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Pharmacogenetic Profiling for Cetuximab Plus Irinotecan Therapy in Patients With Refractory Advanced Colorectal Cancer

Francesco Graziano, Annamaria Ruzzo, Fotios Loupakis, Emanuele Canestrari, Daniele Santini, Vincenzo Catalano, Renato Bisonni, Umberto Torresi, Irene Floriani, Gaia Schiavon, Francesca Andreoni, Paolo Maltese, Eliana Rulli, Bostjan Humar, Alfredo Falcone, Lucio Giustini, Giuseppe Tonini, Andrea Fontana, Gianluca Masi, Mauro Magnani

From the Medical Oncology Unit, Hospital of Pesaro, Pesaro; Institute of Biochemistry "G. Fornaini," and Institute of Biotechnology, University of Urbino, Urbino; Medical Oncology Unit, Azienda USL-6 Livorno, Livorno; University of Pisa, Istituto Toscano Tumori, Pisa; Medical Oncology Unit, University Campus Biomedico, Rome; Medical Oncology Unit, Hospital of Fermo, Fermo; Medical Oncology Unit, Hospital of Macerata, Macerata; Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; and Cancer Genetics Laboratory, University of Otago, Otago, New Zealand

Corresponding author: Francesco Graziano, MD, Medical Oncology Unit, Hospital of Urbino, via Bonconte da Montefeltro, 61029, Urbino, Italy; e-mail: frada{at}tin.it

	ABSTRACT

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Purpose Regulation of epidermal growth factor receptor (EGFR) signaling pathways may play a relevant role in determining the activity of cetuximab therapy in patients with metastatic colorectal cancer (MCRC). We investigated possible associations between genetic variants and clinical outcomes of MCRC patients treated with cetuximab-irinotecan salvage therapy.

Patients and Methods Patients who underwent cetuximab-irinotecan salvage therapy after disease progression during or after first-line bolus/infusional fluorouracil, leucovorin, and oxaliplatin chemotherapy and a second-line irinotecan-based regimen were considered eligible for analysis of polymorphisms with putative influence on cetuximab-related pathways. Epidermal growth factor (EGF) 61A>G, EGF receptor (EGFR) 216G>T, EGFR 497G>A, EGFR intron-1 (CA)_n dinucleotide short (S)/long (L) variant, cyclin-D1 870A>G, immunoglobulin-G fragment-C receptors RIIIa 158G>T, and RIIa 131G>A were studied for a possible association with overall survival (OS) as the primary end point. Additional analyses were addressed at possible associations among polymorphisms and EGFR expression, toxicity, and response.

Results In 110 assessable patients, significant association with favorable OS was observed for EGFR intron-1 S/S and EGF 61 G/G genotypes. In the multivariate model, EGFR intron-1 S/S and EGF 61 G/G genotypes showed a hazard ratio of 0.41 (95% CI, 0.21 to 0.78; P = .006) and 0.44 (95% CI, 0.23 to 0.84; P = .01), respectively. EGFR intron-1 S/S carriers showed more frequent G2-G3 skin toxicity (² test = 12.7; P = .001) and treatment response (² test = 9.45; P = .008) than EGFR intron-1 L/L carriers.

Conclusion Although additional studies are required for confirmation, our findings could optimize the use of cetuximab in MCRC patients.

	INTRODUCTION

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Cetuximab is a chimeric immunoglobulin G1 monoclonal antibody that targets the epidermal growth factor receptor (EGFR) extracellular domain.¹ It competitively inhibits endogenous epidermal growth factor (EGF)/transforming growth factor binding, thereby inhibiting EGFR activation.² Cetuximab has shown activity in metastatic colorectal cancer (MCRC)³ and has been approved for use in conjunction with irinotecan in irinotecan-refractory patients. The approval is currently restricted to patients with EGFR-expressing tumors (by immunohistochemistry, approximately 70% of colorectal carcinomas). Notably, this restriction results from the choice of excluding EGFR-negative patients in clinical trials, which led to this approval. In fact, an affordable marker for selecting patients who benefit from cetuximab therapy has not yet been identified.^2,3 One possibility for optimizing monoclonal antibody therapy may be the application of pharmacogenetics.^4,5

A polymorphic (CA)_n-repeat variant in EGFR gene intron-1, a G>A single nucleotide polymorphism (SNP) in EGFR codon 497 (extracellular subdomain IV), and the –216G>T EGFR promoter SNP within the Sp-1 binding site (Sp-1 is a key regulator of the EGFR promoter activity) are three functional EGFR variants that have been associated with EGFR regulation.^5-11 Modulation of the EGFR ligand EGF and of the downstream EGFR signaling, including the cyclin-D1 gene, also may play a role in modulating cetuximab activity. Functional variants have been described in the EGF 5'-untranslated region (EGF 61G>A),¹² and in exon 4 of the cyclin-D1 gene (870A>G).^13,14 Finally, monoclonal antibodies may exert an indirect antitumor activity by recruiting cytotoxic host effector cells such as monocytes and natural-killer cells,¹⁵ thus determining an antibody-dependent cell-mediated cytotoxicity (ADCC). As observed in experimental models, cetuximab also mediates ADCC against cancer cells.¹⁶ The effectiveness of ADCC may depend on the degree of activation of effector cells after immunoglobulin-G fragment-C receptors (FcR) IIa (CD32a) and IIIa (CD16) engagement. SNPs in FcR-IIIa (158T>G) and FcR-IIa (131G>A) were shown to affect receptors' affinities for the Fc of antibodies and probably ADCC efficiency.^17,18

We studied these genetic variants (Table 1) in patients who underwent cetuximab-irinotecan therapy after disease progression during or after bolus/infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX) and a second-line irinotecan-based regimen. The primary end point of this study was the association between genetic variants and overall survival (OS). Additional analyses were addressed at possible associations between polymorphisms and progression-free survival (PFS), EGFR expression, toxicity, and response.

	PATIENTS AND METHODS

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Analysis of Genetic Variants
A blood sample collected from each enrolled patient before the onset of chemotherapy was used for genotyping. Genomic DNA was extracted using the QiaAmp kit (Qiagen, Valencia, CA). All variants (Table 1) were investigated using a polymerase chain reaction (PCR) –restriction fragment length polymorphism technique, except for EGFR intron-1, for which the CA-repeat number was determined using a 35-cycle PCR, followed by separation with capillary electrophoresis on an ABI 310 system (Applied Biosystems, Foster City, CA). The assays with primers and sequences for studying the genetic variants were performed as described previously.^9-13,17,18

In vitro and in vivo data indicate that the length of the EGFR intron-1 (CA)_n repeat inversely correlates with transcription activity, mRNA amount, and EGFR expression.^5-9 Given the trimodal distribution of the EGFR intron-1 (CA)_n-repeat alleles in the white population,^5,7 and the experimental data indicating enhanced EGFR levels with alleles having less than 17 CA repeats,^5,8,9 EGFR intron-1 less than (CA)₁₇ and (CA)₁₇ repeats defined short (S) or long (L) alleles, respectively. For the purpose of the study, this classification produces three possible EGFR intron-1 (CA)_n genotypes (S/S, S/L, L/L).

Statistical Analyses
The primary end point of the study was the association between pharmacogenetic data and OS. OS was defined as the time from the start of therapy to death from any cause. Patients known to be alive at the time of analysis were not censored at their last available follow-up assessment. PFS was also analyzed and it was defined as the time from the beginning of chemotherapy to first appearance of progression or death from any cause. The analysis was planned at the occurrence of 83 deaths (75% of the total sample patients). This number would allow detection of a relative hazard reduction of at least 50%, assuming a minimum 30% prevalence of the category with a favorable genotype (80% power and 5% type I error for a two-tailed test). Due to the elevated number of deaths and the longest follow-up periods were observed for patients who eventually died, the median follow-up has not been reached. To describe the completeness of follow-up, we used the C index.²¹ It is a simple measure of completeness that is the ratio of the total observed person-time and the potential person-time of follow-up in a study (C values range from 0 to 100, with 100 indicating the highest completeness).²¹ Cox proportional hazards models, as implemented in the PHREG (proportional hazards regression) program in SAS software (SAS Institute Inc, Cary, NC), were used to estimate and test genotypes and baseline clinicopathologic features (age, sex, KPS, carcinoembryonic antigen levels, lactate dehydrogenase levels, number of organs involved, and presence of liver metastasis) for their association with OS and PFS. Polymorphisms were included as dummy variables, using as reference the genotype showing the worst prognosis. Variables that were found to be associated with OS and PFS in the univariate analysis were considered for the multivariate analysis. The assumption of proportional hazards was verified.²² Results are expressed as hazard ratio (HR) with 95% CI. Survival curves were estimated using the Kaplan-Meier method. The ²-test with appropriate degrees of freedom (df) was used for comparing proportions of patients for demographic features, categoric variables, and genotypes. All polymorphisms were examined for deviation from Hardy-Weinberg equilibrium (HWE) by comparing actual allelic distributions with those expected from HWE using a ² test (df = 2). Statistical significance was set at P < .05. Given the exploratory nature of this study, no adjustment for multiple testing was performed. Thus, P values are not strictly definitive, and the analyses are considered hypothesis generating.

Linkage disequilibrium (LD) and haplotype analyses were performed to further define possible clinical effects of genetic variants.²³ Haplotype frequencies were reconstructed in the study population, which was dichotomized according to the median value (below and above) of a given clinical parameter (eg, OS). LD exists between two SNPs of a haplotype if their variants appear together more often than expected (nonrandom inheritance). LD was estimated using r², with r² = 1 indicating complete LD and r² = 0 indicating absence of LD. The SHEsis software platform²³ (http://202.120.7.14/analysis/myAnalysis.php) was used to estimate haplotype frequencies and the presence of LD. Global association of haplotypes with clinical parameters was estimated by comparing haplotype distributions among dichotomized patients using a ² test (df = 7 for EGFR, df = 3 for Fc). The HAP analysis tool (http://diego.cs.UCLA.edu/hap/html/)²⁴ was used to assign haplotypes to individual patients. Patients were subdivided according to their haplotypes and a log-rank test was performed to compare their survival times. Survival curves were estimated using the Kaplan-Meier method.

	RESULTS

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Study Population
One hundred ten white patients were studied (Table 2). After second-line, irinotecan-based chemotherapy,²⁵ irinotecan with weekly cetuximab (400 mg/m² loading dose followed by 250 mg/m²) was administered as weekly (90 to 100 mg/m²) or every 2 weeks (180 mg/m²) infusion in 33 patients and 77 patients, respectively.

Survival and Genotypes
In the univariate model, age, KPS, EGFR intron-1 (CA)_n, and EGF 61A>G were the baseline features, which showed significant association with OS and PFS. We performed an analysis assessing all the first-order interaction in Cox model among age, KPS, and polymorphism. No evidence of interaction was detected. In the multivariate model (Table 3), EGFR intron-1 S/S and EGF 61G/G homozygous genotypes were significantly associated with favorable OS. A borderline association between EGF 61A/G heterozygous genotype and favorable OS was observed. Kaplan-Meier survival curves for EGFR intron-1 (CA)_n and EGF 61A>G variants are shown in Figure 1. Given the clinical relevance of skin toxicity in cetuximab-treated patients, it was added in the model. In the multivariate model, grades 2 to 3 skin toxicity were significantly associated with favorable OS (HR = 0.62; 95% CI, 0.48 to 0.81; P = .0004), whereas the associations between OS and EGFR intron-1 S/S (HR = 0.54; 95% CI, 0.28 to 1.00; P = .05) and EGF 61G/G (HR = 0.55; 95% CI, 0.29 to 1.05; P = .06) genotypes were weakened.

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. (A) Survival curves (vertical marks indicate censored observations) of patients with EGFR intron-1 short/short (S/S), short/long (S/L), and long/long (L/L) genotypes. Median overall survival (OS) times for EGFR intron-1 L/L, S/L, and S/S carriers were 5.6 (95% CI, 4 to 11), 9.8 (95% CI, 7.2 to 10.4), and 13 (95% CI, 10.8 to 14.3) months, respectively (log-rank = 8.69; P = .01). (B) Survival curves (vertical marks indicate censored observations) of patients with EGF 61A/A, A/G, and G/G genotypes. Median OS times for EGF 61A/A, A/G, and G/G carriers were 7.5 (95% CI, 4.9 to 10.2), 10 (95% CI, 9.1 to 12.3) and 11.5 (95% CI, 8.6 to 14.4) months, respectively (log-rank = 8.58; P = .013). (C) Survival curves of patients carrying the eight different EGFR haplotypes. Numbers in brackets represent the P values associated with the comparison of survival curves of the T-L-A haplotype with the other seven EGFR haplotypes (log-rank test).

	DISCUSSION

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The occurrence of skin toxicity was found to be a reliable indicator of cetuximab efficacy.^34,35 This adverse effect was found to be associated with the presence of the EGFR intron-1 short (CA)_n allele in the present as well as in a previous analysis of colorectal cancer patients.⁹ EGFR homodimers seem to be the predominant human epidermal growth factor receptor dimer pair in normal primary keratinocytes and in normal skin tissue.³⁶ Preliminary data suggest that EGFR dimerization in human skin and inhibition of EGFR homodimer signaling rather than EGFR/HER-2 heterodimer signaling may be a key molecular event determining dermatologic toxicity.³⁶ Germline EGFR intron-1 S/S status resulting in EGFR upregulation in tissues may trigger this molecular predisposing condition for skin toxicity. The median number of cetuximab infusions was 12 in EGFR intron-1 L/L carriers (range, six to 36 infusions) and 18 in EGFR intron-1 S/S carriers (range, four to 52 infusions). Cetuximab-induced skin toxicity occurs early,³⁴ and it is therefore unlikely that the observed grade 2 to 3 skin toxicity in EGFR intron-1 S/S carriers is simply the expression of the patients' longer treatment period. Notably, grade 2 to 3 skin toxicity in EGFR intron-1 S/S carriers may explain the slightly lower relative dose-intensity of cetuximab with respect to EGFR intron-1 L/L carriers. The strong association between high-grade skin toxicity and favorable OS obscured the impact of the other variables, including genotypes. However, it should be considered that skin toxicity is not a baseline feature for a possible selection of patients before cetuximab onset. In perspective, the assessment of the EGFR intron-1 status might be considered for planning innovative treatment strategies, such as cetuximab dose escalation according to the entity of the observed skin toxicity.³⁷

The EGF 61G allele is transcriptionally more active than the A allele and is found to be associated with upregulated EGF levels. EGF signaling may promote a number of regulatory factors, which enhance tumor aggressiveness³⁸; therefore, the observed favorable effect of the EGF 61G/G genotype may be counterintuitive. Furthermore, the EGF-ERBB system displays complex tunings and the presence of alternative negative signaling regulators.^39-41 At specific concentrations that vary between experimental systems, EGF has been shown to induce apoptosis and growth inhibition rather than the usual growth-promoting effect.^42-46 According to such findings, it cannot be ruled out that a functional EGF genotype, which upregulates EGF levels, may play a favorable prognostic rather than predictive influence. Indeed, in the present study, the EGF 61G/G genotype was associated with improved OS and not with improved PFS or response rate/skin toxicity. Notably, similar findings have been reported recently by Ali-Osman et al,⁴⁷ who analyzed EGF 61A>G in 332 astrocytoma patients, with the G/G carriers having significantly better survival rates than the A/A carriers.

It is not surprising that our findings are in conflict with results from two previous investigations that include 39 patients from a phase II multicenter trial of cetuximab.^48,49 In these studies, Zhang et al found preliminary associations for Fc-RIIa/Fc-RIIIa⁴⁸ and cyclin-D1 variants.⁴⁹ Notably, they studied the EGFR intron-1 (CA)_n by simply subdividing patients into two groups: 16 carriers of both (CA)_n < 20 alleles, and 18 carriers of any (CA)_n 20 alleles, with five missing cases. Our sample size allowed for a more precise distinction of genotypes, with three distinct groups including two homozygous (S/S and L/L) and the heterozygous S/L genotype. However, Zhang et al may have missed a dose-dependent effect of the EGFR intron-1 (CA)_n variant with both S alleles present (CA repeats < 17) in the EGFR intron-1 S/S genotype (Fig 1A). From a practical perspective, the assessment of the EGFR intron-1 (CA)_n is easy, reproducible, and does not change over time. Potential confounders for its predictive role might be genetic changes in cancer cells. However, loss of heterozygosity does not seem to occur frequently at the EGFR intron-1 locus,^5,50 and EGFR mutations in exons 18, 19, 21, which predict response to TK-EGFR inhibitors in NSCLC, were not found in colorectal carcinomas.³⁵ The frequency of EGFR promoter hypermethylation in colorectal cancer is unknown,⁵¹ whereas the percentage of colorectal tumors with increased EGFR copy number cannot be precisely estimated, and according to series, may be as low as 0.6%⁵² to 12%.⁵³ Notably, Buerger et al⁵⁴ found an interaction between the length of the germline EGFR intron-1 variant and the frequency of EGFR amplification.

We found frequencies of the EGFR intron-1 genotypes that are comparable to those reported in the literature, with short (CA)_n alleles more common in white than in Asian populations and long (CA)_n alleles are less common in white than in Asian populations.⁷ Even if different genetic backgrounds may represent a limitation for reproducibility and applicability of our findings, the EGFR intron-1 S/S genotype seems to confirm its favorable role,^26,27 TK-EGFR with identification of subsets of responsive patients, as domain mutations allowed in gefitinib-treated NSCLC patients.¹ Pharmacokinetic factors do not appear to have influenced our findings; all patients showed progression after second-line irinotecan-based chemotherapy, no apparent pharmacokinetic interactions between cetuximab and irinotecan were described,⁵⁵ and the studied polymorphisms should not influence cetuximab activation/excretion pathways.

A control arm would have been helpful for defining the predictive role of the studied polymorphisms. In reality, EGFR expression is detectable in the majority of colorectal carcinomas and almost all patients with EGFR-expressing tumors receive cetuximab. Therefore, it is difficult to plan an association analysis that includes controls. When we studied EGF and EGFR genotypes in patients treated with first-line FOLFOX chemotherapy¹⁹ and the previously studied genetic variants¹⁹ in the present population of cetuximab-treated patients, no significant association with clinical outcomes was observed (data not shown). These observations may at least in part sustain a predictive effect of the EGFR intron-1 genotype when cetuximab is added in the course of treatments.

In conclusion, the present findings deserve further research; they may represent a valuable tool for optimizing the use of the anti-EGFR cetuximab in MCRC.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Francesco Graziano, Annamaria Ruzzo, Fotios Loupakis, Mauro Magnani

Financial support: Mauro Magnani

Administrative support: Annamaria Ruzzo, Mauro Magnani

Provision of study materials or patients: Francesco Graziano, Annamaria Ruzzo, Fotios Loupakis, Daniele Santini, Vincenzo Catalano, Renato Bisonni, Umberto Torresi, Gaia Schiavon, Alfredo Falcone, Lucio Giustini, Giuseppe Tonini, Andrea Fontana, Gianluca Masi

Collection and assembly of data: Francesco Graziano, Annamaria Ruzzo, Emanuele Canestrari, Irene Floriani, Francesca Andreoni

Data analysis and interpretation: Francesco Graziano, Annamaria Ruzzo, Emanuele Canestrari, Irene Floriani, Eliana Rulli, Bostjan Humar, Mauro Magnani

Manuscript writing: Francesco Graziano, Annamaria Ruzzo, Fotios Loupakis, Irene Floriani, Bostjan Humar, Mauro Magnani

Final approval of manuscript: Francesco Graziano, Annamaria Ruzzo, Fotios Loupakis, Daniele Santini, Vincenzo Catalano, Renato Bisonni, Umberto Torresi, Irene Floriani, Gaia Schiavon, Paolo Maltese, Eliana Rulli, Bostjan Humar, Alfredo Falcone, Lucio Giustini, Giuseppe Tonini, Andrea Fontana, Gianluca Masi, Mauro Magnani

	NOTES

 
Supported by Consorzio Interuniversitario per le Biotecnologie and Fanoateneo.

F.G. and A.R. contributed equally to this study.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted May 4, 2007; accepted October 4, 2007.

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