© 2007 European Society for Medical Oncology
breast cancer |
Role of the HER2 [Ile655Val] genetic polymorphism in tumorogenesis and in the risk of trastuzumab-related cardiotoxicity
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1 Oncopharmacology unit (EA 3836), Centre Antoine Lacassagne, Nice
2 Medical Oncology Department, Centre Antoine Lacassagne, Nice
3 Biostatistics Unit, Centre Antoine Lacassagne, Nice
4 Equipe INSERM ESPRI 2006 and Laboratory of Clinical and Experimental Pathology, Faculté de Médecine, University of Nice–Sophia Antipolis
5 Institute of Signalling, Developmental Biology and Cancer Research UMR CNRS 6543
6 University of Nice-Sophia Antipolis Equipe labellisée Ligue Nationale contre le Cancer
* Correspondence to: Dr G. Milano, Oncopharmacology unit (EA 3836), Centre Antoine Lacassagne, 33 avenue de Valombrose 06189 Nice, France. E-mail: Gerard.milano{at}nice.fnclcc.fr
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Abstract |
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Background: To examine the impact of a frequent her2 gene polymorphism (Ile655Val) on tumor growth and on the pharmacodynamics of treatment by trastuzumab.
Patients and methods: Experimental study: The growth characteristics of cells expressing the Ile or Val isoform were examined in vitro and after injection into nude mice. The effect of trastuzumab was determined in both experimental models. Clinical study: 61 patients with advanced breast cancers and treated by trastuzumab were genotyped for HER2 by PCR–RFLP. The influence of HER2 genotype on the trastuzumab treatment was examined.
Results: Experimental study: HER2-expressing cells acquired the characteristics of tumor cells. The Val isoform-expressing cells showed the highest growth capacity and developed aggressive tumors sensitive to trastuzumab. Clinical study: There was no link between tumor response or survival and HER2 genotype. All cases of treatment-related cardiotoxicity were found in the Ile/Val group and there was no cardiac toxicity in the Val/Val and Ile/Ile patients.
Conclusions: This study establishes a clear-cut difference between the two HER2 isoforms regarding their tumorogenic potential with an advantage for the Val/HER2 isoform. In breast cancer patients treated with trastuzumab, the presence of a Val allele may constitute a risk factor for cardiac toxicity.
Key words: cardiotoxicity, gene polymorphism, HER 2, trastuzumab
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introduction |
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HER2 (erbB-2, neu) is a proto-oncogene which encodes a transmembrane protein with tyrosine kinase activity but with no identified physiological ligand. The HER2 gene is amplified in 30% of invasive breast cancers and correlated with reduced patient survival [1]. Trastuzumab (Herceptin®), a humanized monoclonal antibody which binds to the HER2 extracellular domain, deeply impacts on the treatment of breast cancers [2]. The HER2 gene is subjected to somatic mutations [3] and to germinal polymorphism. The most investigated germinal polymorphism at clinical level concerns codon 655 (GTC/valine to ATC/isoleucine [4], transmembrane domain of the HER2 protein [5]). Xie et al. [6] reported that women with the Ile/Val or Val/Val genotype had a high risk of breast cancer. These results were followed by several studies most of which failed to confirm the initial observations [4, 7, 8]. Wang-Gohrke and Chang-Claude [9] found, however, that carriers of the Val allele were associated with a two-fold increased risk for breast cancer. Millikan et al. [10] have provided evidence that HER2 codon 655 genotype may predispose to early-onset breast cancer. The allele frequency for the Val form was found to be significant in Caucasians and variable among different populations [6]. This background and a lack of knowledge concerning the functional importance of the Val655Ile polymorphism led us to build the present study in order to examine the impact of this polymorphism on tumor development and growth as well as on the pharmacodynamics of trastuzumab treatment in breast cancer patients.
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materials and methods |
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materials
Restriction and DNA-modifying enzymes were obtained from New England BioLabs or from Eurogentec (Liège, Belgium). Anti-HER-2 polyclonal antibody (9E10) was from Biolabs; anti-Akt anti-pAkt polyclonal antibodies were from Cell Signaling; anti-Erk1/2 polyclonal antibody was a home-made polyclonal antibody; anti-pErk1/2 monoclonal was from Sigma; anti-Raf-1, anti-p21 and anti-p27 monoclonal antibodies were from Pharmingen; anti-pTYR (4G10) polyclonal antibody was from Upstate Biotechnology, Lake Placid, NY; anti-pHER-2 (Tyr877:act.ErbB2) and anti-cleaved PARP polyclonal antibodies were from Ozyme; anti-mice and anti-rabbit HRP-coupled antibodies were from DAKO.
preparation of Val655 and Ile655 pcDNA3 vectors using site-directed mutagenesis
The her-2 Val655 gene cloned in pcDNA3 vector (pcDNA3/her-2) was a generous gift from Y. Yarden (Weizmann Institute, Rehovot, Israël). The pcDNA3/her-2 Ile655 vector was obtained from the pcDNA3/her-2 Val655 by site-directed mutagenesis (QuickChange® XL Site-Directed Mutagenesis kit, Stratagen). Briefly, using synthesized 25 nucleotide primers containing in the middle of their sequence the A
G mutation (underlined nucleotide) 5'-CTGACGTCCATCATCTCTGCGGTGG-3' and 3'-CCACCGCAGAGATGATGGACGTCAG-5' and the following steps: 95°C at 50 s, 60°C at 50 s, 68°C at 20 min for 18 cycles. The amplified product was digested by the restriction enzyme DpnI. The introduction of the mutation was checked using PCR–RFLP with the restriction enzyme BsmAI and by sequencing (MWG Biotech).
cell lines and transfections
HEK 293 and CCL39 cells expressing HER2/Val, HER2/Ile or control vector (pcDNA3) were cultivated as described previously [11]. The HER2 stable transfectants (pcDNA3/her-2 Ile655 or Val655 or pcDNA3 empty vector) obtained as described previously, were cultivated in the same medium supplemented with G418 (400 µg/ml; Invitrogen, France) [11]. We generated clones expressing low levels (Ile1 and Val1) or high levels (Ile2 and Val2) of the receptor.
western blot analyses
Cells were lyzed in Laemmli sample buffer or in a buffer described previously [11]. Proteins were separated by electrophoresis on SDS–polyacrylamide denaturing gel (7. 5% or 12%) and analyzed with the antibodies listed above.
in vitro growth experiments
Cells were plated in six-well plates at a density of 100 000 or 200 000 cells/well. One day later (day 1), the cells were counted in two independent wells using a Coulter counter. This count served as the reference number of cells. Cells were starved in serum-free medium. Half of the cells received or not trastuzumab (up to 100 µg/ml [12]). In both conditions, cells were counted in duplicate at day 1 or day 5 following serum starvation.
in vivo growth experiments and analysis of tumor parameters
Animal experiments were performed in accordance with the regulations of our institute's ethics committee. Each of the above cell lines was injected subcutaneously into the flank of 10 six-week-old NMRI nude male mice (Charles River, l'Arbresle, France) (3 x 106 cells). Trastuzumab (10 mg/kg [13]) was administered three times a week during 3 weeks in five independent mice. The appearance and growth of the tumors were followed during 65 days. Tumor length and width were measured and tumor volume was calculated as p/6 x length x width2 until the animal was killed. At that time, animals were killed (after 65 days or when tumor volume reached 2000 mm3) by spinal cord dislocation and tumors were subsequently removed surgically and weighed; one part of the tumor was fixed in paraformaldehyde overnight for von Willebrand factor (vWF) and Ki67 examination by immunohistochemistry.
clinical study
This was a prospective study conducted on a group of 61 consecutive advanced breast cancer patients with a median age of 50.7 years (30.5–83.1). A sample size close to 60 patients was chosen so as to observe between 5 and 10 patients with cardiotoxicity (based on the prediction of risk described by Seidman et al. [14]). HER2 status was assessed on primary tumor by immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue. All patient candidates for treatment by trastuzumab were overexpressing HER2. HER2 overexpression was evaluated by immunohistochemistry (3+ level) and/or by fluorescence in situ hybridization to detect gene amplification. Performance status (the higher the value, the worse the patient status) was 0 in 13 cases, 1 in 34 cases and 2 in 14 cases. Trastuzumab was administered as a loading dose of 8 mg/kg followed by 6 mg/kg every 3 weeks, starting 21 days after the loading dose. Eighty-seven percent of patients were treated for advanced disease by a first-line combination of trastuzumab and paclitaxel. Thirteen percent of patients were given a second or higher line treatment combining trastuzumab and docetaxel. Fifty-nine percent of patients had received previous treatment by anthracyclines (neoadjuvant or adjuvant). The median follow-up of the group of patients was 22.4 months (18.7–29.7). Response to treatment was evaluated by RECIST criteria and toxicity was recorded following WHO recommendations. For all patients, cardiac function (left ventricular ejection fraction, LVEF) was checked at baseline and every 3 months by a multiple-gated acquisition scan. No patient had pre-existing cardiovascular disease. A significant decrease in LVEF (
20% reduction) was considered as cardiotoxicity [14]. Blood samples were obtained at initial patient biological check-up. Genomic DNA was extracted from buffy coat fractions. HER2 genotypes were determined with a PCR–RFLP-based assay [6].
statistics
For the experimental study, comparisons between tumor parameters were performed by ANOVA. Survival estimates were computed using the Kaplan–Meier method and differences between survival times were assessed using the log-rank test. The 
2 test was used to determine associations between patient genotypes, toxicity (hematological, digestive) and response to trastuzumab-based therapy. The link between cardiac toxicity and different tested variables was examined with Fisher's exact test.
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results |
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experimental study
Determination of the functionality of Val or Ile655 HER2 receptors. Figure 1A shows that HER2/Ile and HER2/Val were equally expressed and activated in HEK 293 cells since there was a good correlation between the presence of the receptor and the phosphorylated HER2 on Tyr1248. Expression of HER2 Ile or Val was sufficient to increase Erk phosphorylation (Figure 1A). An increase in basal Erk activity is characteristic of cells transformed by constitutively active members of the Ras pathway [15]. We then stably transfected CCL39 Chinese hamster lung fibroblasts with these vectors because CCL39 cells express undetectable levels of the different HER receptors family members. They also present characteristics of normal cells which can be growth-arrested by serum deprivation and which can form tumors in mice when transfected with oncogenes [15]. These cells were preferred to epithelial breast cancer cells, which are intrinsically tumorigenic. Moreover, all the cell lines tested express basal HER2 levels. Figure 1B shows two independent clones expressing low and high amounts of HER2. The expressed receptors were constitutively activated since phosphorylated forms of the receptors (p-HER2 Tyr1248) were detected. High-expressing clones were kept in order to be as representative as possible of tumors overexpressing HER2, which is a clinical prerequisite for administration of trastuzumab. Cells were serum starved and their capacity to grow in the absence of growth factors—a characteristic of tumor cells—was studied. Figure 1C shows that the presence of HER2 is sufficient to induce cell accumulation superior to control cells in the absence of growth factors at day 1. At day 4, following serum starvation, the number of control cells remained constant whereas the number of HER2 Ile and Val cells continued to increase (Figure 1D, upper panel). They formed multi-levels even when they reached confluence, demonstrating their anchorage independence, again a characteristic of tumor cells (Figure 1D, lower panel). The number of cells was always greater in the case of the HER2/Val cells. These experiments confirm that the expression of HER2 confers the ability to grow independently of growth factors. This is the first demonstration of such potential conferred to normal cells since previous publications established the role of HER2 in tumorigenic breast-derived cells (MCF7).
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HER2 expression both induces activation of growth signaling pathway and protects cells from apoptosis
The increased number of cells detected in the above experiments can be interpreted as an increase in growth potential and/or protection from apoptosis. We investigated the Erk pathway, which has been described as a major prerequisite for growth or for protection against apoptosis. Figure 2A shows that Erk activity was comparable in exponentially growing cells (control or HER2 transfected). However, after growth factor deprivation, Erk activity remained present in HER2 cells whereas it reached undetectable levels in controls. When control cells exhibited 85% caspase 9 cleavage 24 h after serum deprivation, the Ile or Val clones showed a modest caspase cleavage indicating a decrease of apoptosis in the presence of the receptors (Figure 2B). Val- and Ile-expressing cells exhibited comparable Erk activity but Val-expressing cells presented less caspase cleavage in the long term. These characteristics match closely the data shown in Figure 1, which depicts a higher cell number. This difference between Val and Ile cells can be interpreted as an allele-dependent increase in growth capacity as well as an allele-dependent decrease in apoptosis mediated by growth factor deprivation.
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HER2/Val form is more sensitive to trastuzumab in vitro
Figure 3A shows that trastuzumab reduced HER2 phosphorylation in a dose-dependent manner. Such reduction was already maximal following 24 h of drug exposure. Trastuzumab was more effective on the HER2/Val isoform as shown by the decrease of HER2 phosphorylation. HER2 inhibition also affected downstream events, in particular Erk phosphorylation (Figure 3B). Whereas trastuzumab rapidly blocks HER2 phosphorylation, 72 h of exposure to the drug is necessary to obtain maximal inhibition of Erk phosphorylation. The inhibition of Erks is greater in HER2/Val-expressing cells (at 72 h there was 22% of remaining Erk relative intensity for HER2/Ile cells versus 8% for HER2/Val cells).
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only HER2/Val-expressing cells develop tumors in nude mice
Figure 4A shows that HER2/Val cells induced tumor formation in 100% of cases (percentage of mice with a tumor over injected mice, n = 5). Detectable tumors appear 10 days after cell injection. In contrast, a 10% incidence of tumors was obtained for HER2/Ile cells (two tumor-bearing mice over 20 injected mice). The relative lack of growth of the HER2/Ile cells did not result from loss of the HER2 transgene since it was still present in the two tumors that had grown (data not shown). Figure 4B shows that trastuzumab efficiently inhibits the growth of HER2/Val cells but has no effect on control cells (median at 25 days 313 mm3 for the control and 166 mm3 for trastuzumab-treated mice, P < 0.05). This result correlates with the potential of trastuzumab to block Erk activity in vitro. When trastuzumab treatment was stopped the tumor grew again to reach a volume greater than the untreated tumors. This observation was compatible with an increase in Ki67 labeling in Val tumors treated with trastuzumab. In contrast, when examining neo-angiogenesis (von Willebrand factor), Val tumors treated with trastuzumab depicted less intense labeling as compared with untreated ones (data not shown).
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clinical study
Figure 5A and B show an example of a PCR–RFLP experiment for patient genotyping. Distribution of the HER2 polymorphism in the studied patients was: 36 Ile/Ile (59%), 21 Ile/Val (34.4%) and 4 Val/Val (6.6%). These percentages match closely figures described previously in a large number of Caucasians [6]. The distribution of the Ile655Val genotype agrees with that predicted by the Hardy–Weinberg equilibrium (P = 0.72). There were 13 (21.3%) complete responses, 28 (45.9%) partial responses, 10 (16.4%) stabilized diseases and 9 (14.8%) progressive diseases under trastuzumab-based treatment (one case not assessable). No significant link existed between HER2 genotype and either response (objective response at 27.8%, 50% and 33.3% in Ile/Ile, Val/Val and Ile/Val patients, respectively; P = 0.57) or disease-free survival (median 18 months). In addition, hematological and digestive toxicity were not related to HER2 Val655Ile polymorphism. Attention was paid to cardiotoxicity as it is particularly relevant to trastuzumab treatment. Five patients (8.2%) presented a reduction of
20% in their LVEF (Table 1). Table 2 depicts the distribution of different variables according to the presence or absence of cardiac toxicity under trastuzumab-based treatment in the study population. Neither age nor performance status was associated with cardiotoxicity. Associated treatments (irradiation, pre-existing chemotherapy by anthracyclines) were not linked with the development of cardiac toxicity. The Val655Ile genotype was shown to be associated with cardiotoxicity. All cases were found in the Ile/Val group and there was no cardiac toxicity in Ile/Ile homozygous subjects.
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discussion |
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The present data show that only cells carrying the HER2/Val655 form were able to promote tumors in nude mice. This finding may have several implications. The first is to provide fresh argument in the debate over the increased risk for women with either Ile/Val or Val/Val genotypes [4, 6–10, 16, 17] of developing breast cancer. Extrapolation of the present data to the human context may be limited by the fact that HER2 molecular functioning is partly dependent upon the creation of a partnership with other HER family members [18, 19]. The present data may stimulate other experimental studies devoted to comparisons between HER2 homodimers and heterodimers. Another limitation may be the notion of haplotype and linkage desequilibrium applied to the HER2 gene. A recent study by Han and coworkers [20] shows that the HER2 gene includes 29 single nucleotide polymorphisms (SNPs). Six of them, including codon 655, are polymorphic and exhibit >10% rare allele frequency. Han and coworkers [20] have shown that all six SNPs exhibit a strong linkage desequilibrium. This may imply that the putative clinical impact of a given variant can be diluted and carried in part by other variants in linkage desequilibrium within the haplotype. This haplotype-related dilution of the functional impact of a given polymorphism may explain the discrepancies among authors regarding the link between Ile655Val germinal polymorphism of HER2 and the risk of developing breast cancer. Nevertheless, the difference in tumor induction capacity between the Val and the Ile alleles was particularly striking in the present study and this observation should be borne in mind when considering the possible role of HER2 Ile655Val polymorphism in human mammary carcinogenesis. Fleishman and coworkers [5] have concluded that Val alleles would be more inclined to intrinsically activate the protein tyrosine kinase. Our data concur with this model since the Val clones exhibited a higher proliferating capacity in the absence of serum. This may encourage the conducting of retrospective and prospective investigations on the prognostic value of HER2 genotyping at codon 655. The experimental section of the present study suggested, in vitro, a higher sensitivity to trastuzumab for the Val clones. However, we were unable to confirm this differential allele-related effect of trastuzumab in treated animals since only Val clones developed measurable tumors. Thus, the HER2 genotype at codon 655 may not add to the predictive power of HER2 overexpression itself. It is unlikely that variability in a single active tyrosine kinase receptor is responsible for a difference in the antitumor efficacy of the targeted agent as here introduced by the Val655Ile polymorphism of the HER2 gene. The antitumor variability of trastuzumab treatment is probably more related to changes in the HER2 signaling pathway, as occurs following a loss of PTEN [21], or to the presence of alternative receptor signaling for growth and survival, as with IGF-1R [22]. Treatment by trastuzumab is associated with the occurrence of cardiotoxicity [23, 24]. In the present group of 61 patients, five patients (8.2%) presented cardiac toxicity manifested by a
20% reduction of their LVEF. We examined possible variables related to the cardiac events such as patient characteristics (age, performance status) or associated treatments (irradiation, pre-existing chemotherapy by anthracyclines) and none of them were found to be linked to the development of cardiac dysfunction under trastuzumab treatment. There was no cardiac toxicity in the larger group containing Ile/Ile homozygous subjects. In contrast, all cases were found in the Ile/Val group. The fact that no cardiotoxicity was observed in the Val/Val homozygous patients does not necessarily limit the importance of the finding suggesting a possible role for the Val allele as a predisposing factor for cardiotoxicity. This is because there were only four subjects in the Val/Val group and also because the gene dosage from Ile to Val does not necessarily correspond to a proportional modification in protein function. HER2 functions as homodimers and the change in intracellular signaling generated by the Val/Ile complex may be greater than the Val/Val complex due to different steric bulks located in the transmembrane domain of HER2 [5]. There are few experimental arguments which support a major role for HER2 signaling in cardiomyopathies [25]. In all, one can consider that HER2 signaling plays a major role in cardiac function. The present experimental data indicate that Val-expressing cells exhibit a higher growth capacity and are more sensitive to trastuzumab. Thus, the presence of the Val allele may render cardiomyocytes particularly dependent upon HER2 signaling and highly sensitive to trastuzumab. The present data could be of particular importance for breast cancer patients treated with trastuzumab. A series of 61 patients was limited but representative of cardiotoxicity related to trastuzumab treatment and of allelic distribution for codon 655 HER2 polymorphism. Our observation warrants larger retrospective and prospective studies aimed at strengthening these data. This would be fully justified since the introduction of trastuzumab in the adjuvant setting of breast cancer [26, 27] has increased the population concerned with trastuzumab treatment, with a significant economic impact [28].
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Acknowledgements |
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This work was supported by the Ligue Nationale Contre le Cancer (Gilles Pagès équipe labellisée), the Cancéropole from the Région PACA and the Association for International Cancer Research (AICR). We thank Roche-France for financial support to perform these experiments. We also thank Doctor Jean-Claude Chambard for helpful discussions.
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Footnotes |
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These authors co-directed the present work. 
Received for publication January 31, 2007. Revision received March 28, 2007. Accepted for publication April 5, 2007.
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References |
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