COMMUNICATION

ABCB1 G1199A Polymorphism and OvarianCancer Response to Paclitaxel

HENRIK GREEN,1 PETER SODERKVIST,2 PER ROSENBERG,3 GYORGY HORVATH,4 CURT PETERSON1

1Division of Clinical Pharmacology, Faculty of Health Sciences, Department of Medicine and Care,Linkoping University, SE-581 85 Linkoping, Sweden

2Division of Cell Biology, Faculty of Health Sciences, Department of Biomedicine and Surgery,Linkoping University, Linkoping, Sweden

3Department of Oncology, Linkoping University Hospital, SE-581 85 Linkoping, Sweden

4Department of Oncology, Sahlgrenska Academy at Goteborg University, Gothenburg, Sweden

Received 2 May 2007; accepted 2 July 2007

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21169

Corresponde12-29; Fax: þ46-

Journal of Pharm

� 2007 Wiley-Liss

ABSTRACT: P-glycoprotein (P-gp), encoded by the ABCB1 gene, confers multi-drugresistance to a variety of antineoplastic agents, for example, paclitaxel. Recently, theG1199T/A polymorphism in the ABCB1 gene was shown to be important for the functionof P-gp as well as for the resistance to several chemotherapeutic agents in vitro.We analyzed the allelic distribution of the G1199T/A and other polymorphisms in exons11 and 12 of the ABCB1 gene in ovarian cancer patients treated with paclitaxel andcarboplatin in order to evaluate their predictive value in vivo. The SNPs C1236T,G1199T/A, and A1308G were determined using Pyrosequencing in 51 patients withadvanced ovarian cancer and correlated to the progression free survival. The G1199T/ASNP was found to affect the progression free survival. Although only two heterozygous(G/A) patients were found their mean progression free survival was only 2 months ascompared to 19 months for the wild-type patients. This is in accordance with the higherresistance for the 1199A genetic variant found in vitro. Genotyping of the ABCB1 genemay be important for determining the tumor resistance to paclitaxel and provide usefulinformation for individualized therapy. � 2007 Wiley-Liss, Inc. and the American Pharmacists

Association J Pharm Sci 97:2045–2048, 2008

Keywords: ABCB1; paclitaxel; ovarian

cancer; G1199T/A

INTRODUCTION

Crouthamel et al.1 reported that the singlenucleotide polymorphism (SNP) G1199T/A inABCB1 is important for the in vitro resistance to

nce to: Henrik Green (Telephone: þ46-13-22-13-10-41-95; E-mail: henrik.green@imv.liu.se)

aceutical Sciences, Vol. 97, 2045–2048 (2008)

, Inc. and the American Pharmacists Association

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several chemotherapeutic agents including pacli-taxel. The transport activity of P-glycoprotein(P-gp), encoded by the ABCB1 gene, affects thepharmacokinetics of paclitaxel in several ways.Overexpression of P-gp on tumor cells resultingin an enhanced efflux of the drug is a knownin vitro resistance mechanism for paclitaxel.2,3

Clinical resistance and poor response have alsobeen correlated with a high expression of P-gpin tumors.4–6 Studies in knock-out mice and

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Table 1. Patient and Tumor Characteristics

Median age (range) 62 (40–75)FIGO stage

II 4III 41IV 6

HistologySerous 32Mucinous 2Endometrioid 6Undifferentiated 3Unknown 8

Tumor grade (FIGO)Well diff 1Moderately diff 11Poorly diff 36Unknown 3

Median PFS (months) 15

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co-administrations of P-gp inhibitors have shownthat P-gp limits the oral uptake of paclitaxel andmediates the direct excretion of the drug from thesystemic circulation as well as the absorption (orre-absorption) of paclitaxel in the intestine.7–9 Thepenetration of paclitaxel to the cerebrospinalfluid has also been shown to be dependent onthe P-gp.10,11

Recently, we showed that the SNP G2677T/A correlated with the response to paclitaxeltreatment in ovarian cacner,12 although otherstudies have given different results.13,14 Theimportance of P-gp transport for the effects ofpaclitaxel treatment, our previous findings andthe shown impact of the G1199T/A SNP on thein vitro resistance led us to investigate the clinicalimportance of this SNP during paclitaxel treat-ment of ovarian cancer.

PFS, progression free survival.

MATERIALS AND METHODS

To evaluate the relationship between the responseto paclitaxel treatment and the G1199T/A andothers SNPs in exons 11 and 12 of the ABCB1 gene(G1199T/A, C1236T, and A1308G), we retrospec-tively identified the SNPs in 51 epithelial ovariantumors (FIGO stage IIB-IV) included in a previousstudy.12 All three SNPs were investigated due totheir close proximity to each other. After primarysurgery all patients had been treated withpaclitaxel at a dose of 175 or 135 mg/m2 (n¼ 5)in combination with carboplatin for at least fourcycles. The patient and tumor characteristics arepresented in the Table 1.

Eleven tumors were collected from paraffinembedded tissues stored at the Division ofMolecular and Immunological Pathology, Linkop-ing University, and 40 tumors were fresh-frozenand obtained from a bio-bank at the Departmentof Oncology, Sahlgrenska Academy at GoteborgUniversity. This study was approved by theregional ethics committees in Linkoping, Sweden.

DNA Isolation, PCR, and Pyrosequencing

Genomic DNA was isolated using QIAamp1 DNAmini kits (VWR International, Stockholm,Sweden) according to the manufacturer’s protocol.A 392 bp fragment of exons 11 and 12 inthe ABCB1 gene was amplified using theforward primer biotin-GAGTGGGCACAAACCA-GATA and reverse primer GTCATCTCACCATC-

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CCCTCT. The reaction was based on the Hot-StarTaq master mixture (VWR International)and carried out on a Mastercycler gradient(Eppendorf) in a total volume of 25 mL, aspreviously described.12 For the real-time sequen-cing of the PCR products and SNP analysis aPyrosequencing PSQ96MA (Pyrosequencing AB,Uppsala, Sweden) was used. In short, single-stranded DNA was isolated from the PCR reac-tions using the Pyrosequencing Vacuum PrepWorkstation (Pyrosequencing AB) and trans-ferred into a 96-well plate. Three sequencingprimers, CTTTTCGAGATGGGTAA for G1199T/A, TGCACCTTCAGGTTCA for C1236T, and ATA-GAGCCTCTGCATCA for A1308G, were annealedto the single stranded DNA by heating the sampleto 808C for 2 min and then allowing it to cool toroom temperature. The plate was then trans-ferred to the PSQ96MA where the real timesequencing took place by dispensing the nucleo-tides in the following order ACGATGCTGCACTG-TATCATCTATCA. All primers were obtainedfrom Invitrogen (Paisley, UK).

Statistical Analysis

The statistical analysis was performed withthe SPSS software package version 14.0 (SPSS,Inc., Chicago, IL). Kaplan–Meier plots were usedto visualize and study differences in fractions ofpatients with progression-free survival (PFS)between different genotypes.

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ABCB1 SNP G1199T/A AND RESPONSE TO PACLITAXEL 2047

RESULTS

The PCR amplification of exons 11 and 12 ofthe ABCB1 gene resulted in single products ofexpected sizes (as judged by agarose gel electro-phoresis). The pyrograms during real-timesequencing showed peaks corresponding to thetheoretical outcome. The C1236T SNP was foundat an allele frequency of 42% in Hardy–Weinbergdistribution. Only the genetic variant 1308A wasfound in the material and two patients were foundto be G/A heterozygous for the G1199T/A SNP.Both these patients had stage III serous tumorsthat were poorly differentiated. Due to the smallnumber of patients, no significant correlationscould be shown between the tumors’ stage/gradeand the progression free survival.

Patients G/A heterozygously mutated for theSNP G1199T/A were found to have a shorterprogression free survival (mean PFS 2 months)as compared to the wild-type patients (mean PFS19 months), see Figure 1. The SNP C1236T did notcorrelate to the treatment response.

DISCUSSION

As a follow-up of the findings by Crouthamelet al. that the genetic variant G1199T/A inABCB1 is important for the resistance to severalchemotherapeutic drugs in vitro, we investigat-ed the impact of this SNP on the response to

Figure 1. The effect of the ABCB1 SNP G1199T/A onthe progression free survival in patients with advancedovarian cancer treated with paclitaxel in combinationwith carboplatin.

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paclitaxel-carboplatin treatment in ovariancancer. Although we only found two heterozygouspatients (1199G/A), they showed significant clin-ical resistance to paclitaxel-carboplatin treatmentand a shorter progression free survival. This isin accordance with the 2.4 higher IC50 valuefor paclitaxel for this genetic variant and thelower intracellular concentration of doxorubicinfound in HEK-cells expressing the ABCB1 1199Avariant as compared to the wild type transporter.1

The increased drug resistance of cells expressingthe 1199A variant has also been shown for otherchemotherapeutic agents such as vincristine andvinblastine.1,15

The ABCB1 genotype especially the SNPG2677T/A has previously been shown to beimportant for the clinical effect of paclitaxel. Ina study by Green et al. patients homozygouslymutated (T/T or T/A) at position 2677 had a higherchance of responding to treatment. The number ofmutated alleles were also shown to influence theresponse which improved with increasing numberof variant alleles.12 Two groups later publishedresults from similar studies, but with differentoutcome.13,14 However, the differences might beexplained by variations in the composition and/orsubdivision of tumor material and/or patients.16

In vitro a slightly higher efflux of bodipy-FL-paclitaxel has also been shown in HeLa cellsexpressing the wild type P-gp than cells carrying aplasmid containing the 2677T variant.17 In thissystem no effect of the G1199A genetic variantwas seen on the efflux of paclitaxel. However,the fluorescent bodipy modification of the P-gpsubstrates and the usage of only one substrateconcentration may have influenced the abilityto distinguish differences between the geneticvariants of the ABCB1 gene.

In conclusion, this study indicates that the SNPG1199T/A in ABCB1 influences the responseto paclitaxel-carboplatin treatment in ovariancancer. However, the potential importance ofthe ABCB1 genotype as a predictive marker forpaclitaxel treatment is still elusive and futurestudies will hopefully clarify the significance ofthese SNPs.

ACKNOWLEDGMENTS

This study was supported by grants fromthe Swedish Cancer Society, Gunnar Nilsson’sCancer Foundation and the County Council inOstergotland.

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