydroxymetabolite (transition 886 → 585 & 567) and one for the hydroxy metabolites (transition 870 → 585 & 567; transition 870 → 569 & 551) and docetaxel ([M+Na]+, transition 830 → 550). Calibration curves were created ranging between 0.5 and 7500 ng/mL for paclitaxel, 0.5 and 750 ng/mL for 6α-hydroxypaclitaxel, and 0.5 and 400 ng/mL for p-3'-hydroxypaclitaxel. Adduct ion formation was noted and investigated during method development and controlled by mobile phase optimization. In conclusion, a sensitive method for simultaneous quantification of paclitaxel and its metabolites suitable for analysis in clinical studies was obtained.

HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-14244 (URN)10.1002/rcm.2567 (DOI)
Merknad
This is the pre-peer-reviewed version of: Henrik Green, Karin Vretenbrant (Öberg), Björn Norlander and Curt Peterson, Measurement of paclitaxel and its metabolites in human plasma using liquid chromatography/ion trap mass spectrometry with a sonic spray ionization interface, 2006, Rapid Communications in Mass Spectrometry, (20), 14, 2183-2189. which has been published in final form at: http://dx.doi.org/10.1002/rcm.2567 Copyright: John Wiley and Sons, Ltd http://eu.wiley.com/WileyCDA/Brand/id-35.html Tilgjengelig fra: 2007-01-26 Laget: 2007-01-26 Sist oppdatert: 2010-05-28
4. Pharmacogenetics of Paclitaxel in the Treatment of Ovarian Cancer – a Pilot Study in Preparation of Individualized Chemotherapy
Åpne denne publikasjonen i ny fane eller vindu >>Pharmacogenetics of Paclitaxel in the Treatment of Ovarian Cancer – a Pilot Study in Preparation of Individualized Chemotherapy
Vise andre…
2007 (engelsk)Inngår i: Clinical Cancer Research, ISSN 1078-0432Artikkel i tidsskrift (Fagfellevurdert) Submitted
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-14245 (URN)
Tilgjengelig fra: 2007-01-26 Laget: 2007-01-26 Sist oppdatert: 2015-02-03

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