The Pharmacogenomics Journal (2014), 1–10 & 2014 Macmillan Publishers Limited All rights reserved 1470-269X/14 www.nature.com/tpj

REVIEW

Genetic markers for prediction of treatment outcomes in ovarian cancer E Caiola, M Broggini and M Marabese Drug resistance in epithelial ovarian cancer (EOC) limits the efficacy of therapies for this malignancy. This phenomenon might be partially explained by strong inter-individual genomic heterogeneity. Single nucleotide polymorphisms (SNPs) located in specific genes involved in platinum-based drugs inactivation and the metabolism and extrusion of taxanes could be relevant in terms of drugs response prediction. In this paper, we review candidates for genetic markers of treatment outcomes in ovarian cancer. Although an association between SNPs and response to chemotherapy has been detected in several studies, no clear conclusions can be drawn because of conflicting results. Genetic variants in determining response to chemotherapy and clinical outcome need to be clarified in EOC to allow stratification of patients, which would help optimize therapy. The Pharmacogenomics Journal advance online publication, 8 July 2014; doi:10.1038/tpj.2014.32

INTRODUCTION Epithelial ovarian cancer (EOC) represents the sixth most common cause of death due to cancer in women and is the leading cause of death from gynecological malignancies in the developed world. The majority of patients are diagnosed with advanced disease, and this limits the possibility of cure with 5-year survival rates lower than 30%.1,2 The high mortality rate can be associated with the nonspecific nature of the disease symptoms and the lack of screening tools.3 EOC is a very heterogeneous disease from the molecular point of view, and there is an urgent need for biomarkers that may aid stratification of patients who may benefit from therapies.4 With the launch of platinum-based chemotherapy, the response rate of first-line therapy of EOC improved to 70%, and a significant fraction of women survived 5 years. Although initially EOC is a therapy-responsive type of malignancy, chemotherapy fails to completely cure patients.2 Tumor response to chemotherapy changes from population to population and even from patient to patient.5 Many studies demonstrated that drug response is dependent on genetic factors. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation, and they occur at high frequency in the genome. One polymorphic site is present at every 300th nucleotide, which means there are almost 10 million SNPs in the human genome.6 However, although some SNPs have been associated with phenotype and response to therapy, the vast majority of SNPs have not yet been allocated a clear function. Pharmacogenomics, the study of how genetic variation interferes with drug response, aims at improving the definition of patients who may benefit from a particular type of treatment considering their genetic profile. In this review, we analyze the state of the art of EOC pharmacogenomics and present studies that describe SNPs interfering with drug response. A systematic PubMed search was conducted using the following MESH terms: ovarian AND cancer AND polymorphism AND survival OR response NOT review. The selected

hits were further limited to English language papers from 1 January 2007 to 30 June 2013. The search resulted in 170 hits. Among these 170 hits, we focused our attention on 33 papers investigating genes related to nucleotide excision repair (NER; ERCC1, XPA, XPC, XPD, XPG and CSA), base excision repair (BER; XRCC1), drug transporters (ABCB1) and drug-metabolizing enzymes (GSTP1, GSTT1, GSTM1, CYP2C8 and CYP3A4). Among these, six studies were not considered for this review and are listed in Supplementary Table 1 with reasons of exclusion. Among the remaining 137 papers, 58, which analyzed different SNPs in association with outcomes or therapy response in EOC, were used to generate the comprehensive Supplementary Table 2, whereas 79 were excluded for the reasons reported in the same table. Supplementary Table 3 describes all SNPs cited in this review with their ‘NCBI Database of single nucleotide polymorphisms’ identification number.

POLYMORPHISMS IN DNA DAMAGE REPAIR SYSTEMS Platinum-based chemotherapeutic agents have a broad range of activity against ovarian cancer. Cisplatin and its analog carboplatin, the most used platinum compounds, work by binding to DNA and forming DNA adducts, leading to intra-strand or inter-strand cross-links that disrupt the structure of the DNA molecule. Alterations in the DNA structure determine an alert status in cells that triggers different DNA repair systems. There are several DNA repair mechanisms that can be activated when DNA is injured: NER, BER, homologous recombination, Fanconi anemia, mismatch repair, double-strand break repair and direct repair.7 These systems have been suggested to be involved in platinum adduct repair. Mutations in genes associated with some of these mechanisms, such as BRCA1 and BRCA2,8,9 have been correlated to cisplatin/carboplatin sensitivity. Here we consider only those DNA repair systems for which evidence has been published that polymorphisms can potentially have an important role.

Laboratory of Molecular Pharmacology, Department of Oncology, IRCCS–Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy. Correspondence: Dr M Broggini, Laboratory of Molecular Pharmacology, Department of Oncology, IRCCS–Istituto di Ricerche Farmacologiche ‘Mario Negri’, via La Masa 19, 20156 Milan, Italy. E-mail: [email protected]. Received 18 December 2013; revised 29 April 2014; accepted 22 May 2014

Genetic markers for prediction in ovarian cancer E Caiola et al

2 All the proteins belonging to the NER system have been characterized, and the mechanism via which NER repairs the damaged DNA has been elucidated. The reaction starts by recruitment of factors to the damaged locus. Xeroderma pigmentosum complementation group C (XPC) complexes with hHR23B to initiate the repair. Once the damaged site has been identified, TFIIH, XPB (ERCC3) and XPD (ERCC2) form a complex, which mediates the separation of the DNA double helix at the site of the lesion. XPA verifies that DNA helices are in an open frame. Replication protein A then stabilizes the opened DNA complex and places the XPG (ERCC5), an endonuclease that excises the 30 end of the damaged DNA, in suitable position. Excision repair cross-complementation group 1 (ERCC1) complexes with XPF (ERCC4) to form the second endonuclease, which is responsible for the DNA excision at the 50 end of the lesion. This process eliminates 24–32 nucleotides, and the previous damaged site is now available for replication factors, activated to fill in the gap.10,11 BER is a DNA repair system responsible for maintaining genome integrity. Chemotherapeutic agents cause thousands of DNA lesions and strand breaks in tumor cells, which are substrates of BER. BER can be divided into two sub-pathways depending on the type of lesion: short-patch and long-patch BER. In both pathways, a DNA glycosylase cleaves the damaged base creating an apurinic or apyrimidinic site. DNA glycosylase APE1 removes the damaged base by nicking the DNA at both 50 and 30 residues that have to be removed. In short-patch BER, polymerase beta replaces the missing nucleotide, and the gap is sealed by the DNA ligase III/XRCC1/PARP1 complex. In long-patch BER, a small oligonucleotide is synthesized by polymerase beta. An endonuclease then removes the damaged strand, allowing the newly synthesized oligonucleotide to fill the gap, and DNA ligase I seals the gap. Several SNPs have been identified affecting the genes involved in previously cited DNA repair systems, and in the following chapters we will review and discuss the role of SNPs previously associated with EOC outcome and/or response to therapy. In particular, we focus our attention on the most frequently investigated SNPs, to compare, for the same SNP, the results obtained in different studies, and temptatively draw some conclusions about its potential prognostic value in ovarian cancer. Nucleotide excision repair Excision repair cross-complementation group 1. ERCC1 is a ratelimiting protein in the NER pathway. It forms a heterodimeric protein complex with xeroderma pigmentosum group F-complementing protein (XPF) to carry out the 50 incision in the presence of a DNA lesion. Reduction of ERCC1 function may predispose individuals to cancer due to the inefficiency of DNA damage removal. Preclinical research associated resistance to platinumbased drugs with ERCC1 expression, and elevated activity of NER and high levels of ERCC1 mRNA have been found in tumor cell lines resistant to cisplatin.12 Features of the ERCC1 protein in addition to its function in NER have been reported. DNA double-strand breaks, induced by cisplatin and other drugs, can be repaired as stated above, by homologous recombination or nonhomologous end joining. Although the main HR pathway is error free, nonhomologous end joining involves the ligation of free DNA ends in a mechanism resulting in error-prone repair. The role of ERCC1 in double-strand break repair was initially shown in budding yeast and then confirmed in mammalian cells, in which the mutant of ERCC1 caused sensitivity to double-strand breaks. The absence of ERCC1 function attenuates the efficacy of both the homologous recombination and nonhomologous end joining pathways. The role of ERCC1 in these repair mechanisms is to remove the nonhomologous 30 single-stranded flaps at broken ends before rejoining. This process is performed by a sub-pathway of The Pharmacogenomics Journal (2014), 1 – 10

homologous recombination, the error-prone single-strand annealing or by the microhomology-mediated end-joining subpathway belonging to nonhomologous end joining.13 Several SNPs among the 1000 reported for this gene have been identified to affect ERCC1 gene expression and function.14 Among them, the nucleotide 19007 C/T polymorphism (rs11615) in exon 4 is a silent polymorphism encoding the same amino-acid asparagine (Asn118Asn) and affecting ERCC1 mRNA levels,15 whereas the nucleotide 8092 C/A polymorphism (rs3212986) is located in the 30 -untranslated region (UTR) of ERCC1 mRNA influencing mRNA stability.16 These SNPs have been extensively studied and associated with altered outcome in platinum-based chemotherapy in multiple malignancies including EOC with uneven results. At least 14 papers have been published between 2007 and 2013 analyzing the more common ERCC1 polymorphisms (Table 1). Six of them,17–22 including results obtained from more than 2000 patients, suggest that there is no association between outcome and polymorphisms Asn118Asn and 8092 C/A. In addition, White et al.23 investigated a different ERCC1 SNP (rs735482) in more than 10 000 cases and could not establish an association between this variant and outcomes. In contrast, eight more papers were able to assess a role for these SNPs. Smith et al., in 2007, investigated 178 patients treated with platinum chemotherapy with or without paclitaxel for the codon 118 SNP. Among the 103 patients treated with platinum without paclitaxel, the C/C genotype, compared with T-containing genotypes, was associated with greater risk of disease progression (hazard ratio (HR) ¼ 1.95; 95% CI (1.00–3.79); P ¼ 0.051) and death (HR ¼ 2.01; 95% CI (1.06–3.82); P ¼ 0.033). For the 75 remaining patients treated with platinum–paclitaxel combination chemotherapy, the C/C genotype was not associated with poor outcome, suggesting that paclitaxel may help to alleviate ERCC1-related platinum resistance.24 Cancer antigen 125 (CA-125) was introduced into clinical practice with the aim to monitor treatment of ovarian cancer in patients on first-line chemotherapy who have reached clinical complete remission. In patients with known ovarian cancer, a rise in CA-125 is a good indicator of residual disease.25 With this in mind, Steffensen et al.26 tested the role of the codon 118 SNP in a cohort of 159 patients categorized as CA-125 responders or nonresponders, treated with a combination of carboplatin and cyclophosphamide. Patients harboring the T/T genotype had a better response to chemotherapy, being 95% of the patients with this genotype categorized as CA-125 responders compared with 80 and 83% for the C/C and C/T genotype, respectively (P ¼ 0.045). However, the same polymorphism was not associated with a statistically significantly better overall survival (OS).26 Krivak et al. performed SNP analyses by direct pyrosequencing at codon 118 and nucleotide 8092 in ERCC1. DNA was extracted from 233 women who participated in the GOG (Gynecologic Oncology Group) phase III protocol—172 aimed at comparing intraperitoneal vs intravenous treatment with cisplatin plus paclitaxel. Adjusted Cox regression analysis revealed that the codon 118 SNP was not significantly associated with disease progression or death. Women showing the C/A or A/A genotypes compared with the C/C genotype at nucleotide 8092 had an increased risk of disease progression (HR ¼ 1.44; 95% CI (1.06–1.94); P ¼ 0.018) and of death (HR ¼ 1.50; 95% CI (1.07–2.09); P ¼ 0.018). Median progression-free survival (PFS) and OS were 6 and 17 months shorter for a women with the C/A or A/A genotypes vs the C/C genotype, respectively.27 Kim et al. confirmed these data in a Korean sample. The multivariate Cox proportional hazard regression analysis performed on 118 patients who underwent staging laparotomy followed by adjuvant chemotherapy using taxanes and platinum compounds showed that the C/A or A/A genotypes in ERCC1 nucleotide 8092 were prognostic factors for poor PFS (adjusted HR ¼ 1.94; 95% CI (1.07–3.51); P ¼ 0.030). No role was detected for codon 118 SNP.28 In 2011, a couple of papers & 2014 Macmillan Publishers Limited

Genetic markers for prediction in ovarian cancer E Caiola et al

3 Table 1.

Pharmacogenetics studies on codon 118 Asn/Asn and nucleotide 8092 C/A polymorphisms of ERCC1 in EOC N

Reference 17

Ethnicity

Treatment

Marsh et al. Smith et al.24

914 178

Not specified Not specified

Carboplatin Platinum compounds with or without paclitaxel

Steffensen et al.26

159

Not specified

Carboplatin and cyclophosphamide

Krivak et al.27

233

Caucasian 90%; other 10%

Intraperitoneal vs intravenous treatment with cisplatin plus paclitaxel

Kim et al.28

118

Asian

Taxanes and platinum compounds

Khrunin et al.18 Krivak et al.29

104 278

Cisplatin and cyclophosphamide Carboplatin and paclitaxel-based combination

Bo¨smu¨ller et al.19 Peethambaram et al.20 Khrunin et al.21

41 445

Caucasian Caucasian 90%; other 10% Not specified Not specified

30

87

Yan et al.

209

Fleming et al.22

139

Caiola et al.31 Moxley et al.32

332 107

Yakut; Caucasian Asian Caucasian 90%; other 10% Caucasian Caucasian 80%; other 20%

Codon Asn118Asn

Nucleotide 8092 C/A

No differences PFS, C/C vs T/  HR ¼ 1.95; P ¼ 0.051 OS, C/C vs T/  HR ¼ 2.01; P ¼ 0.033 CA-125 responders: T/T ¼ 95%, T/C ¼ 83%, C/C ¼ 80%; P ¼ 0.045 No differences

No differences Not tested Not tested

No differences OS, T/  vs C/C HR ¼ 0.68; P ¼ 0.025

PFS, A/  vs C/C HR ¼ 1.44; P ¼ 0.018 OS, A/  vs C/C HR ¼ 1.50; P ¼ 0.018 PFS, A/  vs C/C HR ¼ 1.94; P ¼ 0.030 No differences No differences

No differences Failed to be assessed

No differences No differences

Cisplatin and cyclophosphamide

No differences

No differences

Cisplatin or carboplatin plus cyclophosphamide or paclitaxel

No differences

Platinum based

PFS, T/T vs C/  HR ¼ 3.34 OS, T/T vs C/  HR ¼ 2.87 R, T/T vs C/  OR ¼ 32.26 No differences

Platinum based Platinum based

No differences No differences

Carboplatin and taxane Platinum based

No differences

No differences No differences PFS, A/A vs C/  , 25 vs 9 months; Po0.06 OS, A/  vs C/C, 45 vs 32 months; P ¼ 0.06

Abbreviations: EOC, epithelial ovarian cancer; HR, hazard ratio; OR, overall response; OS, overall survival; PFS, progression-free survival; R, response.

investigated the role of the two ERCC1 SNPs in EOC patients. Krivak et al. performed pyrosequencing to examine SNPs in codon 118 and C8092A in ERCC1 in the DNA of patients in the GOG phase III protocol (GOG-182) treated with carboplatin- and paclitaxelbased combination chemotherapy. The Kaplan–Meier method and adjusted Cox regression modeling were used to examine associations between ERCC1 polymorphisms and PFS and OS. Patients with the C/T-T/T vs C/C genotype in codon 118 in ERCC1 (n ¼ 278) were at a reduced risk of death (HR ¼ 0.68; 95% CI (0.49– 0.95); P ¼ 0.025). Women with the C/A-A/A genotype as compared with those with the C/C genotype (n ¼ 280) in C8092A in ERCC1 showed a trend suggesting an increased risk of death (HR ¼ 1.29; 95% CI (0.97–1.72); P ¼ 0.077).29 Yan et al. analyzed the outcome of 209 EOC patients treated with cisplatin or carboplatin plus either cyclophosphamide or paclitaxel relative to the same ERCC1 most common SNPs. The codon 118 Asn/Asn polymorphism was significantly associated with response to treatment. Patients harboring the T/T genotype had a significantly decreased response to platinum-based treatment (overall response (OR) ¼ 32.26; 95% CI (3.66–250.00)). Cox multivariate analysis highlighted that patients with the T/T genotype had an increased risk of disease progression (HR ¼ 3.34; 95% CI (1.77– 6.29)) and death (HR ¼ 2.87; 95% CI (1.38–5.96)) when compared with those patients harboring the C/C genotype. In contrast, the C8092A polymorphism was not associated with OS, PFS or response. The possible role of the combined genotypes of polymorphisms at codon 118 and C8092A was also investigated. The results of the haplotype analysis revealed that patient carrying T/T at codon 118 and C/C in position 8092 had a shorter survival time than patients with the C/C-C/C genotypes.30 & 2014 Macmillan Publishers Limited

Recently a couple of papers were published on this topic. Caiola et al. analyzed the role of codon 118 and nucleotide 8092 in ERCC1 in 332 ovarian cancer samples divided into early (I/II) and late stages (III/VI). The codon 118 polymorphism was not statistically significantly correlated to any clinical outcome in early- and advanced-stage patients. When compared with the A/A genotype, the group with the nucleotide 8092 polymorphism C genotype showed a 40% (95% CI (63–2%); P ¼ 0.042) improvement in PFS and a 43% (95% CI (65–7%); P ¼ 0.025) improvement in OS. However, the assumption of proportional hazard was questionable as the crossing survival curve was present and the results were not confirmed in the multivariate analysis.31 In the final paper considered for this review, 107 advancedstage patients were genotyped, 45 of them were platinum sensitive and 62 platinum resistant. No differences in genotype were observed between platinum-sensitive and platinum-resistant patients. Polymorphisms in the ERCC1 codon 118 did not correlate with clinical response to platinum or with survival. Women homozygous A/A for the ERCC1 8092 SNP had a significant increase in PFS compared with A/C and C/C patients (PFS, A/A vs C/  , 25 vs 9 months; Po0.06). Both A/A and A/C genotypes conferred improved survival on the major (C/C) genotype (OS, A/  vs C/C, 45 vs 32 months; P ¼ 0.06).32 The two most frequently investigated ERCC1 SNPs are the silent polymorphisms encoding Asn118Asn and one located in the 30 -UTR. Fourteen studies were analyzed for this review. No unambiguous role could be discerned for these SNPs even when different ethnicities of patients enrolled in the studies were taken into consideration. The Pharmacogenomics Journal (2014), 1 – 10

Genetic markers for prediction in ovarian cancer E Caiola et al

4 Other NER genes In addition to ERCC1, other NER gene members have been investigated for polymorphic sites possibly affecting clinical outcome. For this review, 10 papers have been considered in which one or more NER gene SNPs were studied (Table 2). Among them, five authors17,18,21,23,28 focused on XPD gene polymorphisms. They did not reveal any associations with outcome. Saldivar et al. tested the roles of SNPs at XPA 50 -UTR, XPC poly-AT, XPD Asp312Asn, Lys751Gln and XPG Asp1104His in 146 women who had been treated with platinum-based combinations or single-agent platinum chemotherapy. The median PFS was significantly shorter in patients with the XPA 50 -UTR heterozygous variant genotype (A/G) than in women harboring the wild-type genotype (A/A; 11.3 vs 13.9 months; P ¼ 0.05). Similar results were obtained for survival: the median OS for patients with the XPA heterozygous variant genotype was significantly shorter than for those with the wild-type genotype (21.5 vs 37.9 months; P ¼ 0.03). The median survival time for patients with the XPG Asp1104His homozygous variant genotype (C/C) was significantly shorter than that for patients with the wildtype genotype (G/G; 8.3 vs 24.6 months; P ¼ 0.006). The absence of statistical significant difference between therapy response and genotype may be due to the small sample size.33 Peethambaram et al. investigated 445 samples mainly from patients treated with platinum-based therapy (95%) for eight common XPD haplotype using the Illumina (San Diego, CA, USA) platform. Among cases with high-grade serous disease, three SNPs were associated with outcome (recurrence or death): rs238417 (HR ¼ 0.74; 95% CI (0.59–0.92); P ¼ 0.006), rs238416 (HR ¼ 0.79; 95% CI (0.63–0.98); P ¼ 0.04) and rs238415 (HR ¼ 0.75; 95% CI (0.61–0.93); P ¼ 0.01). Among high-grade serous cases treated with platinum-based therapy, results were similar to those in the overall

Table 2.

group: rs238417 and rs238415 associate with outcome, whereas in the case of rs238416 the association was lost. Variation in this DNA repair gene appears related to outcome of high-grade serous disease, which may be due to differential responses to platinuminduced DNA damage.20 Fleming et al. investigated at least one SNP for each gene involved in the NER pathway. Screening was performed in 139 patients with advanced-stage papillary serous ovarian cancer who underwent primary cytoreductive surgery followed by platinumbased chemotherapy. Patients with the A/- genotypes of XPC intrinsic polymorphism rs3731108 were associated with a prolonged PFS (21.3 months), whereas patients with the G/G genotype were not ( PFS 13.4 months; HR ¼ 0.63; 95% CI (0.42– 0.95); P ¼ 0.03). Patients carrying G/  genotypes in XPC rs1124303 SNP presented with a prolonged PFS compared with patients with the T/T genotype (PFS 22.8 vs 14.9 months; HR ¼ 0.47; 95% CI (0.24–0.94); P ¼ 0.03). The XPC poly(AT) (PAT)  /  and  / þ genotypes vs the þ / þ genotype were associated with a prolonged PFS of 17 vs 11.6 months (HR ¼ 0.56; 95% CI (0.36– 0.89); P ¼ 0.01). These data were also adjusted for BRCA gene status and cytoreductive surgery status, in a multivariate analysis. The XPC SNPs remained significantly associated with prolonged PFS. Finally, the XPF/ERCC4 rs12926685 C/  genotypes vs the T/T genotype were associated with a prolonged PFS of 16.7 vs 12.4 months (HR ¼ 0.63; 95% CI (0.41–0.95); P ¼ 0.03).22 Caiola et al. analyzed the role of His46His, Asp1104His XPG polymorphisms in 332 ovarian cancer samples divided into early (stage I/II) and late (stage III/VI) stage disease. The analysis revealed that, for the Asp1104His XPG polymorphism, the OS for patients with early stage and the PFS for those with late stage diseases were significantly different when the C/C genotype was compared with C/G and G/G genotypes. The correlation detected

Pharmacogenetics studies on NER gene polymorphisms in EOC

Reference

N

Ethnicity

Treatment

Marsh et al.17 Saldivar et al.33

914 146

Not specified Caucasian 85%; other 15%

Carboplatin Platinum or platinum based

Kim et al.28

Taxanes and platinum compounds Cisplatin and cyclophosphamide Platinum based

118

Asian

18

Khrunin et al.

104

Caucasian

Peethambaram et al.20

445

Not specified

Khrunin et al.21

87

Yakut; Caucasian

Fleming et al.22

139

Caucasian 90%; other 10%

White et al.23

10,084

Caiola et al.31 Kang et al.34

332 213

Cisplatin and cyclophosphamide Platinum based

Not specified

Not specified

Caucasian Asian

Platinum based Cisplatin or carboplatin plus cyclophosphamide or paclitaxel

SNPs investigated

SNPs associated with outcome

XPD Lys751Gln XPA 50 -UTR XPC poly-AT XPD Asp312Asn, Lys751Gln XPG Asp1104His XPD Lys751Gln

None XPA 50 -UTR (PFS, OS) XPG Asp1104His (OS)

XPD Asp312Asn, Lys751Gln

None

XPD Lys751Gln, rs10853773, rs1799787, rs238417, rs238416, rs238415, rs50872, rs50871, rs1618536, rs3810366, rs11878644 XPD Asp312Asn, Lys751Gln

XPD rs238417, rs238415, rs238416

XPA rs1800975 XPB rs4150441, rs4150434 XPC Ala499Val, rs3731108, rs1124303, rs1126547, poly-AT XPD Asp312Asp, Lys751Gln, rs11878644 XPF Ser662Pro, rs11075221, rs12926685, rs4781560 XPG His46His, Cys529Ser CSB rs17332991 XPD Lys751Gln, rs1799787, rs238417, rs238416, rs238415, rs50872 XPG His46His, Asp1104His XPC Ala499Val, poly-AT, Lys939Gln XPD Asp312Asn, Lys751Gln

XPC rs3731108, rs1124303, poly-AT XPF rs12926685

None

None

None XPG Asp1104His XPC Lys939Gln

Abbreviations: EOC, epithelial ovarian cancer; NER, nucleotide excision repair; OS, overall survival; PFS, progression-free survival; SNP, single nucleotide polymorphisms; UTR, untranslated region. Intronic and 50 flanking SNPs are reported as dbSNP accession number.

The Pharmacogenomics Journal (2014), 1 – 10

& 2014 Macmillan Publishers Limited

Genetic markers for prediction in ovarian cancer E Caiola et al

5 by univariate analysis (OS early stages: HR ¼ 0.38; 95% CI (0.15– 0.94); P ¼ 0.037; PFS late stages: HR ¼ 1.94; 95% CI (1.03–3.68); P ¼ 0.041) was confirmed in the multivariate analysis (OS early stages: HR ¼ 0.37; 95% CI (0.14–0.96); P ¼ 0.041; PFS late stages: HR ¼ 2.12; 95% CI (1.10–4.12); P ¼ 0.026).31 Kang et al. investigated XPC SNPs (Ala499Val, poly-AT, Lys939Gln) and XPD polymorphisms (Asp312Asn, Lys751Gln) in 213 patients who received cisplatin or carboplatin in combination with paclitaxel or cyclophosphamide. The median PFS of patients harboring the Lys-only genotype and Gln/  genotype of the XPC Lys939Gln polymorphism were 25 and 12 months, respectively (P ¼ 0.039). The mean OS of patients was 31.1 and 27.8 months, respectively (P ¼ 0.048). Cox multivariate analysis suggests that patients with EOC with the Gln allele had an increased risk of death (HR ¼ 1.75; 95% CI (1.06–2.91)) compared with those with the Lys-only genotype. There were no associations between the XPC polyA, XPD Asp312Asn and Lys751Gln polymorphisms and survival of patients with EOC who underwent therapy with platinum-based chemotherapy.34 In nine papers, five authors were able to detect a role for NER gene variants, but the same SNP was never found to be associated with patient outcome in more than one study. The role of these SNPs needs to be elucidated with consideration of the ethnicity of the patients included in the studies. Base excision repair X-ray repair cross-complementing protein 1. The X-ray repair cross-complementing protein 1 (XRCC1) facilitates the repair of single-strand breaks in cells, due to its ability to interact with multiple enzymatic components of repair reactions. Enzymatic activity is not known for this protein, but XRCC1 interacts with enzymatic components at each stage of DNA strand break repair.35 Seven papers, published between 2007 and 2013, analyzed XRCC1 polymorphisms (Table 3). In four of these, there was no association with outcome.17,18,28,34 Three studies conducted on Chinese individuals presented positive results on XRCC1 and outcomes in ovarian cancer. Cheng et al. included 310 patients in a prospective study that aimed at investigating Arg194Trp, Arg280His and Arg399Gln XRCC1 polymorphisms in patients treated with platinum-based therapy. The study showed a lower survival rate in patients with the XRCC1 399 Arg/Arg genotype than in those with Gln/Gln, with a significant increased risk of death (HR ¼ 1.69; 95% CI (1.07–2.78); Po0.05). There was no

Table 3.

significant association between other XRCC1 gene polymorphisms and ovarian cancer death.36 Miao et al. tested the association between the same three amino-acid substitution polymorphic variants in 195 patients with primary ovarian cancer treated with a combination of bleomycin, etoposide and cisplatin. This study showed that the variant Trp/Trp of Arg194Trp XRCC1 SNP was associated with a significantly increased death risk compared with individuals with Arg/Arg (HR ¼ 1.56; 95% CI (1.04– 3.15); Po0.05). The Arg399Gln XRCC1 polymorphism was also associated with an increased risk of death: the Gln/Gln genotype was linked to a worse prognosis than the Arg/Arg genotype (HR ¼ 1.98; 95% CI (1.09–3.93); Po0.05). No significant association was found between the XRCC1 Arg280His gene polymorphism and ovarian cancer death risk.37 Li et al. conducted a prospective study to determine whether survival time was associated with polymorphisms in XRCC1 (Arg194Trp, Arg280His and Arg399Gln) in 335 Chinese ovarian cancer patients treated with platinumbased chemotherapy. Patients with the polymorphism Trp/Trp in Arg194Trp XRCC1 presented with a longer survival time and a significantly lower risk of death than those with the Arg/Arg genotype (HR ¼ 0.61; 95% CI (0.34–0.96); Po0.05). Patients harboring the Gln/Gln genotypes in the Arg399Gln XRCC1 polymorphism had a 0.44-fold risk of death compared with those with the Arg/Arg genotype (HR ¼ 0.44; 95% CI (0.28–0.91); Po0.05). There was no significant association between the XRCC1 Arg280His gene polymorphism and risk of death. Polymorphism interactions between XRCC1 Arg194Trp and XRCC1 Arg399Gln have been estimated. The combination of the XRCC1 194 Trp and 399 Gln alleles was associated with a decreased risk of death (HR ¼ 0.43; 95% CI (0.21–0.98); Po0.05) when compared with the combined homozygotes (194 Arg/Arg and 399 Arg/Arg).38 The seven papers considered here focused on the three most frequently studied variants of the XRCC1 gene. Results from the only prospective study conducted on XRCC1 SNPs seem to indicate that the presence of Arg in the SNPs Arg399Gln and Arg194Trp confers a favorable OS. All significant associations were observed in Asian populations.

POLYMORPHISMS IN DRUG TRANSPORTERS AND DRUGMETABOLIZING ENZYMES For many drugs, polymorphisms in genes that encode drugmetabolizing enzymes and drug transporter proteins are among

Pharmacogenetics studies on XRCC1 polymorphisms in EOC

Reference

N

Ethnicity

Treatment

XRCC1 SNPs investigated

XRCC1 SNPs associated with outcome

Marsh et al. Kim et al.28 Khrunin et al.18

914 118 104

Not specified Asian Caucasian

Carboplatin Taxanes and platinum compounds Cisplatin and cyclophosphamide

None None None

Cheng et al.36

310

Asian

Platinum-based

Miao et al.

195

Asian

Cisplatin, bleomycin and etoposide

Arg399Gln Arg194Trp, Arg399Gln Arg194Trp, Arg280His, Arg399Gln Arg194Trp, Arg280His, Arg399Gln Arg194Trp, Arg280His, Arg399Gln

Li et al.38

335

Asian

Cisplatin, carboplatin, vinorelbine, paclitaxel and docetaxel

Arg194Trp, Arg280His, Arg399Gln

Kang et al.34

213

Asian

Cisplatin or carboplatin plus cyclophosphamide or paclitaxel

Arg194Trp, Arg280His, Arg399Gln

17

37

OS, Arg399Gln: Arg/Arg vs Gln/Gln HR ¼ 1.69; Po0.05 OS, Arg194Trp: Trp/Trp vs Arg/Arg HR ¼ 1.56; Po0.05 OS, Arg399Gln: Gln/Gln vs Arg/Arg HR ¼ 1.98; Po0.05 OS, Arg194Trp: Trp/Trp vs Arg/Arg HR ¼ 0.61; Po0.05 OS, Arg399Gln: Gln/Gln vs Arg/Arg HR ¼ 0.44; Po0.05 None

Abbreviations: EOC, epithelial ovarian cancer; NER, nucleotide excision repair; OS, overall survival; PFS, progression-free survival; SNP, single nucleotide polymorphisms.

& 2014 Macmillan Publishers Limited

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Genetic markers for prediction in ovarian cancer E Caiola et al

6 the most clinically important genotypic variations. They can influence drug disposition by impacting on the absorption, distribution and excretion of drugs.39 The cytotoxic activity of platinum derivatives and taxanes, the drugs of choice for ovarian cancer, is modulated by these proteins. Permeability glycoprotein (PGP), an energy-dependent drug efflux pump,40 is a transmembrane transporter belonging to the superfamily of ATP-binding cassette (ABC) transporters,41 encoded by the ABCB1 gene. It transports exogenous and endogenous substrates from the inside of the cell to the outside.42 PGP is expressed in both non-malignant tissue, such as the intestine and the blood–brain barrier, and malignant tissue, such as ovarian cancer.42 PGP transports a broad range of compounds, including natural products such as paclitaxel.43 In vitro studies have shown that high levels of PGP expression are correlated with paclitaxel resistance in several cell lines, and that the degree of overexpression correlates with the degree of resistance.44 Clinical studies of ovarian cancer patients showed that increased PGP expression correlates with reduced response and PFS following treatment.45,46 It is plausible that the heterogeneity in response to chemotherapy may be explained by inter-individual variability in ABCB1 gene expression, which may at least in part be influenced by SNPs. In contrast to the mechanism of multidrug resistance to mainly natural products, platinum-based drugs are not generally substrates of PGP. There is an extensive body of evidence implicating increased levels of cytoplasmic thiol-containing species as responsible for resistance to cisplatin or carboplatin. These molecules, such as the tripeptide glutathione, are rich in the thiol-containing amino acids cysteine and methionine, and can detoxify platinum species because platinum binds avidly to sulfur.47 The enzymes involved in this process are glutathione-S-transferases (GSTs), in particular the proteins encoded by the GSTP1, GSTM1 and GSTT1 genes, which catalyze the conjugation of glutathione with electrophilic compounds including the cytotoxic agents cisplatin and carboplatin.48 It has been suggested that high GST activity results in rapid drug metabolism that diminishes the cytotoxic effects of platinum drugs on tumor cells, and hence is associated with an unfavorable response. For example, GSTP1 expression has been shown to be associated with reduced response and survival.49,50 The GST genes GSTP1, GSTM1 and GSTT1 have been found to harbor functional polymorphisms that are present in the general population with a variable ethnic distribution.51 The cytochrome P450 gene superfamily consists of a group of proteins that metabolize a wide variety of endogenous and exogenous compounds. Paclitaxel is metabolized to inactive compounds in the liver mainly by CYP2C8 and CYP3A4.52,53 These enzymes give rise to the formation of 6-hydroxypaclitaxel,

Table 4.

3-p-hydroxypaclitaxel and 6,3-p-dihydroxypaclitaxel. When tested for cytotoxicity in different cancer cell lines, these metabolites had a very low activity.54 Polymorphic variants of cytochromes 2C8 and 3A4 have been discovered, and they have been considered to confer altered activity on these enzymes.55 These polymorphisms could at least partially explain the observed differences in paclitaxel response rate, toxicity, pharmacokinetics and pharmacodynamics. ABCB1 gene polymorphisms One of the first polymorphisms identified for the ABCB1 gene is the triallelic G2677T/A (rs2032582), a non-synonymous SNP, which causes the substitution of alanine by serine or threonine at position 893 of the PGP protein. The homozygous mutant genotype of this polymorphism has been reported to lead to low PGP levels, causing a reduced cellular clearance of chemotherapeutic drugs like paclitaxel56,57 and potentially a better response to such drug in patients. The other two most studied polymorphisms in the ABCB1 gene are the synonymous C1236T (rs1128503) and C3435T (rs1045642) SNPs, located at exons 12 and 26, respectively. Seven papers have been published between 2007 and 2013 analyzing ABCB1 polymorphisms (Table 4). Johnatty et al. retrospectively investigated the role of G2677T/A, C1236T and C3435T polymorphisms in 309 patients enrolled in The Australian Ovarian Cancer Study, who received at least four cycles of adjuvant paclitaxel and carboplatin. Analyses of the C3435T and C1236T SNPs revealed no significant associations with PFS or OS in either unadjusted or adjusted models. However, patients carrying the 2677T/A minor allele in hetero- or homozygosity displayed a reduced risk of disease progression compared with wild-type G/G patients (HR, GA/GT vs GG ¼ 0.62; 95% CI (0.44–0.86); P ¼ 0.004; HR, TT/TA vs GG ¼ 0.60; 95% CI (0.40–0.89); P ¼ 0.013) in an unadjusted model. After adjustment for International Federation of Gynecology and Obstetrics stage and residual disease, this association was not statistically significant. Subgroup analysis based on extent of residual disease revealed that among cases who had undergone optimal debulking (residual disease o1 cm), the heterozygote and homozygote carriers of the minor 2677T/A allele showed a reduction in risk of disease progression compared with G/G homozygotes in both unadjusted (HR, GA/GT vs GG ¼ 0.55; 95% CI (0.37–0.82); P ¼ 0.004; HR, TT/TA vs GG ¼ 0.43; 95% CI (0.25–0.74); P ¼ 0.002) and adjusted (HR, GA/GT vs GG ¼ 0.51; 95% CI (0.30–0.89); P ¼ 0.02; HR, TT/TA vs GG ¼ 0.60; 95% CI (0.41–0.88); P ¼ 0.01) models. The association was not statistically significant in the suboptimal debulking group. To validate this observation, the authors also assessed the effect in a validation data set derived from the SCOTROC1 trial previously

Pharmacogenetics studies on nucleotide G2677T/A, C1236T and C3435T polymorphisms of ABCB1 in EOC N

Reference 17

Ethnicity

Treatment Carboplatin and docetaxel or paclitaxel Carboplatin and paclitaxel

Marsh et al.

914

Not specified

Johnatty et al.58

309

Not specified

Kim et al.28

118

Asian

Grimm et al.59 Bergmann et al.60 Peethambaram et al.20 Tian et al.61

106 119 445

Caucasian Caucasian Not specified

511

Caucasian 90%; other 10%

Taxanes and platinum compounds Platinum based Carboplatin and paclitaxel Taxanes and platinum compounds Carboplatin and taxane

G2677T/A

C1236T

C3435T

No differences

No differences

No differences

Optimally debulked patients’ PFS, GA/GT vs GG HR ¼ 0.51; P ¼ 0.02 PFS, TT/TA vs GG HR ¼ 0.60; P ¼ 0.01 No differences

No differences

No differences

Not tested

No differences

No differences No differences Not tested

Not tested No differences No differences

No differences No differences Not tested

No differences

Not tested

No differences

Abbreviations: EOC, epithelial ovarian cancer; HR, hazard ratio; PFS, progression-free survival.

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Genetic markers for prediction in ovarian cancer E Caiola et al

7 described by Marsh et al.17 They found a statistically significant reduction in the risk of disease progression in patients carrying the 2677T/A allele with no or microscopic residual disease (unadjusted HR, T/A carriers vs G/G ¼ 0.70; 95% CI (0.46–1.04); P ¼ 0.039; stageadjusted HR, T/A carriers vs G/G ¼ 0.62; 95% CI (0.37–1.03); P ¼ 0.033). Analysis of the G2677T/A SNP with regards to OS revealed no significant survival advantage associated with this SNP in either the total cohort or the residual disease subgroup.58 In the other six papers, a possible role of ABCB1 polymorphisms in EOC was investigated.17,28,59–61 In none of them, a positive correlation was detected. Peethambaram et al. analyzed the role of C1236T and other poorly characterized SNPs in the ABCB1 gene in a cohort of 445 patients with ovarian cancer treated in first line with platinum compounds and taxanes. There was no significant association between C1236T polymorphisms and patient outcome, but some other SNPs (rs12334183, rs11760837, rs10274587, rs2032588, rs2235023, rs13237132, rs1010264990, rs1989830, rs1202172, rs13229143, rs4148732) were found to be predictive factors for ovarian cancer in their cohort.20 Except for one SNP in one paper, no association with any outcome was detected in the three polymorphisms described in the seven papers evaluated for this review.

investigated, patients with the null genotype displayed a higher rate of overall response than those with a non-null genotype in the multivariate regression analysis (overall response, non-null vs null ¼ 0.29; 95% CI (0.17–0.67); P ¼ 0.037). Patients with the null genotype benefitted from significantly longer OS than those with the non-null genotype (HR, non-null vs null ¼ 1.65; 95% CI (1.79– 3.42); P ¼ 0.038). Significant results regarding patient outcome were not delineated from the analysis of polymorphisms at GSTP1 Ile105Val and GSTM1.28 Khrunin et al. evaluated a possible correlation between polymorphisms in the GSTP1, GSTT1 and GSTM1 genes and PFS or OS in a prospective study with 104 women of Eastern Slavonic origin who suffered from EOC and received cisplatin and cyclophosphamide. Patients with the homozygous Ile/Ile genotype had an increased PFS compared with Val allele carriers (PFS, Ile/Ile vs Val carriers, 15 vs 8.0–8.5 months; P ¼ 0.0001). The association remained significant when optimally debulked patients were excluded from the analysis (P ¼ 0.005).18 In addition, three other studies did not support any role for polymorphisms at GSTT1, GSTP1 and GSTM1.17,20,21 Overall, the six studies considered here did not reveal a clear role for any variant associated with GST. In three papers there were associations, but they were inconsistent.

GSTP1, GSTM1 and GSTT1 gene polymorphisms A single nucleotide substitution (A4G) at position 592 of the GSTP1 gene, which results in replacement of isoleucine with valine at amino-acid position 105 (Ile105Val, rs1695), reduces GSTP1 activity, probably due to decreased enzyme stability.62 A significant percentage of individuals exhibit genetic polymorphisms with a homozygous deletion (null genotype) of the GSTT1 and GSTM1 genes, causing the absence of the two enzymes.51 For this review, six papers have been considered in which GSTP1, GSTT1 and GSTM1 gene polymorphisms were studied (Table 5). Nagle et al. investigated the role of the three GST gene variants in a population of 454 women affected by ovarian cancer and treated (75%) with platinum-based chemotherapy. Patients carrying the 592G allele in the GSTP1 gene presented with a significantly better OS than women with the A/A genotype (adjusted HR, G/A, G/G vs A/A ¼ 0.77; 95% CI (0.61–0.99); P ¼ 0.04). When the three SNPs were combined to explore whether there was a correlation between the presence of one or more ‘enzyme low function’ polymorphisms and a benefit in terms of OS, a trend was evident, which was not statistically significant.62 Kim et al. found a significant association between GSTT1 polymorphism and overall response. Among the 118 women affected by EOC

CYP2C8 and CYP3A4 gene polymorphisms Only four studies analyzed CYP2C8 and CYP3A4 variants (Table 6). For the CYP2C8 enzyme, the Lys399Arg (CYP2C8*3, rs10509681) variant and other less common polymorphisms have been analyzed, but none of them was associated with patient outcome.17,20,60 Regarding the CYP3A4 gene, only one report describes a statistically significant correlation between genotype and overall patient survival.63 Two SNPs were analyzed, A-392G (CYP3A4*1B, rs2740574) located in the promoter region of the gene, and the intronic variant CYP3A4*22 (rs35599367) recently discovered by Wang et al.64 The CYP3A4*1B -392G minor variant seemed to be associated with increased activity of the enzyme, whereas the CYP3A4*22 C/C genotype affected mRNA processing, resulting in decreased protein expression. One hundred and twenty women with serous ovarian cancer treated with first-line platinum compounds and paclitaxel were genotyped for these two SNPs. Mean survival rates were statistically different depending on CYP3A4*1B genotype. G allele carriers had a significantly worse survival than patients with the A/A genotype (age-adjusted HR, G carriers vs A/A ¼ 2.15; 95% CI (1.03–4.52); P ¼ 0.043). Patients were grouped according to CYP3A4*1B -392G allele or CYP3A4*22 C/C genotype, both of which have been reported to increase enzyme expression

Table 5.

Pharmacogenetics studies on polymorphisms of GSTP1, GSTT1 and GSTM1 in EOC N

Reference 62

Nagle et al.

Marsh et al.17

Ethnicity

Treatment

454

Caucasian

914

Kim et al.

118

Not specified Asian

Khrunin et al.18

104

Caucasian

Peethambaram et al.20 Khrunin et al.21

445

Not specified Yakut; Caucasian

28

87

GSTP1 Ile105Val

GSTT1 deletion

GSTM1deletion

Platinum-based chemotherapy (340) Carboplatin and docetaxel or paclitaxel Taxanes and platinum compounds

OS, G/A, GG vs A/A HR ¼ 0.77; P ¼ 0.04 No differences

No differences

No differences Not tested

No differences

Cisplatin and cyclophosphamide Taxanes and platinum compounds Cisplatin and cyclophosphamide

PFS, Ile/Ile vs Val carriers, 15 vs 8.0– 8.5 months; P ¼ 0.0001 Not tested

OR, non-null vs null HR ¼ 0.29; P ¼ 0.037 OS, non-null vs null HR ¼ 1.65; P ¼ 0.038 No differences

No differences

Not tested

No differences No differences

No differences No differences Not tested No differences

Abbreviations: EOC, epithelial ovarian cancer; HR, hazard ratio; OR, overall response; OS, overall survival; PFS, progression-free survival.

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Genetic markers for prediction in ovarian cancer E Caiola et al

8 Table 6.

Pharmacogenetics studies of CYP2C8 and CYP3A4 variants in EOC N

Reference 17

Marsh et al. Bergmann et al.60 Peethambaram et al.20 Assis et al.63

914 119 445 120

Ethnicity

Treatment

CYP2C8

Not specified Caucasian Not specified Caucasian

Carboplatin and docetaxel or paclitaxel Carboplatin and paclitaxel Taxanes and platinum compounds Paclitaxel and cisplatin or carboplatin

No differences No differences No differences Not tested

CYP3A4 No differences Not tested No differences CYP3A4*1B OS, G/A, G/G vs A/A HR ¼ 2.15; P ¼ 0.043 CYP3A4*1B and CYP3A4*22 OS, high-activity profile vs low HR ¼ 2.29; P ¼ 0.038

Abbreviations: EOC, epithelial ovarian cancer; HR, hazard ratio; OS, overall survival.

and activity. Patients with high CYP3A4 activity had significantly diminished survival as compared with those with low CYP3A4 activity (age-adjusted HR, CYP3A4 high activity vs low activity ¼ 2.29; CI (1.05–5.02); P ¼ 0.038). Therefore, individuals with the CYP3A4*1B G carrier or CYP3A4*22 C/C genotypes (CYP3A4 high activity) experienced lower OS than individuals with the CYP3A4*22 T carrier or CYP3A4*1B A/A genotype (low activity).63 Except for one SNP in one paper involving a relatively small cohort of patients (n ¼ 120), no association with outcome was found for the polymorphisms considered in the studies selected for this review.

CONCLUSIONS Despite the increasing number of studies and the advances in molecular technologies, clinical advance remains a challenge for pharmacogenomics. Among the reasons are the small number of patients in pharmacogenomic studies and the difficulty of incorporating rapid genetic tests into routine clinical practice. Genetic variants in genes involved in the distribution, metabolism, accumulation or repair of lesions are likely to influence the response of drugs used in the treatment of EOC. Platinum-containing drugs are the first-line therapy of choice in EOC. Platinum-mediated lesions are predominantly repaired by the DNA repair machinery.65 Therefore, it is not surprising that SNPs known to alter the stability, expression or function of genes associated with DNA repair have been found to be linked to clinical outcome. Platinum drugs are often associated with paclitaxel. Polymorphisms in genes implicated in its metabolism and transport could be potentially relevant in defining paclitaxel activity, but data published so far do not support this hypothesis. The fact that often contradictory results have been obtained indicates that a more comprehensive analysis of gene SNPs associated with DNA repair, metabolism and transport should be performed in the same study. The current lack of evidence for a link between drug treatment choice, dose adjustment or modification of schedule on the one side and specific polymorphisms on the other could be due to several facts. First, the majority of the studies were retrospective and hence not designed to specifically assess the role of a given SNP in clinical outcome. Second, the most commonly used first-line treatment of EOC involves drugs (platinum–taxane), the effects of which are modulated by different enzymes making difficult the definition of the role of a single genetic variant. Third, several of the studies, although well performed, lacked sufficient numbers of patients to allow determination of the true impact of a variant, which may well be present in only a low percentage of cases. The impact of pharmacogenomics on clinical therapy decisions in oncology with the aim to optimize drug treatment is still weak. There are examples in clinical malignancies other than EOC in which genetic polymorphisms have been demonstrated to be The Pharmacogenomics Journal (2014), 1 – 10

important for dose selection. An example is irinotecan, a topoisomerase I inhibitor, which is used in the treatment of particularly colorectal cancer. For this drug, Food and Drug Administration approved the dose adjustment based on the presence of a UGT1A1*28 gene variation associated with decreased drug metabolism and consequently increased toxicity.66 Some clinical evidence suggests a correlation between CYP2D6 status and tamoxifen efficacy.67 According to certain guidelines for the treatment of breast cancer, tamoxifen should be replaced by aromatase inhibitors in postmenopausal patients genetically recognized as poor metabolizers. This alternative to tamoxifen might decrease the increased risk of relapse due to the reduced formation of the active tamoxifen metabolite. Analysis of polymorphisms is relatively easy to perform, does not require invasive analysis, and the tremendous advance in analytical technology will certainly allow in the future the analysis of several SNPs in the same sample. Genome-wide association studies should be performed in a defined patients’ population with the power to detect important variants associated with response to validate SNPs for subsequent studies. Efforts should be invested in defining possible associations of SNPs with outcome. It is not difficult to imagine that in the future, these studies will be possible in collaborative efforts of multicentre studies. New targeted therapies may well become available soon for the treatment of EOC. The anti-angiogenic drug bevacizumab, targeting VEGFR (vascular endothelial growth factor receptor), is one possible candidate. It is already known that there are variants in genes including VEGFR that can modulate bevacizumab activity.68 Studies incorporating this information should be performed in early phase trials.

ACKNOWLEDGMENTS Professor Andreas Gescher kindly revised the manuscript. We would like to apologize to all authors whom we have not been able to cite owing to space restrictions.

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