Pharmacogenetic predictors for EGFR-inhibitor-associated ... - Nature

The Pharmacogenomics Journal (2013) 13, 181–188 & 2013 Macmillan Publishers Limited. All rights reserved 1470-269X/13 www.nature.com/tpj

ORIGINAL ARTICLE

Pharmacogenetic predictors for EGFR-inhibitorassociated skin toxicity S Parmar1, C Schumann2, S Ru¨diger2, S Boeck3, V Heinemann3, V Ka¨chele4, A Seeringer1, T Paul1, T Seufferlein5 and JC Stingl1 1 Institute of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Ulm, Germany; 2Department of Internal Medicine II, University of Ulm, Ulm, Germany; 3 Department of Internal Medicine III, Ludwig-Maximilians-University of Munich, Munich, Germany; 4Department of Internal Medicine I, University of Ulm, Ulm, Germany and 5Department of Internal Medicine I, Martin Luther University Halle-Wittenberg, Halle, Germany

Correspondence: Professor J Stingl (formerly Kirchheiner), Institute of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Helmholtzstr. 20, D-89081 Ulm, Germany. E-mail: [email protected]

The aim of this study was to investigate pharmacogenetic determinants of skin rash associated with epidermal growth factor receptor (EGFR) inhibitor treatment. A total of 109 prospectively sampled cancer patients, receiving the first treatment with an EGFR inhibitor, were genotyped for functional EGFR polymorphisms and tagging variants in genes involved in receptor downstream signaling. Skin rash was absent in 26 (23.9%) patients and associated with shorter overall survival compared with patients presenting skin rash (P ¼ 0.005). The EGFR polymorphisms, 497G/A (P ¼ 0.008), and the haplotypes of the promoter variants, EGFR–216G/T and –191C/A (P ¼ 0.029), were associated with the appearance of skin rash. In addition, a common haplotype in the PIK3CA gene was associated with skin rash (P ¼ 0.045) and overall survival (P ¼ 0.009). In conclusion, genetic variation within the EGFR gene and its downstream signaling partner PIK3CA might predict EGFR-inhibitor-related skin rash. The Pharmacogenomics Journal (2013) 13, 181–188; doi:10.1038/tpj.2011.51; published online 13 December 2011 Keywords: EGFR; skin rash; pharmacogenetics; polymorphism

Introduction

Received 5 August 2011; accepted 7 November 2011; published online 13 December 2011

The human epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. Extracellular ligand binding leads to receptor dimerization and subsequent autophoshporylation of intracellular tyrosine residues. The EGFR is capable to stimulate a variety of downstream cell signaling pathways that influence cellular proliferation, apoptosis, migration and cell survival.1 EGFR function can be found aberrant in various human epithelial cancers, such as non-small-cell lung cancer (NSCLC), pancreatic cancer, colorectal cancer and others. Several mechanism lead to aberrant EGFR activation, including receptor overexpression, mutations of receptor tyrosine kinase and ligand-independent activation.2 Hence, targeting EGFR has become a promising approach in cancer therapy and, at present, two monoclonal antibodies, cetuximab and panitumumab, and two small-molecule tyrosine kinase inhibitors, erlotinib and gefitinib, have been licensed. Several clinical trials demonstrated the effectiveness of EGFR-targeted therapies in the management of several solid cancers. Furthermore, treatment with EGFR inhibitors is generally well tolerated, and associated with lower systemic adverse reactions, than conventional chemotherapy.3 However, EGFR is also expressed in almost all normal epithelial cells, such as skin and gastrointestinal tract, and, in consequence, the most frequent adverse effects of these agents are skin toxicity and diarrhea.

Pharmacogenetics of EGFR inhibitors S Parmar et al

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The skin toxicities associated with EGFR inhibitors (EGFRI) include papulopustular rash (also referred as acneiform), xerosis, hair and nail change, and pruritus.4–6 The rash is the most common skin toxicity, characterized by an early onset, typically occurring in the first treatment week and reaching its maximum after two-to-three weeks,3,6 affecting the majority of patients (45–100%) with a generally mild manifestation.5 The appearance of rash is comparable between small-molecule tyrosine kinase inhibitors and monoclonal antibodies and is, therefore, depicted as a class effect of EGFRIs.7 In addition, numerous studies suggested a positive association between the occurrence and severity of rash with patient outcomes in various cancer diseases.7,8 The EGFR gene is polymorphic, and candidate markers are the CA repeat in intron 1; the promoter variants, -216G/T and -191C/A; and the only frequent missense polymorphism EGFR 497G/A, which results in an amino acid substitution from arginine to lysine at position 521 of the corresponding protein (Arg521Lys). To date, a possible association of genetic variation in the EGFR gene with the occurrence and severity of skin rash has been studied in mainly retrospective reports, with partially conflicting results.9–16 Besides genetic variation within the drug target, germline variation within the downstream pathways also might contribute, at least in part, to the likelihood to develop EGFRI-related toxicity. Interestingly, it has been reported that patients with higher baseline pAkt levels in the skin have a reduced risk to develop EGFRI-induced skin toxicity.17 We, therefore, hypothesized that genetic variation in the PI3K/AKT1 pathway might be associated with the risk for EGFRI toxicity. We undertook this prospective pharmacogenetic study to correlate genetic variation within the EGFR/PI3K/AKT1 pathway with the occurrence of EGFRIassociated toxicity.

Patients and methods Patients From September 2008 to November 2010, 109 patients, treated with EGFRI, were included in this multicentre prospective study. Inclusion criteria were histologically confirmed cancer disease and first-time treatment with an EGFRI (monoclonal antibody or small molecule). Written informed consent was obtained from all participating subjects before inclusion. The study was reviewed and positively voted by the ethical review boards of the University Ulm and the LMU Munich. Severity of skin rash was rated according to the common toxicity criteria of the National Cancer Institute (NCI CTC version 3.0 criteria):18 grade 1, macular or popular eruption or erythema without associated symptoms; grade 2, macular or popular eruption or erythema with pruritus or other associated symptoms, and localized desquamation or other lesions covering o50% of body surface area; grade 3, severe, generalized erythroderma or macular, papular or vesicular eruption, and desquamation covering X50% body surface

The Pharmacogenomics Journal

Table 1

Patient characteristics

Characteristic

No. of patients (%)

Applied EGFRI Erlotinib Gefitinib Cetuximab Panitumumab

84 4 19 2

Combination chemotherapy No Yes

75 (68.8) 34 (31.2)

Disease NSCLC Pancreatic Cancer CRC

60 (55.1) 43 (39.4) 6 (5.5)

Median age, years (range)

68 (43–86)

Sex Male Female

73 (67) 36 (33)

Smoking history Never Former Present Unknown

34 54 13 8

(31.2) (49.5) (11.9) (7.3)

Clinical stage IIIa IIIb IV Unkown

2 12 90 5

(1.8) (11) (82.6) (4.6)

Skin rash (grade) 0 1 2 3

26 38 40 5

(23.9) (34.9) (36.7) (4.6)

Diarrhea (grade) 0 1 2 3 4

71 22 10 5 1

(65.1) (20.2) (9.2) (4.6) (0.9)

(77.1) (3.7) (17.4) (1.8)

CRC, colorectal cancer; EGFRI, epidermal growth factor receptor inhibitors; NSCLC, non-small-cell lung cancer.

area; grade 4, generalized exfoliative, ulcerative, or bullous dermatitis. Appearance and severity of skin toxicity and diarrhea were rated weekly (or biweekly) within the first four weeks after treatment initiation, and highest reported toxicity grade was used for analysis. In addition, pharmacological interventions against skin toxicity and/or diarrhea were recorded. Patient characteristics are summarized in Table 1. The sample consists of patients diagnosed for NSCLC (n ¼ 60),


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pancreatic cancer (n ¼ 43) and colorectal cancer (n ¼ 6). The majority of the patients received oral erlotinib daily (n ¼ 84); NSCLC patients were treated with 150 mg, whereas pancreatic cancer patients received 100 mg. Eighteen patients were treated with 250 mg/m2 cetuximab weekly (or biweekly) and one patient received 500 mg/m2 biweekly. Only a few patients were treated with 250 mg gefitinib daily (n ¼ 4) or 6 mg/kg panitumumab biweekly (n ¼ 2). DNA isolation and genotyping Genomic DNA was isolated from peripheral whole-blood samples using QIAamp DNA Blood mini kit (Qiagen, Hilden, Germany). EGFR intron 1 CA repeat was determined using primers published by Nomura et al.19 The reverse primer was labeled with 6-FAM at the 50 -End. PCR amplification was performed in a 25 ml reaction volume containing 100 ng of genomic DNA, 2.5 ml 10 PCR buffer, 2 mM magnesium chloride, 2 mM of each dNTP, 0.4 mM of each primer and 2.5 units of Taq polymerase (Invitrogen, Karlsruhe, Germany). Samples were initially denatured at 94 1C for 2 min; afterwards, 35 cycles were run as follows: 94 1C for 30 s, 58 1C for 45 s and 72 1C for 60 s. A final extension was performed at 72 1C for 7 min. Afterwards, product size was confirmed by agarose gel electrophoresis. 2 ml of the 1:20 diluted PCR product were denatured in a mixture of 0.5 ml Genescan 500 Rox (Applied Biosystems, Darmstadt, Germany) size standard and 20 ml Hi-Di formamide at 95 1C for 2 min. Samples were separated on an ABI 3130xl genetic analyzer (Applied Biosystems) and analyzed by Gene mapper software Version 4.0 (Applied Biosystems). As previously proposed in literature, patients were subdivided according to the number of CA repeats on both alleles, using 16 repeats as cutoff value.9,13 Alleles with p16 repeats were classified as short (s), whereas alleles with 416 repeats were classified as long (l), resulting in the following allele combinations: s/s, s/l and l/l. All other genotypes were determined using the KASPar single nucleotide polymorphism (SNP) genotyping system (KBioscience, Hoddesdon, UK). Allelic discrimination was carried out on ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s protocol. Selection of tagging variants for PIK3CA and AKT1 Tagging variants were selected within the exonic and intronic regions of the genes and the 10-kb flanking region, as proposed by Petterson et al.20 using Haploview version 4.2.21 Marker selection was based on the allele frequencies from the Centre d’Etude du Polymorphisme Humain samples, applying an r2 cutoff of 0.8 and a minor allele frequency of 15% using the tagging algorithm implemented in Haploview. The haplotype pairs of each individual were determined using Phase software (Version 2.1).22 Ten runs were performed, and the most likely haplotype pairs were used for further analysis. Only haplotypes with a minor allele frequency X5% were considered for further analysis. Statistical analysis All statistical tests were conducted using the SPSS software version 17.0. (SPSS, Chicago, IL, USA). Fisher exact test for

3 2 or 2 2 tables was used to analyze the association between genetic variants and toxicity (grade 0 vs grade X1). Analysis was performed comparing the respective three genotype groups of wild-type, heterozygous and homozygous carriers of the respective variant. For calculating odds ratios, logistic regression analyses were performed referring to previously reported allele functionality, that is, EGFR -216 G/G versus G/T and T/T, EGFR -191 C/C versus C/A and A/A, EGFR 497G/G versus G/A and A/A.9,10,19,23 The association between haplotypes and skin toxicity was assessed according to the number of respective haplotype alleles carried by each patient (zero, one or two), and, additionally, haplotype carriers and non-carriers were compared. Overall survival (OS) was defined as the time from treatment start to the date of death. Patients who were alive at the last available follow-up were censored at the last date they were known to be alive, based on the date of last contact. The association between overall survival and each variant/haplotype was estimated using Kaplan–Meier plots and assessed using the log-rank test. Univariate and multivariate Cox proportional regression models were calculated. The multivariate models included the variables, gender, age (Median), smoking history (never vs present þ former), tumor type (lung vs gastrointestinal tumors), clinical stage (III vs IV), former chemotherapy (yes/no), former surgery (yes/no) and combination or monotherapy (yes/no). All tests were two-sided and the level of significance was set at 0.05, unadjusted P-values are presented throughout the manuscript. Results Frequency of skin rash and diarrhea Among the 109 patients included into this prospective pharmacogenetic study, skin rash was absent in 26 (23.9%) patients; 38 patients (34.9%) experienced grade 1 and 45 patients (43.3%) developed grade 2 or greater skin toxicity. Diarrhea was less frequent. A total of 71 (65.1%) patients did not develop diarrhea; 22 (20.2%) individuals experienced grade 1 and 16 individuals (14.7%) developed grade 2 or greater diarrhea (Table 1). Frequency of skin toxicity and diarrhea was comparable between the different EGFRIs (no significant group differences, Fisher exact test). No significant associations between prevalence of skin rash or diarrhea with age, gender, tumor type or clinical stage were detected. Skin toxicity and overall survival Kaplan–Meier analysis revealed a favorable outcome for patients who experienced skin toxicity compared with those who did not (log-rank P ¼ 0.005; Figure 1a). In addition, a significant association of patients’ OS with intensity of skin toxicity was observed (log-rank P ¼ 0.001; Figure 1b). Age (median), gender, combination chemotherapy, previous non-EGFRI chemotherapy, cancer type (being associated with erlotinib drug dose), clinical stage and the particularly applied EGFRI (erlotinib þ gefitinb vs



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Figure 1 Kaplan–Meier curves for overall survival by grade of rash, comparing (a) patients who presented skin rash with patients without skin rash. (log-rank P ¼ 0.005) and (b) each skin rash severity grade (log-rank P ¼ 0.001).

cetuximab þ panitimumab) were not significantly associated with overall survival in the univariate Kaplan–Meier analysis (log-rank P4 0.05 for all variables). EGFR polymorphisms Correlations between EGFR polymorphisms and toxicity are summarized in Table 2. The EGFR 497G/A polymorphism was associated with skin rash. Among patients carrying the EGFR 497 G/G, G/A and A/A genotypes, skin toxicity was observed in 86, 61.7 and 100% of patients (Fisher exact P ¼ 0.008). In particular, skin rash was more frequent among carriers of the G/G genotype compared with carriers of at least one A-Allele (OR ¼ 3.24 (1.27–8.31); P ¼ 0.014). Interventions against skin toxicity were more frequent among carriers of the EGFR 497 G/G versus G/A or A/A genotype (78.9% vs 59.4%; Fisher exact P ¼ 0.037). The promoter polymorphism EGFR-216G/T showed a trend toward an association with skin toxicity (Fisher exact P ¼ 0.071, dominant model). The frequency of skin toxicity was 67.3, 81.3 and 91.7% among patients with EGFR-216 GG, GT and TT genotype. No association with skin toxicity was observed for the EGFR-191 C/A polymorphism (Fisher exact P ¼ 0.62). Both the promoter polymorphisms were in linkage disequilibrium (D0 ¼ 1.0), and the corresponding haplotypes, as described by Rudin et al.,13 were additionally assessed. Among carriers of zero, one or two EGFR-216/-191 GC haplotype alleles, skin toxicity was observed in 94.7, 76.6 and 61.5% of patients (Fisher exact P ¼ 0.029; Table 2). However, only a trend to decreased odds to develop skin toxicity was observed in the logistic regression analysis by comparing carriers and noncarriers of this haplotype (OR ¼ 0.14 (0.02–1.14); P ¼ 0.066). Treatment interventions against skin toxicity tended to be less frequent among carriers of EGFR-216/-191 GC (65.6% vs 89.5%; Fisher exact P ¼ 0.053): this was particularly true for the treatment with oral histamine antagonists (4.4% vs 26.3%; Fisher exact P ¼ 0.008) and treatment discontinuation due to skin toxicity (1.1% vs 15.8%; Fisher exact P ¼ 0.016).


In addition, an interaction of the promoter variant -216G/ T and the exonic 497G/A variant with the sensitivity to EGFRIs has been suggested.24 We, therefore, also assessed the most frequent haplotypes arising from the -216G/T, -191C/A and 497G/A variant. Carriers of the haplotype GCA had a reduced risk to develop skin toxicity with an odds ratio of 0.24 (0.09–0.62; P ¼ 0.003) and, consistently, also had a reduced demand for interventions against skin toxicity (55.3 vs 80.6%; Fisher exact P ¼ 0.006). The number of intron 1 CA repeats ranged from 14 to 22. The most common alleles were those with 16 (43.1%) and 20 (23.4%) or 18 (15.1%) repeats. Skin rash was observed among 86.4, 70.7 and 79.3% of patients in the s/s, s/l and l/l groups, respectively (Fisher exact P ¼ 0.36). No significant association between any of the EGFR polymorphisms with diarrhea was observed. Likewise, no significant association between EGFR haplotypes and the occurrence of diarrhea was observed. PIK3CA and AKT1 polymorphisms We analyzed a possible association of genetic variation in PIK3CA and AKT1 with development of EGFRI-related toxicity. Tagging SNPs were derived spanning the 10-kb flanking region and were analyzed if the minor allele frequency was X15% with an r2 cutoff value of 0.8. We identified three tagging SNPs in AKT1 and five in PIK3CA, and the specific haplotype pairs for each individual were determined. The selected tagging polymorphisms and respective haplotypes are summarized in Tables 3 and 4. Among the PIK3CA-tagging SNPs, only rs2459693 showed a significant association with skin toxicity (Fisher exact P ¼ 0.023). However, the association was mainly with the heterozygous group, and no significant association was observed either in the dominant or in the recessive model (Fisher exact P ¼ 0.36 and P ¼ 1.0). Five PIK3CA haplotypes with a frequency 40.05 were defined by the combination of tagging SNPs. The PIK3CA haplotype H4 was significantly associated with skin toxicity. Among carriers of H4 (n ¼ 15), 53% exhibited skin toxicity


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Table 2

Association of EGFR polymorphism and skin toxicity

Polymorphism/ haplotype

Genotype/ No. of haplotype patients

Skin toxicity (%)

Fisher exact Pa

EGFR -216 G/T

GG GT TT

49 48 12

67.3 81.3 91.7

0.147 0.071

EGFR -191 C/A

CC CA AA

79 30 —

74.7 80.0

0.62

EGFR 497 G/A

GG GA AA

57 47 5

86.0 61.7 100.0

EGFR intron 1 CA repeat

s/s s/l l/l

22 58 29

86.4 70.7 79.3

0.36 0.27

EGFR -216/-191 GC haplotype

0 1 2

19 64 26

94.7 76.6 61.5

0.029b 0.040b

EGFR -216/-191 TC haplotype

0 1 2

49 48 12

67.3 81.3 91.7

0.147 0.071

EGFR -216/-191 GA haplotype

0 1 2

79 30 —

74.7 80.0 —

0.62

EGFR -216/-191/ 497 GCG haplotype

0 1 2

54 43 12

75.9 79.1 66.7

0.62 1.0

EGFR -216/-191/ 497 TCG haplotype

0 1 2

54 49 9

70.4 80.4 88.9

0.45 0.183

EGFR -216/-191/ 497 GCG haplotype

0 1 2

62 45 2

87.1 60.0 100.0

0.003b 0.003b

EGFR -216/-191/ 497 GAG haplotype

0 1 2

79 30 —

74.7 80.0

0.008b 0.014b

0.62

a

First row: Fisher exact test for 3x2 table, second row: dominant model. significant association.

b

compared with 79.8% among patients without H4 (Fisher exact P ¼ 0.045; Table 5). The corresponding OR to develop skin toxicity was 0.29 (0.09–0.90) (logistic regression P ¼ 0.032). Consistently, carriers of H4 tended to have a reduced demand for treatment intervention due to skin toxicity (53.3 vs 72.3%; Fisher P ¼ 0.145). Especially, the demand for topic glucosteroids was reduced (26.7% vs 60.6%; Fisher P ¼ 0.023), and, in those patients, no therapy break due to skin toxicity was required. None of the tagging SNPs were significantly associated with diarrhea. Among the PIK3CA haplotypes, H5 was significantly associated with diarrhea. Among patients carrying H5, 66.7% experienced diarrhea as compared with 29.9% among non-carriers (Fisher exact P ¼ 0.02). No significant association with skin toxicity or diarrhea was observed for the AKT1-tagging variants or the corresponding haplotypes.

Table 3

Genotyped tagging variants for PIK3CA and AKT1

Gene PIK3CA PIK3CA PIK3CA PIK3CA PIK3CA AKT1 AKT1 AKT1

SNP ID

Alleles

MAF

rs9831234 rs2699905 rs6443624 rs2677760 rs2459693 rs3001371 rs2498794 rs1130214

A/G G/A C/A C/T T/C C/T T/C G/T

0.17 0.24 0.22 0.53 0.44 0.33 0.49 0.27

MAF, minor allele frequency; SNP ID, rs number as given in the NCBI database.

Association with overall survival None of the studied EGFR polymorphisms or haplotypes was associated with OS. For the PIK3CA haplotype H4, a significant association with survival time was observed. Patients who carried the haplotype H4 (associated with lower skin toxicity) had a shorter OS (log-rank P ¼ 0.009; Figure 2). The corresponding unadjusted and adjusted HR were 2.37 (1.21–4.62; P ¼ 0.012) and 2.28 (1.05–4.98; P ¼ 0.038).

Discussion The introduction of EGFRI into clinical practice has led to a considerable progress in the treatment of various solid tumors. However, considerable variability in clinical outcomes has originated a demand for clinical and molecular biomarkers affecting the sensitivity and resistance to EGFR inhibitors.1,25 Several studies reported a relationship between the manifestation of EGFRI-related skin toxicity and clinical outcome for various solid tumors, including NSCLC26–29 squamous cell carcinoma of the head and neck,30 colorectal cancer31,32 and pancreatic cancer.29,33,34 Therefore, EGFRI-related skin toxicity may serve as a clinical marker predicting the sensitivity or resistance to EGFR inhibitors. If genetic biomarkers predicting skin rash would be known, this could serve as a molecular diagnostics for tailoring EGFRI therapy. Similar to prior study data, we observed a clear positive association between the presence of skin toxicity and the overall survival in our patients with different tumor entities. In particular, patients who developed skin toxicity (of any grade) showed a more favorable outcome compared with patients who did not. The pathophysiology of EGFRI-associated skin rash is not completely understood. The EGF receptor has a crucial role in skin homeostasis, and receptor inhibition results in numerous consequences, including alterations in keratinocyte growth and migration, abnormal survival, proliferation, differentiation and increased synthesis of proinflammatory cytokines, followed by the recruitment of immune cells, which seems to result in the typical skin phenotype.7,35 Therefore, it has been hypothesized that genetic variation



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within the EGFR gene, leading to altered expression or activity of the receptor, might contribute to the likelihood to develop skin toxicity.9–14,25 We studied the association between EGFR variants as well as genes from its intracellular signal transduction pathway and skin rash in patients treated with EGFR inhibitors. We observed a significant association between the EGFR 497G/A variant and the occurrence of skin toxicity. Patients carrying the common 497 G/G genotype presented significantly more often with skin toxicity compared with patients who carried at least one variant A-Allele. The A-Allele of EGFR 497G/A encodes a lysine at position 521, and lower EGF or TGF-a binding affinity was reported for the EGFR 521Lys isoform.36 A decrease in EGFR phosphorylation and subsequent c-Myc activation for this variant has been shown in tissue sections of colorectal carcinoma patients.37 Thus, the A-Allele of EGFR 497G/A may be less sensitive to pathway inhibition compared with the G-allele, where this pathway is more active. This hypothesis is supported by observations suggesting that elevated EGFR expression is correlated with an increased sensitivity against EGFRIs.23,38 Our observation is in accordance with a recent study in cetuximab-treated squamous cell carcinoma of the head and neck patients.10 Other studies with gefitinib or erlotinib did not find such an association.9,11–13,15 In our sample, this effect was observed, in the cetuximab as well in the subgroup of patients treated with erlotinib or gefitinib (Fisher exact for the erlotinib- or gefitinib-treated patient subgroup P ¼ 0.002). Additionally, we observed that carriers of the EGFR-216/191 GC haplotype experienced less skin toxicity. The T-allele of EGFR-216 G/T is known to be associated with an increase in EGFR promoter activity and protein expression,23,24 thus, leading to a stronger EGF-receptor-inhibiting effect. Our finding is consistent with other studies reporting an association of the EGFR-216 T-Allele with an increased risk for skin toxicity,11,12 but not in line with others who did not observe a similar association.9,13,15,16 Cancer cell lines with the -216/497 GA or TA haplotype alleles showed a weak association with resistance to EGFRI, whereas the TG haplotype seemed to be associated with sensitivity.24 When we combined the genotype information of EGFR-216G/T, -191C/A and -497G/A by assessing the corresponding haplotypes, we observed a lower prevalence of skin toxicity among carriers of the frequent GCA haplotype. Therefore, it might be promising to study also a potential association

Table 4

between the functional EGFR variants beside their individual contribution. Similar to other investigators, we did not find an association between the intron 1 CA repeat and EGFRI-related toxicity.9–12,16 However, two studies observed an association between this polymorphism and skin toxicity in gefitinibor erlotinib-treated patients.13,15 A possible reason for the discrepancy might be interethnic differences in distributions of the intron 1 CA repeat and the EGFR promoter polymorphisms.19 The development of EGFRI-related skin rash might also be modulated by changes within the EGFR downstream signaling pathways. Indeed, certain evidence exists that the baseline pAKT levels may be a predictive marker for EGFRIrelated skin toxicity.17 In a prospective study, non-cancerous skin biopsies were collected at baseline, as well as one month after erlotinib treatment initiation and the investigators reported a reduced occurrence of skin toxicity among

Figure 2 Kaplan–Meier curve for overall survival comparing carriers and non-carriers of PIK3CA haplotype H4 (log-rank P ¼ 0.009).

PIK3CA Haplotypes

Haplotype PIK3CA_H1 PIK3CA_H2 PIK3CA_H3 PIK3CA_H4 PIK3CA_H5

rs9831234 A/G

rs2699905 G/A

rs6443624 C/A

rs2677760 C/T

rs2459693 T/C

Frequency

A A G A A

G A G G G

C C A C C

C T T T C

C T T T T

0.41 0.24 0.17 0.07 0.06

Letters in bold denote the variant allele.



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Table 5

Association of PIK3CA haplotypes with skin toxicity

Haplotype

No. of haplotype alleles

No. of patients

Skin toxicity (%)

Fisher exact Pa

PIK3CA H1

0 1 2

38 52 19

81.6 67.3 89.5

0.116 0.36

PIK3CA H2

0 1 2

58 49 2

81.0 69.4 100.0

0.29 0.26

PIK3CA H3

0 1 2

77 28 4

75.3 78.6 75.0

0.92 0.81

PIK3CA H4

0 1 2

94 15 —

79.8 53.3

0.045b

PIK3CA H5

0 1 2

97 12 —

74.2 91.7

0.29

a

First row: Fisher exact test for 3X2 table; second row: comparison of haplotype carriers and non-carriers. b Significant association.

patients with elevated baseline pAkt levels.17 Moreover, it has been suggested that AKT1 represents an important factor for keratinocyte differentiation and stratification.39 Additionally, inhibition of PI3K leads to an impaired keratinocyte differentiation.40,41 We hypothesized that genetic variation in the AKT1 gene and the catalytic subunit of its upstream regulator phosphatidylinositol-3-kinase might be associated with the risk to develop EGFRI-associated skin toxicity. A potential association between skin toxicity and genetic variation in the PIK3CA gene was detected in our sample. In particular, skin toxicity was less frequent among carriers of the PIK3CA haplotype H4. Although the molecular basis for this observation is not yet known, our results suggest that genetic variation within the PIK3CA gene may modulate PI3K activity. We speculate that functional variants that are in linkage disequilibrium with the tagging variants may modulate the activity or expression of PIK3CA and, therefore, the signaling in the EGFR/PI3K/AKT1 pathway. Further studies are warranted to understand the molecular basis for this observation. No significant association between the assessed AKT1tagging variants and haplotypes with the risk for EGFRI-related toxicity was observed. A recent study also assessed the impact of two genetic variants (differing from the tagging SNPs we assessed) within the AKT1 gene and treatment response of gefitinib-treated NSCLC patients.9 The authors observed no association with the prevalence of skin toxicity or diarrhea, whereas one of the variants was significantly associated with time to tumor progression and overall survival. We also assessed these variants but did not detect any association with toxicity or survival (data not shown). A limitation of our study might be the heterogeneity of tumors and treatments. With the inclusion criteria being

any treatment with an EGFRI, resulted in a heterogeneous group of patients with different drugs, dosage regime, treatment duration and tumor types. But, given that the EGFRI-related skin toxicity seems to be a class effect, it was, in particular, our intention to evaluate the potential use of EGFR germline genetic variants as predictive marker, irrespective of the applied EGFRI or the cancer type. The limited sample size, short duration of follow-up and the heterogeneity of our sample limit conclusions on possible associations between genetic variations and patient outcomes. In addition, it should be considered that we were not able to stratify for somatic mutations, such as KRAS or EGFR mutations, given that these factors were not routinely assessed for all patients. Nevertheless, a consistent correlation between skin toxicity and survival was observed. In addition, poorer survival among patients carrying the PIK3CA haplotype H4, which was correlated with less skin toxicity, was observed. In conclusion, genetic variation within the EGFR gene as well as in downstream molecules of EGF receptor mediated effects might be predictive for the risk to develop EGFRIrelated skin toxicity. We detected for the first time an association between genetic variants in the PIK3CA gene and the prevalence of skin toxicity and patient outcomes after EGFRI treatment. Future studies are needed characterizing the functional effects of the haplotypes defined by the tagging SNPs in these genes. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was financially supported by a research Grant from the Wilhelm Sander Foundation (Grant No 2008.017.1). We would like ¨ hner and Marianne Habdank for to thank Professor Dr Konstanze Do technical support in genotyping the EGFR CA repeat. SP is a fellow of the International Graduate School in Molecular Medicine, Ulm.

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